The present invention relates to an efficient hydrolyzate production process from lignocellulosic feedstock. Further, the present invention is mutant xylanases relates to the use of the manufacturing method and mutant xylanase mutant xylanases.
BACKGROUND
[0002]
　Xylanase is an enzyme that hydrolyzes randomly beta-l, 4 bonds of xylan constituting the plant cell wall. Enzyme, a) the lignocellulosic feedstock saccharification, b) pulp bleaching, c) the animal feed additive, d) application to a wide range of applications such as detergent auxiliaries and e) Baking modifier is expected.
[0003]
　Saccharification of a) lignocellulosic feedstock: from lignocellulosic feedstock, saccharification method lignocellulosic feedstock to produce monosaccharides as a fermentation substrate using an enzyme is known. However, cellulase usable in the sugar reduction method, it is high prices of the enzyme hemicellulase (xylanase, etc.) has become an obstacle to the practical use of the sugar reduction method. Therefore, as an effective means for reducing the cost of the sugar reduction method, it has been proposed to reuse the enzyme used in the sugar reduction method (for example, Japanese 2006-87319 (Patent Document 1), International Publication No. 2011-065449 pamphlet see (Patent Document 10) and WO 2011-125056 pamphlet (Patent Document 11)).
[0004]
　Xylanase is an enzyme that divides the hemicellulose (composed mainly beta-l, 4-xylan) which is one of the main components of lignocellulosic feedstock. In the saccharification method therefore lignocellulosic feedstock, is one of important enzymes. However, xylanase is known to have low stability.
　On the other hand, the saccharification of lignocellulosic feedstock, an acidic region pH 4.0 ~ pH 6.0, high temperature of 40 ° C. ~ 60 ° C., and requires a process over several days. Therefore, the stability of the low level of xylanase, are preventing enzyme reuse.
[0005]
　Trichoderma reesei (hereinafter, abbreviated as "T.reesei".) From thermostable xylanase variants (e.g. WO 2007/115391 pamphlet (Patent Document 2) and WO 2007/115407 pamphlet (Patent Document 3) see.) shows a 50 ° C. ~ 80% more than 30 minutes after the heat treatment at 70 ° C. for residual activity.
　Further, (see for example, Japanese 2004-121257 (Patent Document 4).) Bacillus sp derived from thermostable xylanase also, 70 ° C., are known to exhibit a residual activity of 90% or more after heat treatment of 30 minutes there.
[0006]
　b) pulp bleaching: The use of xylanase in pulp bleaching process, it is known that it is possible to reduce the amount of bleaching agent.
　Generally, pulp bleaching in the paper industry, delignification (pH10 ~ pH12,80 ℃) from the pulp by preceding oxygen treatment step, consisting of the subsequent bleaching step. The reason for this manner the bleaching process at a later stage, lignin few percent after a delignification with oxygen is to remain in the pulp as a coloring component. In addition to the delignification process and the bleaching process, by adding a step of reacting xylanase, it is possible to cut the hemicellulose chains which are bound to lignin and cellulose. Thus, lignin can be effectively removed, effect of reducing the amount of bleaching agent used in the bleaching process can be expected.
[0007]
　For efficient step of reacting the xylanase, 70 ℃ ~ 80 ℃, it is necessary to use a xylanase with a pH10 characteristics that can withstand the processing of a few hours before and after.
　T. thermostable xylanase mutants derived reesei (e.g., Patent Document 2, Patent Document 3, WO 2001/92487 pamphlet (see Patent Document 5) and WO 2003/046169 pamphlet (Patent Document 6)) is , before and after the optimum reaction temperature of 70 ° C., optimum reaction pH is 7-8, the possibility of use in the pulp bleaching process is shown.
[0008]
　c) animal feed additive: animal feed because it is rich in vegetable fibers, by addition of xylanase, since capable of degrading plant cell walls in animal feed, it is possible to improve the absorption efficiency of the plant nutrients by the animal.
　When pelleted animal feed using a xylanase, stability is determined for xylanase to withstand approximately 10 minutes at about 70 ° C. ~ about 90 ° C.. Further, in order to act xylanase in animal digestive organs, before and after 40 ° C., it is necessary to exhibit high activity in an environment of about pH 4.8.
　Many xylanase derived from a filamentous fungus such as Trichoderma or the genus Acremonium sp is optimum pH pH 3 ~ pH 5, the operating temperature range is around 40 ° C..
[0009]
　Patent Documents 2 and 3, in the thermostable xylanase mutants have been described in WO 2001/27252 pamphlet (Patent Document 7) and WO 2005/108565 pamphlet (Patent Document 8), optimum pH is as mutants around pH 5 ~ pH 5.5.
[0010]
　d) Detergent adjuncts: By using a xylanase as a detergent adjunct, can be removed fuzz clothing.
　Since the recent drum type washing machine has a water-saving, fine fuzz-prone by overlapping washing times. When the fuzz occurs, prone to re-contamination of clothing.
　The use of xylanase as a detergent adjunct, can be removed this fuzz. Therefore, it becomes possible to prevent re-contamination. In addition, the main component of stains derived from vegetables and fruits that were attached to the garment is a cell wall derived from the bound vegetables and fruits of the dye. Therefore, by using the xylanase during washing, can be effectively cleaned even when a water-saving drum type washing machine.
[0011]
　When using xylanase as a detergent adjunct, it is necessary to use a xylanase with an alkaline resistance and detergent resistance. Also, when using a xylanase laundry cleaning, it is necessary to use a xylanase acting stably in a high temperature range of 50 ℃ ~ 70 ℃.
　T. reesei derived heat resistance alkali resistance xylanase mutants (e.g., see Patent Document 2, Patent Document 3, Patent Document 5 and Patent Document 6.) is optimum temperature 62 ° C. ~ 75 ° C., the optimum pH is pH 7 ~ pH 8 with the properties such.
　Xylanase variants shown in Patent Document 8, although the optimum pH is in the pH5 acidic side, a optimum temperature 70 ° C., 60 ° C. in pH 8 ~ pH 9, for 10 minutes to maintain the 100% activity ing.
　Derived from the genus Bacillus of the heat-resistant alkali-resistant xylanase (e.g., see Patent Documents 4 and JP 2007-54050 (Patent Document 9).) Both have the optimum temperature range to 50 ° C. ~ 70 ° C., Itaru suitable pH has the property that pH7 ~ pH8. In 4 ° C.-5 ° C., 1 day to 2 days, the pH9 with 100% activity is maintained.
[0012]
　e) Bread modifier: bread by using a xylanase as a modifier, it is possible to improve the quality of bread.
　Xylanase has the nature to degrade hemicellulose part of the flour. By hemicellulose portion is degraded by a xylanase, moisture attached to this moiety is released in the dough, it changes the nature of the dough occurs. As a result, by the particle structure and loaf volume of bread is improved, leading to better quality retention of the bread.
　During preparation of the dough, in addition with a large physical impact and pressure loads in stirring the material kneading step, it requires 1-2 hours of time 35 ° C. - 40 ° C. in the fermentation process.
Summary of the Invention
Problems that the Invention is to Solve
[0013]
　However, reuse of saccharifying enzymes mentioned saccharification of (a) the lignocellulosic feedstock, from the viewpoint of effective utilization of sugar production costs and lignocellulose resource, there is room for improvement.
　The saccharification enzyme recycling described in Patent Document 1, by the enzyme to bind lignocellulosic residues, saccharification activity of the enzyme has been shown to reduce. Therefore, there is provided a limitation on input of enzyme and substrate.
　In particular, the amount of enzyme, in the Examples of Patent Document 1, the lignocellulose is described as degradable amounts greater than 96% at 12 hours, are mentioned points require introduction of large amounts of enzyme. Accordingly, the economic standpoint, it is necessary to re-use of the enzyme over a long period of time.
　Moreover, the lignocellulose concentration as a substrate as low as about 1%, less sugar concentration to be produced. Therefore, considering the use of sugars to such as ethanol fermentation process, capital investment to correspond to the concentration or the like before volumetric efficiency and ethanol fermentation saccharification tank is required, and the industrial process in an economical point of view it is hard to say.
　Therefore, in the saccharification of lignocellulose containing hemicellulose, a high concentration of lignocellulose can decompose, and since it is considered that a long period is needed enzymes to withstand reuse in, as described above, the stability of the xylanase low becomes a problem.
[0014]
　On the other hand, Patent Document 10, enzymes such as cellulase and hemicellulase have been described to maintain the activity even after adsorption to the residue. Moreover, the saccharification enzyme to the lignocellulose was recovered by suction after the reaction, there is described how to reuse in the next glycation.
　However, in Patent Document 10, advance a lignocellulose as a saccharification raw material, heat treatment under acidic conditions, are subjected to degradation of hemicellulose in lignocellulosic. Therefore, since it becomes necessary to install equipment of the pressure vessel or the like having a heating costs and acid resistance, is not preferable in economical point of view.
　Therefore, although the degradation of hemicellulose by xylanase is desired, since over glycation long, as described above, its low stability becomes a problem.
[0015]
　In Patent Document 11, by increasing the saccharification enzyme amount used in initial reaction, it reduces additional charged amount of enzyme amount corresponding to the lost activity when the enzyme recycling, to be able to reduce the cost of the enzyme as a whole Has been described.
　However, in practice, because of the added-on 1/3 things enzyme amount of initial charged amount of enzyme, not the preferred method in economical point of view. Further, in Examples of Patent Document 11, it is described that discard the reaction residue. It is a major factor which can not reduce the amount of enzyme to add administered enzyme adsorbed to the residue is lost.
　Further, in Examples of Patent Document 11, it is described the use of cellulose contained in lignocellulose, that is, the utilization of glucose only. The straw of preprocessed used in Examples of Patent Document 11 includes the hemicellulose and the like 35% or more. Considering the effective utilization and economic point of view the lignocellulose resource, xylose in the hemicellulose is also necessary to utilize as a monosaccharide. However, in this case, assuming the enzyme reuse after prolonged reaction that assumes to ethanol fermentation saccharification, low stability of the xylanase is a problem.
　As these solutions, there may be mentioned the use of thermostable xylanase thermal stabilization xylanase and from heat Fungi improve existing xylanase, at the present time, reported cases of prolonged enzyme recycled using such xylanase It is not.
[0016]
　(A) T. described in Patent Documents 2 and 3 mentioned saccharification of lignocellulosic feedstock reesei derived thermostable xylanase mutants, conditions (acidic region, long-term use at high temperatures) required during the saccharification of lignocellulosic feedstock or is not clear whether it is intended to satisfy.
　Similarly, for the thermostable xylanase of Bacillus sp from described in Patent Document 4 mentioned in (a), results for residual activity at the time of heat treatment in pH7.2 around neutral are shown. Therefore, given the benefit of several days heat resistant xylanase under acidic conditions in order to perform the saccharification of lignocellulose, the activity of the heat resistant xylanase is likely to decrease.
[0017]
　(B) T. mentioned in the pulp bleaching reesei derived thermostable xylanase variants regarding Patent Document 2, Patent Document 3, Patent Document 5 and Patent Document 6, the T. after treatment for 30 minutes at pH5,60 ℃ ~ 80 ℃ Data is shown for thermostable xylanase residual activity from reesei. However, the T. assuming the conditions (a few hours at pH10,70 ℃ ~ 80 ℃) required during pulp bleaching Stability of thermostable xylanase from reesei are not shown.
[0018]
　(C) Xylanase from filamentous fungi such as genus Trichoderma or genus Acremonium described in animal feed additives, the thermal stability to withstand the pelletizing is not ready.
　Also, Patent Document mentioned (c) 2, Patent Document 3, some thermostable xylanase mutants have been described in WO 01/68664 and WO 03/025576, optimum pH of around pH 5 ~ pH 5.5 mutations that contains the body. However, since significant thermal deactivation occurs in any of the mutants 60 ° C. or higher high-temperature range, it can not be used as an animal feed additive.
[0019]
　(D) Patent Document 2 mentioned detergent adjuncts, Patent Document 3, T. disclosed in Patent Document 5 and Patent Document 6 Regarding reesei derived heat resistance alkali resistance xylanase mutants, information regarding the stability of the resistant alkaline xylanase mutants in a basic region in the time (1-2 hours) required for general cleaning is nil, detergents it is not clear whether it is possible to use as an adjuvant.
　Also, xylanase variants described in Patent Document 8 described in (d) and, heat resistance alkali resistance xylanase from Bacillus genus described in Patent Document 4 and Patent Document 9, for use as detergent adjuvants in the land cleaning It is not clear whether withstand.
[0020]
　Patent Document 2 mentioned (e) Bread modifiers, Patent Document 3, T. described in Patent Documents 5 and 6 thermostable xylanase mutants derived reesei is, whether withstand the large physical impact and pressure loads applied during the bread were not disclosed.
　In addition, the bread step, 35 ° C.-includes the 1-2 hours fermentation process performed at 40 ° C., it can correspond is also required in this process.
[0021]
　As shown above, the range of applications that can utilize xylanase diverse. Therefore, the conditions required for the xylanase is also a wide range. For example, pH 4 ~ pH 10, temperature 40 ℃ ~ 80 ℃, enzymes such as use time over several days can be cited deactivated easily severe conditions.
　To meet such diverse needs, although mutant xylanases and novel xylanase wide have been reported, xylanases seen yet that in severe conditions where the enzyme is easily deactivated, may act sufficiently stable it can be said that not been issued.
[0022]
　Moreover, mutant xylanases obtained for the purpose of improving the heat resistance, there is a problem that the initial rate of reaction is significantly decreased. This is due to mutations such as on the amino acid sequences added for improving heat resistance, flexibility in the structure of the whole protein is presumed to be because lowered.
[0023]
　In this context, the acidic region (pH 4 ~ pH 6), the basic region (pH 8 ~ pH 10) or a high temperature range such as (40 ℃ ~ 80 ℃), under severe conditions where the enzyme is deactivated easily, fixed time stable activity together showing the development of never initial rate of reaction as compared to the corresponding wild-type xylanase is significantly reduced xylanase has been awaited.
[0024]
　The present invention used a thermostable xylanase, and to provide an efficient method for saccharification inexpensive lignocellulosic. Also in enzyme deactivation easily severe conditions, stable with showing activity, mutant xylanases with substituted amino acid residue not to initial rate of reaction as compared to the corresponding wild-type xylanase is significantly reduced it is an object of the present invention to provide a. Furthermore, the present invention as well as provide a manufacturing method capable of producing the mutant xylanase at a low cost, and to provide a variety of uses of the mutant xylanase.
Means for Solving the Problems
[0025]
　The present invention is as follows.
[1] hydrolyzate manufacturing method of lignocellulose comprising contacting the lignocellulosic material and the thermal stability xylanase.
[2] The lignocellulosic feedstock is pulp hydrolyzate production method according to [1].
[3] [1] or from the saccharification reaction solution containing hydrolyzate of lignocellulose obtained by the saccharification product manufacturing method according to [2], and recovering the thermostable xylanase, recovered heat stability xylanase If, hydrolyzate production method comprising the method comprising producing a hydrolyzate contacting the lignocellulosic feedstock, the.
[4] The saccharification reaction liquid was solid-liquid separated by centrifugation or microfiltration membranes, by ultrafiltering separated liquid by ultrafiltration membrane, separating the thermostable xylanase and saccharification of lignocellulose, It recovered, hydrolyzate production method according to [3].
[5] to produce the centrifugal separation or a thermostable xylanase recovered in the solid-liquid separated solid and ultrafiltration membrane microfiltration membranes, saccharified by contacting the lignocellulosic feedstock, according to [4] hydrolyzate method of manufacturing.
[6] The heat stability xylanase, as compared to the corresponding wild-type xylanase, the initial rate of the reaction is 70% xylanase activity after heat treatment at 50 ° C. and 24 hours compared to the xylanase activity before the heat treatment Te is 50% or more, and hydrolyzate process according a variant xylanase from [1] to any one of [5] having a substituted amino acid residue.
[7] the variant xylanase, in SEQ ID NO: 1 amino acid sequence as set forth in the Sequence Listing, 29 th asparagine residue is replaced with a leucine residue, the 58th lysine residue is replaced by an arginine residue, 27 At least th tyrosine residue of tyrosine residues is replaced with other different amino acid residues, and 44 th asparagine residue of a substituted amino acid residue substituted with other different amino acid residue to the asparagine residue hydrolyzate manufacturing method according to a variant xylanase [6] with.
[8] are replaced with 27th tyrosine residue of the variant xylanase, other different amino acid residue and a tyrosine residue is a phenylalanine residue, is and replaced with 44th asparagine residue, asparagine hydrolyzate production method according to [7] for use in the hydrolyzate production of mutant xylanases is different from the amino acid residue is a serine residue and residues.
[9] the variant xylanase, in SEQ ID NO: 2 amino acid sequence described in the Sequence Listing, at least 154 amino acid residues according to a variant xylanase has been replaced with another amino acid residue [6] hydrolyzate method of manufacturing.
[10] of the variant xylanase, 33rd substituted amino acid residues asparagine residue is replaced with an aspartic acid residue, the 36th substituted amino acid residue glycine residue is replaced with an arginine residue, 90 th substituted amino acid residue threonine residues is substituted with a serine residue, 132 th substituted amino acid residue glutamine residue is substituted with an arginine residue, 154th leucine residue is replaced with a methionine residue substituted amino acid residue, 174th serine residue is substituted amino acid residue substituted with threonine residue, 195th substituted amino acid residues proline residue is replaced with a histidine residue, the 197th serine residue substituted amino acid residue substituted with an asparagine residue, and the 217th substituted amino acid residue glycine residue is replaced with glutamic acid residue Hydrolyzate manufacturing method according to use mutant xylanases that contains at least a group hydrolyzate production [9].
[11] The mutant xylanases, 30 th substituted amino acid residue isoleucine residue is replaced with a valine residue, 33rd substituted amino acid residues asparagine residue is replaced with an aspartic acid residue, 36 th used the substituted amino acid residues glycine residue is replaced with an arginine residue, and 154th leucine residue is a mutant xylanase comprising at least a substituted amino acid residue substituted with a methionine residue to the hydrolyzate produced hydrolyzate production method according to [9].
[12] The mutant xylanases, 30 th substituted amino acid residue isoleucine residue is replaced with a valine residue, the 59th substituted amino acid residue serine residue is substituted with a threonine residue, 154 th substituted amino acid residue leucine residue is replaced with a methionine residue, the 239th tyrosine residue substituted amino acid residue substituted histidine residues, and the 242 th cysteine residues were substituted with serine residues hydrolyzate production method according to [9] using the mutant xylanase comprising at least a substituted amino acid residue in saccharified production.
[13] In SEQ ID NO: 1 amino acid sequence as set forth in the Sequence Listing, 29 th asparagine residue is replaced with a leucine residue, the 58th lysine residue is replaced by an arginine residue, the 27th tyrosine residue the tyrosine residue is replaced with other different amino acid residues, and 44 th mutant xylanases with at least a substituted amino acid residue substituted with other different amino acid residues asparagine residue and the asparagine residue.
[14] In the amino acid sequence of SEQ ID NO: 1 of the Sequence Listing, are replaced with 27th tyrosine residue, other different amino acid residue and a tyrosine residue is a phenylalanine residue, and 44 th asparagine residue is substituted with group mutant xylanase according to the asparagine residue is different from the amino acid residue is a serine residue [13].
[15] In SEQ ID NO: 2 amino acid sequence described in the Sequence Listing, at least 154 th leucine residue, mutant xylanases has been replaced with another amino acid residue.
[16] In SEQ ID NO: 2 amino acid sequence described in the sequence listing of a replacement amino acid residue 33 asparagine residue is replaced with an aspartic acid residue, the 36th glycine residue is replaced with an arginine residue amino acid residues, 90th-substituted amino acid residue threonine residue is substituted with serine residue, 132 th substituted amino acid residue glutamine residue is substituted with an arginine residue, 154th leucine residue is methionine substituted amino acid residue substituted with residues, 174th substituted amino acid residues serine residue is substituted with a threonine residue, a substituted amino acid residue 195 proline residue is replaced with a histidine residue, 197 th substituted amino acid residue serine residue is replaced with an asparagine residue, and the 217th glycine residue is substituted with a glutamic acid residue Is mutated xylanase according to at least comprising, [15] the substituted amino acid residues.
[17] In SEQ ID NO: 2 amino acid sequence described in the sequence listing of a replacement amino acid residue 30 isoleucine residue is replaced with a valine residue, 33 th asparagine residue is substituted with an aspartic acid residue amino acid residue, the 36th glycine residue contains at least substituted amino acid residue substituted with arginine residue, and the 154th of substituted amino acid residues leucine residue is replaced with a methionine residue [15] variant xylanase described.
[18] In SEQ ID NO: 2 amino acid sequence described in the Sequence Listing, 30 th substituted amino acid residue isoleucine residue is replaced with a valine residue, a substituted amino 59th serine residue is substituted with a threonine residue residues, 154th substituted amino acid residues leucine residue is replaced with a methionine residue, the 239th substituted amino acid residue tyrosine residues is replaced with a histidine residue, and 242 th cysteine residues serine mutant xylanases described the substituted amino acid residues are substituted with residues contains at least have [15].
[19] [13] to a nucleic acid represented by the nucleotide sequence encoding the amino acid sequence of the mutant xylanase according to any one of [18].
Expression vectors containing a nucleic acid according to [20] [19].
Transformants containing the expression vector according to [21] [20].
[22] E. coli transformant according to Bacillus subtilis, yeasts, actinomycetes or filamentous fungi cells from the host cell [21].
[23] The filamentous fungus, Trichoderma spp., Acremonium spp transformant according to belonging to Humicola genus or Aspergillus genus [22].
[24] The filamentous fungus, Trichoderma viride, Acremonium cellulolyticus, transformant according to a Humicola insolens or Aspergillus niger [22] or [23].
[25] culturing the transformant according to any one of [21] [24], from at least one of the culture of cultured transformant and the transformant, [13] method for producing a mutant xylanases comprising recovering the mutant xylanase of any one of - [18].
[26] [25] mutant xylanase produced by the production method according to.
[27] [13] - [18] and a composition containing a variant xylanase of any one of [21].
[28] [13] - [18] and a variant xylanase of any one of [21], the bleaching process of pulp comprising contacting the pulp.
[29] [13] - [18] and [21] a detergent comprising a variant xylanase of any one of.
[30] [13] - [18] and [21] an animal feed comprising the variant xylanase of any one of.
[31] [13] - [18] and the pan modifier made comprising the variant xylanase of any one of [21].
The invention's effect
[0026]
　According to the present invention, using thermostable xylanase, it is possible to provide an efficient method for saccharification inexpensive lignocellulosic. Also in enzyme deactivation easily severe conditions, stable with showing activity, mutant xylanases with substituted amino acid residue not to initial rate of reaction as compared to the corresponding wild-type xylanase is significantly reduced it is possible to provide a. Furthermore, according to the present invention, the mutant xylanase as well as provide a manufacturing method which can be manufactured at low cost, it is possible to provide various uses of the mutant xylanase.
DESCRIPTION OF THE INVENTION
[0027]
　Thermostable xylanases according to the present invention, the degree of xylanase activity after a certain time heat treatment, which does not change the heat treatment prior to the xylanase activity, or a decrease of xylanase activity after heat treatment is less than the heat treatment before the xylanase activity it may be one.
　As an example, Aspergillus (Aspergillus) genus Trichoderma (Trichoderma) sp, Au Leo Basi de um (Aureobasidium) genus Schizophyllum commune (Schizophyllum commune) fungal genera such as Bacillus (Bacillus) or the genus Clostridium (Clostridium) genus Streptomyces (Streptomyces ) and the xylanase obtained from bacteria of the genus or the like.
　Among the wild-type xylanase above, it is preferably 50 ° C. and 24 hours of xylanase activity after heat treatment at least 50% compared to the xylanase activity before the heat treatment.
　The thermal stability xylanase according to the present invention, the filamentous fungi, by introducing a mutation into wild-type xylanase, including bacteria, the thermal stability may be mutant xylanases with improved if necessary. In such a variant xylanase, as compared to the corresponding wild-type xylanase, the initial rate of reaction is not less than 70%, xylanase activity after heat treatment at 50 ° C. and 24 hours compared to the xylanase activity before the heat treatment is 50% or more, and those having a substituted amino acid is more preferred.
　Nucleic acid according to the present invention are those represented by the nucleotide sequence encoding the amino acid sequence of the variant xylanase.
　Expression vectors according to the present invention are those comprising a nucleic acid represented by the nucleotide sequence encoding the amino acid sequence of the variant xylanase.
　Host cells according to the present invention has been transformed with an expression vector comprising a nucleic acid represented by the nucleotide sequence encoding the amino acid sequence of the variant xylanase.
　Method for producing a mutant xylanases of the present invention, culturing the host cells, from at least one of the culture of the cultured host cell and the host cell, is intended to include harvesting the mutant xylanase . Incidentally, the variant xylanase of the present invention also includes mutant xylanase produced by the production method of the variant xylanase.
　The composition according to the present invention are those containing the variant xylanase.
　Method for producing a saccharification of lignocellulose according to the present invention comprises contacting the a variant xylanase, a lignocellulosic feedstock.
　Bleaching process of pulp according to the present invention, the a variant xylanase, in which comprises contacting the pulp.
　Detergent according to the present invention, an animal feed or bakery modifiers are those containing the variant xylanase.
[0028]
　In the present invention, the amino acid sequence and nucleotide sequence encoding a variant xylanase, or for primer individual sequences, matters described on the basis of these mutual complementary relationship, unless otherwise specified, and each sequence, It is also applied to a sequence complementary to each sequence. In applying the matters of the present invention for complementary the sequence to each sequence for the complementary sequence recognized sequences within the skill in the art of the skilled person, corresponding to herein as complementary sequences to the listed sequences, to be replaced the entire specification.
[0029]
　The term "step" in this specification includes not only separate steps, even if that can not be clearly distinguished from other steps if it is achieved the intended purpose of the present process, it is included in this term .
　Numerical range expressed by using "to" in the specification are indicative of the range including the respective minimum and maximum values of the numerical values described before and after "to".
　In this specification, the amount of each component in the composition, if substances corresponding to the component in the composition there are a plurality, unless otherwise specified, the total amount of the plural substances present in the composition means.
　The following describes the present invention.
[0030]
(1) Definition
DEFINITIONS initial velocity of xylanase activity and reaction]
　The term "xylanase activity" in the present invention, randomly hydrolyze predominantly beta-l, 4 bonds of xylan constituting the plant cell walls, the reducing end oligosaccharides having (hereinafter, also referred to as "reducing sugar".) indicates that to produce a.
　The "initial rate of reaction" in the present invention, showing the initial rate of reaction of the xylanase activity.
　Initial rate of reaction can be confirmed in the following manner. First, as a substrate, it was mixed vigorously Birchwood xylan (manufactured by Sigma-Aldrich) in 1% 100mM sodium citrate buffer (pH 4.5) (w / w), and centrifuged for 15 min at 5000 × g, Preparing the supernatant excluding the residual xylan present in sodium citrate buffer. Then, the supernatant liquid as a substrate solution, and mixed so as to be 0.1% xylanase against said substrate solution (w / w), and reacted with stirring at 45 ° C. 30 min, the resulting reaction solution DNS method the amount of reducing sugars in (Bailey et al., 1992) by measuring in can confirm the initial rate of reaction of the xylanase activity.
[0031]
Definitions of wild-type activity comparable]
　The term "wild-type activity equivalent" as used herein, when one of the initial rate of the reaction of the wild-type xylanase, the initial rate of reaction of the mutant xylanase 0. 7 refers to a (70%) more.
[Range definitions enzyme acts stably]
　"enzyme ranges acting stable" as used herein, the temperature is less than 40 ° C. greater than 30 ° C., and the pH refers to 8 smaller range greater than 6.
　As used herein, the term "enzyme deactivation easily stringent conditions" acidic range (pH 4 ~ pH 6), referred to the basic region (pH 8 ~ pH 10) and the high temperature region of (40 ℃ ~ 80 ℃).
[0032]
[Residual activity and stability of the Definitions
　As used herein, "residual activity", in the range that the enzyme acts in a stable, predetermined time, the initial rate of the reaction after exposure to the enzyme, exposing the previous reaction of divided by the initial velocity, say those shown in percentage. Specific measurement method, 50 ° C. and pH4.5 for 16 hours, 24 hours, 48 hours, 72 hours of heat treatment, at the same time and allowed to stand on ice for 5 minutes to measure the initial rate of the reaction, before the heat treatment the initial velocity of the enzyme was also measured, dividing to percentage display. In addition to the, 50 ° C. and pH 8, pH 9, after pH10 at 1 hour heat treatment, 60 ° C. and pH 8, pH 9, after pH10 at 1 hour heat treatment, 70 ° C. and first reaction after 5 minutes heat treatment at pH5.5 measured similarly residual activity also speeds.
　As used herein, stability is determined by the degree of residual activity when the enzyme is exposed to severe conditions likely the enzyme is inactivated.
[0033]
(2) mutant xylanases of the present invention
　the mutant xylanases of the present invention, as compared to the corresponding wild-type xylanase, the initial rate of the reaction is 70% or more, 50 ° C. and 24 hours of heat treatment after the xylanase activity There is 50% compared to xylanase activity before the heat treatment, and those having a substituted amino acid residue.
　Mutant xylanases of the present invention, by having a substituted amino acid residue, even in the severe conditions the enzyme was inactivated easily, with exhibits stable activity, initial rate of reaction as compared to the corresponding wild-type xylanase It never is greatly reduced.
[0034]
　The mutant xylanases of the present invention, as compared to the corresponding wild-type xylanase, the initial rate of reaction is not less than 70%, compared to 50 ° C. and 24 hours of heat treatment after the xylanase activity before heat treatment xylanase activity It is 50% or more, and as long as it has a substituted amino acid residue, and is not particularly limited.
[0035]
　The mutant xylanases of the present invention, as compared to the corresponding wild-type xylanase, and preferably that the initial velocity of the reaction is 70% or more.
　If the initial velocity of the reaction is 70%, the amount of mutant xylanases, since never be more than the use of wild-type xylanase corresponding to mutant xylanase, industrial utilization preferred.
[0036]
　The mutant xylanases of the present invention, as compared to 50 ° C. and 24 hours of heat treatment after the xylanase activity before heat treatment xylanase activity, preferably at least 50%, more preferably 70% or more.
　If 50 ° C. and 24 hours of xylanase activity after heat treatment at least 50% compared to the xylanase activity before the heat treatment is preferable because the enzyme can be used within the scope that act stably. Specifically, if it takes a long time of the enzyme reaction and the enzyme reuse such saccharification of lignocellulose, there is no need to add large amounts of enzyme in order to maintain the reaction beginning of initial reaction rate, the cost as a result without fear of such increase, also preferable from an economic point of view.
[0037]
　The mutant xylanases of the present invention, its origin is not particularly limited. For example, Bacillus subtilis, Clostridium spp, actinomycetes or filamentous fungi, include mutant xylanases derived from basidiomycete. From the viewpoint of industrial use, among the filamentous fungus, Trichoderma spp., Acremonium spp., Mutant xylanases from Humicola genus or Aspergillus genus are preferred. Further, from the viewpoint of mass production, Trichoderma viride, Acremonium cellulolyticus, and more preferably mutant xylanase derived from Humicola insolens or Aspergillus niger.
[0038]
　Even under severe conditions where the enzyme is deactivated easily, with exhibits stable activity, from the viewpoint never initial rate of reaction as compared to the corresponding wild-type xylanase is greatly reduced, the following two variants xylanase may be mentioned as further preferred variant xylanase.
[0039]
　First, as the first preferred variant xylanase include mutant xylanases from Trichoderma filamentous fungi.
[0040]
　The first preferred variant xylanase, further, as compared to the corresponding wild-type xylanase, the initial rate of the reaction is 70% or more, 50 ° C. and 24 hours of heat treatment after the xylanase activity before the heat treatment xylanase activity from the viewpoint of 50% or more as compared with is preferably a variant xylanase of Trichoderma viride.
[0041]
　The first preferred variant xylanase, further, as compared to the corresponding wild-type xylanase, the initial rate of the reaction is 70% or more, 50 ° C. and 24 hours of heat treatment after the xylanase activity before the heat treatment xylanase activity in the amino acid sequence of from the viewpoint of easily achieving 50% or more, sequence listing SEQ ID NO: 1 compared to 29 th asparagine residue is replaced with a leucine residue, the 58th lysine residue arginine residue is replaced by a group, 27th tyrosine residue is replaced with other different amino acid residue and a tyrosine residue, and 44th asparagine residue is replaced with other different amino acid residue to the asparagine residue mutant xylanases with substituted amino acid residues.
[0042]
　SEQ ID NO: 1 amino acid sequence as set forth in the Sequence Listing is the amino acid sequence encoding the xylanase II of Trichoderma viride.
[0043]
　Further, as the second preferred variant xylanase include mutant xylanases derived from Acremonium spp fungi.
[0044]
　The second preferred variant xylanases as compared to the wild-type xylanase further corresponding initial velocity of the reaction is 70% to 50 ° C. and 24 hours of heat treatment after the xylanase activity before heat treatment xylanase activity from the viewpoint of 50% or more than, preferably a variant xylanase of Acremonium cellulolyticus.
[0045]
　SEQ ID NO: 2 amino acid sequence described in Sequence Listing is the amino acid sequence encoding a xylanase I of Acremonium cellulolyticus.
[0046]
　The second preferred variant xylanases as compared to the corresponding wild-type xylanase, the initial rate of reaction is not less than 70%, xylanase activity after heat treatment at 50 ° C. and 24 hours compared to the xylanase activity before the heat treatment from the viewpoint of easily achieving 50% Te, preferably contains a substituted amino acid residues at least 154 th leucine residue is replaced with a methionine residue.
[0047]
　Specific examples of the mutant xylanases of the present invention, for example, following those having substituted amino acid residue represented by clone number 1 clone number 17 as shown in Table 1 can be mentioned, but the mutant xylanases of the present invention these the present invention is not limited to.
[0048]
[Table 1]

[0049]
　Among the above Table 1, as compared to the corresponding wild-type xylanase, the initial rate of the reaction is 70% or more, 50 ° C. and compared to over 50% at 24 hours of heat treatment after the xylanase activity before heat treatment xylanase activity from the viewpoint of mutant xylanases TVX01 (clone # 1), mutant xylanases ACX01 (clone # 15), preferably mutant xylanases ACX02 (clone # 16) or mutant xylanase ACX03 (clone No. 17).
[0050]
　The variant xylanase TVX01, in SEQ ID NO: 1 amino acid sequence as set forth in the Sequence Listing, 29 th asparagine residue is replaced with a leucine residue, the 58th lysine residue is replaced by arginine residues, the 27 th tyrosine residues are substituted with a phenylalanine residue, and 44th asparagine residue contains a substituted amino acid residue substituted with serine residues. Mutant xylanases TVX01, compared to the corresponding wild-type xylanase, the initial rate of the reaction is 70% or more, 50 ° C. and compared to over 50% at 24 hours of heat treatment after the xylanase activity before heat treatment xylanase activity It preferred from the viewpoint that it is.
[0051]
　Mutant xylanases TVX01 according to the present invention is preferably one having an activity in the range of 30 ° C. ~ 90 ° C., and more preferably those having an activity in the range of 30 ℃ ~ 70 ℃. Also, preferably one having an activity in the range of pH 3 ~ pH 9, more preferably to have activity in the range of pH 4 ~ pH 7.
[0052]
　The variant xylanase ACX01 is substituted amino acid residue 33 asparagine residue is replaced with an aspartic acid residue, the 36th substituted amino acid residue glycine residue is replaced with an arginine residue, 90 th threonine substituted amino acid residues residues are substituted with serine residue, 132 th substituted amino acid residue glutamine residue is substituted with an arginine residue, a substituted amino acids 154 th leucine residue is replaced with a methionine residue residues, 174th substituted amino acid residues serine residue is substituted with a threonine residue, 195th substituted amino acid residues proline residue is replaced with a histidine residue, 197th serine residue asparagine residue substituted amino substituted amino acid residue substituted with groups, and the 217 th glycine residue is replaced with glutamic acid residue It contains a group. Mutant xylanases ACX01, compared to the corresponding wild-type xylanase, the initial rate of the reaction is 70% or more, 50 ° C. and compared to over 50% at 24 hours of heat treatment after the xylanase activity before heat treatment xylanase activity It preferred from the viewpoint that it is.
[0053]
　Mutant xylanases ACX01 according to the present invention is preferably one having an activity in the range of 30 ° C. ~ 80 ° C., and more preferably those having an activity in the range of 30 ℃ ~ 65 ℃. Also, preferably one having an activity in the range of pH 2 ~ pH 8, more preferably to have activity in the range of pH 2 ~ pH 5.
[0054]
　The variant xylanase ACX02 is substituted amino acid residues 30 isoleucine residue is replaced with a valine residue, 33rd substituted amino acid residues asparagine residue is replaced with an aspartic acid residue, the 36th glycine residue contains a substituted amino acid residue substituted amino acid residue substituted, and the 154th leucine residue is substituted with a methionine residue to the arginine residue. Mutant xylanases ACX02, compared to the corresponding wild-type xylanase, the initial rate of the reaction is 70% or more, 50 ° C. and compared to over 50% at 24 hours of heat treatment after the xylanase activity before heat treatment xylanase activity It preferred from the viewpoint that it is.
[0055]
　Mutant xylanases ACX02 according to the present invention is preferably one having an activity in the range of 30 ° C. ~ 80 ° C., and more preferably those having a reaction activity in the range of 30 ℃ ~ 65 ℃. Also, preferably one having an activity in the range of pH 2 ~ pH 8, more preferably to have activity in the range of pH 2 ~ pH 5.
[0056]
　The variant xylanase ACX03 is substituted amino acid residues 30 isoleucine residue is replaced with a valine residue, the 59th substituted amino acid residue serine residue is substituted with a threonine residue, 154th leucine residue substituted amino acid residue group substituted by a methionine residue, the 239th substituted amino substituted amino acid residue tyrosine residues is replaced with a histidine residue, and the 242 th cysteine ​​residues were substituted with serine residues It contains residues. Mutant xylanases ACX03, compared to the corresponding wild-type xylanase, the initial rate of the reaction is 70% or more, 50 ° C. and compared to over 50% at 24 hours of heat treatment after the xylanase activity before heat treatment xylanase activity It preferred from the viewpoint that it is.
[0057]
　Mutant xylanases ACX03 according to the present invention is preferably one having an activity in the range of 30 ° C. ~ 80 ° C., and more preferably those having an activity in the range of 30 ℃ ~ 65 ℃. Also, preferably one having an activity in the range of pH 2 ~ pH 8, more preferably to have activity in the range of pH 2 ~ pH 5.
[0058]
　The mutant xylanases of the present invention, for example, mutant xylanases consisting mutant xylanase TVX01 and homology each amino acid sequence found also encompassed mutant xylanase in the present invention.
　The amino acid sequence homology is observed as long amino acid sequences showing e.g. mutant xylanase TVX01 equivalent of about xylanase activity. Preferably the amino acid sequence 80% or more homology of the mutant xylanase TVX01, more preferably 90% or more homology, more preferably include mutant xylanases having homology of 95% or more. By showing a homology of 80% or more, it is considered that the three-dimensional structural similarity of xylanase is increased, by introducing the mutation point revealed in the present invention, mutant xylanases showing the effect of the present invention and about equal there are advantages such as can be developed.
　Other variants xylanase TVX01, mutant xylanases ACX01, it is the same for mutant xylanase ACX02 and mutant xylanases ACX03.
[0059]
　As the mutant xylanase TVX01 in the present invention, as long as it exhibits the effect of the order equal to the mutant xylanase TVX01, amino acid residue is inserted, and deleted or replaced with an amino acid sequence encoding a variant xylanase TVX01 mutation type xylanases are also included in the mutant xylanase TVX01 in the present invention.
[0060]
　If the amino acid residue is inserted, which is deleted or substituted, position of the insertion, deletion or substitution, so long as it does not impair the effects shown in the present invention, can be arbitrarily determined. Insertion, one or more amino acid residues or 2 amino acid residues as the number of deleted or substituted amino acid residues may be mentioned, for example, an amino acid residue to 10 amino acid residues, preferably one amino acid residue 1-5 amino acid residues and the like. Specifically, at the same time having a quadruple mutation point, in SEQ ID NO: 1 amino acid sequence as set forth in the Sequence Listing, 47 th what glycine residue has been substituted with a cysteine ​​residue, 52 th glutamine residue lysine residue those substituted groups, 59th valine residue which is substituted with an isoleucine residue, 67th asparagine residue which is substituted with an aspartic acid residue, 69th asparagine residue is substituted with an isoleucine residue things, that 80 serine residue is substituted with alanine residue, which 97th asparagine residue has been substituted with an aspartic acid residue, which 105th leucine residue is substituted with a methionine residue, 109th those threonine residue is substituted with alanine residue, which 120th threonine residue is substituted with an arginine residue, 143 th thread What Nin residue has been substituted with an isoleucine residue, the 151st what asparagine residue has been substituted with serine residue, which 161st serine residue is substituted with a leucine residue, 186th serine residue threonine residue such as those substituted groups.
[0061]
　Other variants xylanase TVX01, mutant xylanases ACX01, it is the same for mutant xylanase ACX02 and mutant xylanases ACX03. Specifically, in addition to mutation point having the ACX01, in SEQ ID NO: 2 amino acid sequence described in the sequence listing, 133 serine residue to asparagine residues, 176 th glutamine residue has been substituted with an arginine residue things and the like.
[0062]
　Similarly, the mutant xylanase ACX02, many variants with all mutation point showed comparable properties and ACX02, specifically, in addition to mutation point having the ACX02, of SEQ ID NO: 2 in the amino acid sequence described, 90th threonine residue serine residue, the 132 th glutamine residue is an arginine residue, the 133rd serine to asparagine residues, 174th serine residue is threonine residues to, substituted 195th proline residue is a histidine residue, the 176 th glutamine residue is an arginine residue, the 197th serine residue is an asparagine residue, the 217th glycine residue to a glutamate residue such as the ones and the like.
[0063]
　In addition, many mutants with all mutation point of the mutant xylanase ACX03 is a ACX03 showed comparable properties, specifically, in addition to mutation point having the ACX03, in SEQ ID NO: 2 amino acid sequence described in the Sequence Listing , 176 th glutamine residues like those substituted with an arginine residue.
[0064]
　Mutant xylanases of the present invention can be synthesized according to known methods. As a method for producing a mutation in a gene, for example, site-directed mutagenesis (Kramer, W. And frita, H.J., Methods in Enzymology, vol.154, P.350 (1987)), recombinant PCR method ( PCR Technology, Stockton Press (1989), a method of chemically synthesizing the DNA for a particular part, a method of hydroxylamine processes the gene, ultraviolet irradiation treatment a strain carrying the gene, or with a chemical agent such as nitrosoguanidine or nitrous acid and a method of treatment. among the methods for obtaining the mutant xylanases of the present invention, preferred methods include the production method of the mutant xylanases described below.
[0065]
(3) mutant xylanase production method of
　the production method of the mutant xylanase according to the present invention (hereinafter, simply referred to as "manufacturing method".) It is, culturing the transformant, the transformant and the transformant cultured from at least one of the culture of the body, it is intended to recover the variant xylanase.
　Here, the transformants, indicating those transformed with an expression vector comprising a nucleic acid represented by the nucleotide sequence encoding the amino acid sequence of the variant xylanase.
　Method for producing a mutant xylanases of the present invention, producing the mutant xylanases by culturing a transformant which is transformed with an expression vector comprising a nucleic acid represented by the nucleotide sequence encoding the amino acid sequence of the variant xylanase it is intended to. By this manufacturing method, even in the severe conditions the enzyme was inactivated easily, with exhibits stable activity, as compared to the corresponding wild type xylanase mutant xylanases never initial velocity greatly reduced reaction, it can be manufactured at low cost.
[0066]
　The following is a description of each step that can be included in the manufacturing method, a manufacturing method of the mutant xylanase according to the present invention, transformed with an expression vector comprising a nucleic acid represented by the nucleotide sequence encoding the amino acid sequence of the variant xylanase from at least one of the conversion has been culturing the transformant (host cell culture step), and transformant and cultures of the transformant cultured, recovering the variant xylanase (mutation it suffices include type xylanase recovery step), may further contain other steps, if necessary.
[0067]
A. Transformant culture step
　transformant culture step is a step of culturing a transformant which is transformed with an expression vector comprising a nucleic acid represented by the nucleotide sequence encoding the amino acid sequence of the variant xylanase.
[0068]
[Transformants]
　In the manufacturing method according to the present invention is not particularly limited as long as it is transformed with the expression vector and the transformant containing a nucleic acid represented by the nucleotide sequence encoding the amino acid sequence of the variant xylanase .
　The transformant, for example, E. coli, Bacillus subtilis, yeasts, actinomycetes, cells from filamentous fungi, etc. include those as host cells, among this, it is possible to secretory production of target enzyme extracellularly Bacillus subtilis, yeast, those of actinomycetes or filamentous fungi cells from the host cell 1. Technical preferable.
[0069]
　As yeast, for example, Saccharomyces (Saccharomyces) genus Hansenula (Hansenula) spp, or can be mentioned those belonging to Pichia (Pichia) sp., An example of a preferred yeast are Saccharomyces cerevisiae (Saccharomyces cerevisiae).
[0070]
　As the filamentous fungus, e.g., Humicola (Humicola) genus Aspergillus (Aspergillus) genus Trichoderma (Trichoderma) sp., Or Acremonium (Acremonium) belongs what is listed in the genus, preferred examples of the filamentous fungus Humicola insolens (Humicola insolens), Aspergillus niger (Aspergillus niger) or Aspergillus oryzae (Aspergillus oryzae), or Trichoderma viride (Trichoderma viride), or Acremonium cellulolyticus (Acremonium cellulolyticus). Further, from the viewpoint of industrial use, Trichoderma viride, Acremonium cellulolyticus, it is Humicola insolens or Aspergillus niger more preferable.
[0071]
[Nucleic]
　the nucleic acid represented by the nucleotide sequence encoding the amino acid sequence of the variant xylanase.
　As a method of synthesizing a nucleotide sequence encoding the amino acid sequence of the variant xylanase, a method of introducing a mutation point on the nucleotide sequence encoding the corresponding wild-type xylanase, chemically synthesizing the entire nucleotide sequence containing the mutation point the method and the like. Hereinafter, a method for introducing a mutation point on the nucleotide sequence encoding the wild-type xylanase corresponding will be described with reference to the nucleotide sequence encoding the xylanase II nucleotide sequence and Trichoderma viride encoding xylanase I of Acremonium cellulolyticus, nucleic acid according to the present invention is not limited thereto.
[0072]
[Introduction of mutation point of the nucleotide sequence encoding the wild-type xylanase]
　Examples of the base sequence encoding wild-type xylanase, the nucleotide sequence encoding the xylanase I of Acremonium cellulolyticus described in SEQ ID NO: 3, and nucleotide sequences mentioned encoding xylanase II of Trichoderma viride set forth in SEQ ID NO: 4.
　The nucleotide sequences encoding these wild-type xylanase as a template, as a method for generating a mutation in a gene, for example, site-directed mutagenesis (Kramer, W. And frita, H.J., Methods in Enzymology, vol. 154, P.350 (1987)), recombinant PCR method (PCR Technology, Stockton Press (1989 ), a method of chemically synthesizing the DNA for a particular part, a method of hydroxylamine processes the gene, the strain carrying the gene ultraviolet radiation processing, or a method of treatment with a chemical agent such as nitrosoguanidine and nitrous acid, such as by a commercially available mutagenesis kits. mutations can be introduced into the nucleotide sequence
　as the position and type of introducing a mutation, in particular the present invention is not limited. Show mutation point of various clone shown as examples in the clone number 1 to the clone number 17 in Table 2 below, but the invention is not limited to this as a position and type for introducing mutations.
[0073]
[Table 2]

[0074]
[Expression vector]
　The expression vector, the but are not particularly limited as long as it contains a nucleic acid represented by the nucleotide sequence encoding the amino acid sequence of the mutant xylanase, such as to improve the transformation efficiency and translation efficiency it is more preferable aspect, a plasmid vector or phage vector showing the configuration as shown below.
[0075]
[Basic structure of expression vector]
　expression vector comprises a nucleotide sequence encoding the mutant xylanase, not particularly limited as long as it can transform the host cells. If necessary, in addition to the base sequence, the base sequence constituting the other areas (hereinafter, simply referred to as "other regions".) May be contained. Other regions, for example, the transformant is, the or control area required to produce the mutant xylanase, and the like region required for autonomous replication.
　Further, from the viewpoint of facilitating the selection of the transformant, it may further comprise a nucleotide sequence encoding a selection gene can be a selectable marker.
　The control area needed to produce the variant xylanase, a promoter sequence (including an operator sequence to control transcription.), Ribosome binding sequence (SD sequence) include transcription termination sequences and the like.
[0076]
[Expression vectors when was the host cell yeast]
　If the yeast as a host cell, the expression vector, in addition to the nucleotide sequence encoding the variant xylanase, from the viewpoint of production efficiency of the mutant xylanase, the promoter preferably it contains sequences. The promoter sequence may be any one as long as it can express the mutant xylanase in transformants that yeast as a host cell.
[0077]
　Such as alcohol dehydrogenase (ADH1) promoter, phosphoglycerate kinase (PGK1) promoter, peptide chain elongation factor (TEF) promoter, glycerol 3-phosphate dehydrogenase (GPD) promoter, galactokinase (GAL1) promoter, metallothionein (CUP1) promoter, inhibitory acid phosphatase (PHO5) promoter, a promoter sequence, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter is used.
　Incidentally, from the promoter sequence it is not limited to yeast as a host cell.
　Such as cytomegalovirus (CMV) promoter, may be used as the promoter of the foreign. It can be appropriately selected depending from and the type of enzyme used.
[0078]
　Further, the expression vector may have a secretory signal. Thus, if the transformants produced the mutant xylanase, it is possible to secrete the mutant xylanase extracellularly.
　The secretion signal as long as the yeast serving as a host cell capable of secreting the mutant xylanase, not particularly limited. From the viewpoint of secretion efficiency, alpha-factor signal sequence, invertase signal sequence, the acid phosphatase signal sequence, it is preferable to use a glucoamylase signal sequence.
[0079]
　The expression vector having a promoter sequence and secretion signal as described above, and specific examples thereof include pRS423, pRS424, YEplac195 like.
[0080]
[Expression vectors when a filamentous fungus as a host cell]
　If filamentous fungi as host cells, the expression vector, in addition to the nucleotide sequence encoding the variant xylanase, from the viewpoint of production efficiency of the mutant xylanase preferably contains a promoter sequence. The promoter sequence, a filamentous fungus may be used so long as it can express the mutant xylanase in transformants that the host cell.　　　
[0081]
　Suitable expression vectors against filamentous fungi, van den Hondel, C. A. M. J. J. et al. (1991) In: Bennett, J . W. and Lasure, L. L. (Eds.) More gene Manipulations in Fungi. Academic Press, pp. It is described in the 396-428.
　Further, pUC18, pBR322, pUC100, pSL1180 (Pharmacia, Inc.), can also be used generally another expression vector used, such as pFB6 and Aspergillus (Aspergillus) pRAX, Trichoderma (Trichoderma) pTEX.
[0082]
[Expression vectors when a prokaryote as a host cell]
　If E. coli, Bacillus subtilis, prokaryotic organisms, such as actinomycetes and host cells, expression vectors, in addition to the nucleotide sequence encoding the variant xylanase, a mutant from the viewpoint of production efficiency of the type xylanase, and preferably it contains a promoter sequence. In addition to may contain a ribosome binding sequence and a transcription termination sequence and the like of the promoter sequence.
[0083]
　Examples of the promoter sequence, trp promoter of tryptophan operon derived from Escherichia coli, lac promoter of lactose operon, and PL promoter and PR promoter derived from lambda phage, derived from Bacillus subtilis gluconate synthase promoter (gnt), alkali protease promoter ( apr), neutral protease promoter (npr), alpha-amylase promoter (amy) and the like.
　Furthermore, uniquely modified or designed promoter sequence as tac promoter may also be utilized.
[0084]
　The ribosome binding sequence, although E. coli or sequences from B. subtilis, it is not particularly limited as long as it is a sequence which functions in a desired host cell such as Escherichia coli and Bacillus subtilis.
　As the ribosome binding sequence, for example, of the sequence complementary to the 3 'terminal region of 16S ribosomal RNA, it includes a continuous consensus sequence four or more base such sequences were generated by DNA synthesis.
　The transcription termination sequence is not always necessary, those ρ factor independent, available for example lipoprotein terminator, trp operon terminator or the like.
　Arrangement order on the expression vectors for these control regions, and it is not particularly limited, considering the transfer efficiency 5 'end upstream promoter sequences from ribosome binding sequence, the gene encoding the protein of interest, transcription termination sequences it is desirable that the line up of the order.
[0085]
　Specific examples of the expression vector referred to herein, pBR322 has autonomously replicable region in E. coli, pUC18, Bluescript II SK (+ ), pKK223-3, pSC101 or autonomous replication in B. subtilis and a region capable pUB110, pTZ4, pC194, ρ11, φ1, can be used as the expression vector φ105 like.
　As examples of autonomous replication expression vector in the two or more host can be used as the expression vector pHV14, TRp7, YEp7 and pBS7 like.
[0086]
Preparation method of transformant]
　transformant according to the present invention can be prepared by known methods. For example, a nucleotide sequence encoding a mutant xylanases of the present invention, if necessary to construct the expression vector containing the said other region, and a method of transforming the expression vector into a desired host cell It is. Specifically, Sambrook, J. , Et. al. , "Molecular Cloning A Laboratory Manual, 3rd Edition", Cold Spring Harbor Laboratory Press, molecular biology as described in (2001), etc., it is a known common methods in the field of biotechnology and genetic engineering it can.
[0087]
　Transformant according to the present invention, the not only incorporate the expression vector into a host cell, the lower as needed frequently used in the host cell codon, so as to frequently used codons, silent mutations it is also possible to perform prepared together to introduce and the like.
　Thus, it may be possible to increase the production of the variant xylanase derived proteins incorporated into expression vectors.
[0088]
　Examples of methods for introducing a silent mutation, the following listed in Table 3 as an example, but the method for introducing a silent mutation, secrete the xylanase gene and the xylanase gene on an expression vector in codon usage in the host cell extracellularly as long as it matched the codon of the signal sequence for causing, that approach, mutation point, the kind of base to be changed is not particularly limited.
　The following Table 3 T. In viride, the mutant xylanase ACX02, shows the location and nature of the base to change the base addition silent mutations in order to express at high frequency.
　In Table 3, "the position of the base" sequence name "ACX02" indicates the position of the base shown in SEQ ID NO: 4. "Locating base" sequence name "A.cellulolyticus signal sequence" denotes a position of a base represented by SEQ ID NO: 73.
[0089]
[table 3]

[0090]
[Transformant culture method]
　conditions of culture of the expression vector transformant obtained by transformation with is similar to the culturing conditions of the host cell before the transformation, it can be used known conditions .
　Carbon source as the medium, nitrogen source, it is either possible use of inorganic and other if nutrients contain appropriate amounts medium synthetic medium or a natural medium. The components used in the medium may be a known. For example, meat extract, yeast extract, malt extract, peptone, NZ amine and organic nutrient sources such as potato, glucose, maltose, sucrose, carbon sources such as starch and organic acids, ammonium sulfate, a nitrogen source such as urea and ammonium chloride, phosphorus salts, magnesium, inorganic nutrient sources such as potassium and iron, a combination of vitamins can be suitably used.
　In the culture of the transformant transformed with the expression vector containing the selection marker, for example, if the selectable marker is a drug resistance using a medium containing the drug to the corresponding said selectable marker when auxotrophic uses media without nutrients corresponding thereto. The pH of the medium may be selected in the range of pH 4 ~ pH 8.
　Culture in a liquid medium containing the medium, the transformant cultured with shaking, aeration agitation culture can be carried out continuously cultured using conventional culture methods, such as fed-batch culture.
　Culture conditions, the transformant, the medium may be appropriately selected depending on the type of cultivation method, and growing the transformant is not particularly limited as long as the variant xylanase of the present invention can be produced.
　Culture temperature is 20 ℃ ~ 45 ℃, preferably carried out aerobically cultured at 24 ℃ ~ 37 ℃.
　Culture period may be cultured until the content of proteins with variant xylanase activity of interest in the range of 1 to 7 days is maximum.
[0091]
B. Mutant xylanase recovery process
　variant xylanase recovery step, from at least one of the culture of the cultured transformant and the transformant is a step of recovering the variant xylanase.
[0092]
　After culturing the transformant transformed, a process for recovering the variant xylanase of the present invention can be used the conventional methods in the art.
　If the variant xylanase of the present invention is secreted into the transformed outside the transformed, can a culture of transformant centrifuged to obtain a crude enzyme solution easily by performing filtration. Also, if the variant xylanase of the present invention is accumulated in the body transformation the transformed, the transformant was cultured and collected by means such as centrifugation, the recovered transformant in buffer suspended, lysozyme treatment, freezing and thawing, by disrupting the cell membrane of the transformant according to the known methods such as sonication, the crude enzyme solution may be recovered.
[0093]
　The crude enzyme solution was concentrated by ultrafiltration method, it is possible to use as concentrated enzyme was added and preservatives. Further, after concentration, it can be obtained a powder enzyme of the mutant xylanase by a spray drying method.
　The crude enzyme solution having recovered xylanase activity, when in need of separation and purification, for example, an organic solvent precipitation method salting-out, due to alcohols by such ammonium sulfate, membrane separation method such as dialysis and ultrafiltration, or ion exchange chromatography, reverse-phase high performance chromatography, can be performed in combination affinity chromatography, a well-known chromatographic separation methods such as gel filtration chromatography as appropriate.
[0094]
(4) Use of the mutant xylanase
　and the variant xylanase of the present invention, even in deactivation tends conditions described above enzymes, having long-term stable activity. Therefore, the variant xylanase of the present invention, it is possible to use a wide range of applications.
　The compositions of the present invention contains the mutant xylanases may also contain optional ingredients suitable for the intended application as needed.
　The compositions of the present invention, even in deactivated easily under enzymes, containing the variant xylanase acting stably for a long period of time. Therefore compositions of the present invention can be used in a variety of applications.
　The content of the mutant xylanase may be appropriately determined depending on the use of the composition is not particularly limited.
[0095]
　Wherein said variant xylanase of the present invention can be used for various applications, and preferably be used as follows.
[0096]
Manufacturing method for saccharification of lignocellulosic raw material]
　Production method of saccharification of such lignocellulose present invention comprises contacting the a variant xylanase, a lignocellulosic feedstock.
　Method for producing a hydrolyzate of such lignocellulose present invention, even in deactivated easily under enzymes, for using the variant xylanase that can act stably for a prolonged period of time, to deactivate the enzyme can be done in easy conditions, obtained the saccharification of efficiently lignocellulose.
[0097]
　The lignocellulosic feedstock may be used known ones if low lignocellulosic feedstock lignin content.
　The low lignin content, because average lignin content of the lignocellulosic feedstock of about 30% by weight, based on the total weight of the lignocellulosic feedstock, indicating that lignin content is less than 30 wt%. Preferably not more than 20% by weight lignin content, and more preferably is suitable lignin content is 10 mass% or less of the lignocellulosic feedstock.
　Such lignocellulosic feedstock, softwood, hardwood, forest remainder, construction waste, pruning waste, Sawdust, kenaf, rice straw, from lignocellulosic material agricultural discarding, etc., such as straw, alkali extraction, alkali cooking etc. chemical pulping process, by a method such as organosolv, cellulose highly lignin removal, such as pulp fibers as a main component hemicellulose and the like. Preferably, hardwood kraft pulp, softwood kraft pulp, mechanical pulp, herb-derived pulp such as kenaf, recycled pulp, or (including pulp fiber content that is recovered from the pulp and paper mills) paper sludge, or mixtures thereof. In particular, hardwood kraft pulp, a softwood kraft pulp more preferable.
　These lignocellulosic feedstock may be obtained from the pulp manufacturing enterprises其s general.
[0098]
　Wherein the mutant xylanase, as a method of contacting the lignocellulosic feedstock, adding the variant xylanase lignocellulosic feedstock, a method that allows the reaction to proceed while stirring, a method of advancing the shaking reaction, mutant xylanases and after sufficiently mixing the lignocellulose, and a method that allows the reaction to proceed on standing. Preferably, from the viewpoint of reaction efficiency, adding the variant xylanase lignocellulosic feedstock, and a method that allows the reaction to proceed while stirring.
　There is no particular limitation on the reaction vessel can be used for the reaction. Preferably, it is possible to stir as lignocellulosic raw material and the variant xylanase charged is sufficiently mixed, it is preferable that the a reaction vessel having a temperature control function to maintain the optimum temperature of the mutant xylanase.
　The reaction temperature is not particularly limited as long as the temperature capable of acting in the variant xylanase. For example 60 ° C. from 40 ° C. and the like, preferably is 55 ° C. from 40 ° C..
　PH of the liquid in the saccharification reaction vessel is not particularly limited as long as pH that can act in the variant xylanase. For example pH4 from pH7 and the like, with preference given to pH6 from pH4.
[0099]
　In the method for producing a saccharification of lignocellulose according to the present invention, in addition to the variant xylanase of the present invention, other enzymes can also be used in combination with the variant xylanase as needed.
　Other enzymes may be used in combination cellulase, xylosidase, mannanase, pectinase, galactosidase, glucuronidase, an enzyme such as arabinofuranosidase. Saccharification of lignocellulose terms of efficiently producing, cellulase, it is preferably used in combination with the variant xylanase.
[0100]
　The cellulase is not limited as long as it can decompose cellulose to glucose, it can be used known ones. The cellulase and examples thereof include those having endoglucanase, at least one activity of the activity of cellobiohydrolase and β- glucosidase. Further, from the viewpoint of enzyme activity, it is preferred that an enzyme mixture having each of these activities.
[0101]
　Origin of the cellulase is not limited, it is possible to use a filamentous fungi, basidiomycetes, cellulase bacteria and the like. For example, Trichoderma spp., Acremonium spp., Filamentous fungi of the genus Aspergillus, such as, basidiomycete of Irpex genus, etc., Aeromonas spp, Clostridium spp, Bacillus spp, Pseudomonas spp, Penicillium spp, Ya any of various origin, such as bacteria of Humicola genus, etc. the cellulase of the origin as a template, those produced by gene recombination can be used as a mixture singly or in combination. It is also generally cultures cellulase preparation or the cells that are commercially available and that the use of filtrate directly.
　As the cellulase, among this, such as Trichoderma (Trichoderma) from the genus cellulase or Acremonium (Acremonium) from the genus cellulase are preferred from the viewpoint of having a strong cellulolytic force.
[0102]
　Examples of commercially available cellulases, Accellerase1000 (Genencor, Inc.), Accellerase1500 (Genencor, Inc.), AccelleraseXC (Genencor Inc.) (manufactured by Genencor, Inc.) AccelleraseXY, (manufactured by Genencor, Inc.) Accellerase DUET, (manufactured by Genencor, Inc.) Accellerase TRIO, Celluclast (Novozymes Co., Ltd.), Cellic CTec (manufactured by Novozymes Co., Ltd.), (manufactured by Novozymes Co., Ltd.) Cellic HTec, (manufactured by Novozymes Co., Ltd.) Cellic CTec2, Cellic HTec2 (manufactured by Novozymes Co., Ltd.), manufactured by Acremonium cellulase (Meiji Seika Pharma Co., Ltd. (Manufactured by Meiji Seika Pharma Co., Ltd.) meicelase, cellulase Amano A (made by Amano Enzyme Co., Ltd.), (manufactured by Amano Enzyme Co., Ltd.) cellulase Amano T, cellulase Daiwa (Daiwa Kasei Co., Ltd.), cell Riser ( Nagase Seikagaku Kogyo Co., Ltd.), Driselase (manufactured by Kyowa Hakko Co., Ltd.), cellulase Onozuka (manufactured by Yakult Pharmaceutical industry Co., Ltd.), it can be used Cellulosin (manufactured by Hankyu Bioindustry Co., Ltd.), and the like.
[0103]
　Mixing ratio cellulase of the mutant xylanase, if the mixing ratio such that the amount of reducing sugars produced is maximized may be any ratio of, preferably the variant xylanase, 20% cellulase ~ It includes mixing at a ratio of 60%.
[0104]
　As a substrate to be charged to the reaction vessel, and the lignocellulosic feedstock, the variant xylanase and overall enzyme obtained by summing the other enzyme (hereinafter, simply referred to as "enzyme".) And the concentration of, but is not particularly limited.
　Lignocellulosic feedstock of liquid transfer, the operation such as charging, it is preferable that the solid content concentration of 8 mass% to 30 mass%.
　The enzyme to be used may be charged an amount sufficient to decompose efficiently substrate in accordance with its activity. The amount of enzyme may be appropriately adjusted by an enzyme such as the type.
　The hydrolyzate produced by the production method of preparation and saccharification of lignocellulosic feedstock by saccharifying enzyme reuse of saccharification of lignocellulosic feedstock of the present invention may be a hydrolyzate derived from lignocellulose. The saccharified, specifically, monosaccharide, and a disaccharide or oligosaccharide. As the monosaccharide, glucose, xylose, arabinose, fructose, mannose, galactose, and the like.
　Also, the glycated comprises, chemicals and fuels may be used for the production of plastics, and other products or intermediates, also be used as a fermentation raw material for the production of these substances by using microorganisms good.
　Chemicals and fuels, plastics, and as other products, ethanol and isopropanol, acetone, acetate, 1,3-propanediol, butanediol, glycerol, ethylene glycol, amino acids, organic acids, furfural, polyhydroxyalkanoates acids (Polyhydroxyalkanoates ), animal feed and xylose, and the like.
　In particular, ethanol, isopropanol, can be suitably used in the fermentative production of lactic acid.
[0105]
Manufacturing Method of the mutant xylanase for reuse]
　the production method of the mutant xylanase for reuse of the present invention, glycation comprising saccharification of lignocellulose obtained by the production method of the hydrolyzate of the lignocellulose the reaction solution (hereinafter, simply referred to as "saccharification reaction".), recovering the variant xylanase of the present invention is intended to include (hereinafter. may be referred to as "recovery process").
　Thus, it is possible to produce a variant xylanase of the present invention at a low cost.
[0106]
　Wherein in the recovery step, as the recovery methods, it may be used a known method. For example, the solid-liquid separation, then, a method in which the recovery of the enzyme by using a membrane device or a known device capable of recovering the enzymes.
　As a method for solid-liquid separation include a method of centrifugation or coarse filtration the saccharification reaction solution.
　Centrifugation conditions separation or coarse filtration may be applied to a method generally used in the art as, for example, may be the case 500 × g ~ 10000 × g centrifugation.
　In the case of coarse filtration, opening size of 0.1 [mu] m ~ 2 mm, stainless steel filter may be filtered through a ceramic filter or resin filtration membrane.
　It may also be carried out microfiltration by microfiltration membranes. In this case, the use of microfiltration membranes having an average pore diameter of 0.01 [mu] m ~ 10 [mu] m is preferred.
　As a method of performing microfiltration by microfiltration membranes, pressure filtration, vacuum filtration, crossflow filtration, and the like centrifugal filtration. Among them, by performing cross-flow filtration, it can be reduced membrane fouling.
[0107]
　When recovering the enzyme from the solid-liquid separated liquid, for example, a method using a resin column, and a method using a membrane device.
　As a method of using the resin column, for example, can be cited ion exchange chromatography, reverse-phase high performance chromatography, affinity chromatography, a well-known chromatographic separation methods gel filtration chromatography.
　As the film unit, e.g., ultrafiltration membranes may be recovered using a membrane device comprising a dialysis membrane or the like. Among them, the use of ultrafiltration membrane having an average pore size 0.001 [mu] m ~ 0.01 [mu] m is more preferable.
　The ultrafiltration membrane flat membrane type, multi broadsword membrane, there is a type of hollow-fiber and the like, may be in any format. The flat membrane type, pressurizing the inside of the tank in order to ensure proper filtration rate. Nitrogen gas is pressurized, the helium gas, it is preferable to use air or the like. Further, it is preferable to install a impeller into the reaction vessel as necessary. By performing the stirring of the liquid by the impeller, it is possible to prevent clogging of the membrane surface, to maintain a better filtration rate. On the other hand, multi broadsword membrane, in the case of hollow fiber by infusion from substrate feed tank to the reaction vessel using a pump, it is possible to hold an appropriate filtration pressure and the linear velocity, to maintain a better filtration rate .
　For filtration method, submerged membrane system, an ultrafiltration method, and a microfiltration method, or the like. Incidentally, pressure filtration, vacuum filtration, crossflow filtration, centrifugal filtration, can be used in either method of ultrafiltration methods and microfiltration method. The filtration operation, constant pressure filtration, constant flow filtration, but is roughly divided into a non-constant pressure non steady flow filtration is not particularly limited in the present invention.
　In addition, for the recovery material of film used in step according to the present invention, cellulose acetate, aromatic polyamide, polyvinyl alcohol, polysulfone, polyvinylidene fluoride, polyethylene, polyacrylonitrile, ceramic, polypropylene, polycarbonate, polytetrafluoroethylene (Teflon (registered trademark)), and others. Among these, in view of the reaction in the use or the acidic conditions of cellulases, made of a non-cellulosic and acid-resistant material, the use of films such as polyacrylonitrile or polysulfone is preferable.
　Incidentally, saccharification reaction liquid used in the recovery step, without performing a pretreatment such as the solid-liquid separation, may be used saccharification reaction solution immediately after preparation.
　Produced by the production method of the mutant xylanase for reuse of the present invention, the mutant xylanase for reuse, as well as the mutant xylanase described above of the present invention can be used in various applications . Moreover, mutant xylanases for the recycling can be used in the following monosaccharides manufacturing method.
[0108]
[Monosaccharide production method]
　monosaccharide production method of the present invention, the saccharification reaction solution containing hydrolyzate obtained lignocellulose by the method for producing a saccharification of lignocellulose (hereinafter, when simply referred to as "saccharification reaction" is. from) recovering the variant xylanase of the present invention (hereinafter, may be referred to as "recovery process".), and recovered mutant xylanase, by contacting the lignocellulosic feedstock single to produce a sugar (hereinafter, simply referred to as "re-saccharification step.") and is intended to include.
　Thus, it is possible to effectively utilize the mutant xylanase according to the present invention.
[0109]
　Examples recovery step applies aforementioned recovery process.
　Wherein in the re-saccharification step, the enzyme solution containing the recovered mutant xylanase, add the lignocellulosic feedstock and water described above, performs pH, again saccharification reaction with stirring controlling the temperature. The conditions of pH and the reaction temperature may be in the same conditions as described in the method for producing a saccharification of lignocellulosic feedstock.
　Further, in the re-saccharification step, in addition to the lignocellulosic feedstock and water added administered, in addition the recovery process, it is preferable to add the solid obtained in the solid-liquid separation. Thus, it is possible to effectively utilize the mutant xylanases adsorbed on lignocellulose and the lignocellulose such as the unreacted contained solids in which solid-liquid separation by membrane devices or resin column.
　Re saccharification process according to the present invention may be added to the variant xylanase or the solid separated solids, both may be added.
[0110]
　In monosaccharide production method according to the present invention, the recovery step and re-saccharification step may be repeated. Thus, it is possible to reduce the catalyst cost in the period in which the recovered activity of the mutant xylanase is maintained.
　In the monosaccharides manufacturing method according to the present invention, to repeat the recovery process and re-saccharification step may be added a new mutant xylanases during the re-saccharification step. There is no particular limitation on the amount of mutant xylanases add, from an economic point of view, it is preferable that the first 50 mass% or less of the amount of mutant xylanases used in saccharification reactions. Further, it becomes 20 mass% or less of the amount of mutant xylanases used in the initial glycation reaction, more preferably from an economic point of view, and the first 10% of the amount of mutant xylanases used in the saccharification reaction below It becomes still more preferable.
　The monosaccharide production method of the present invention, the monosaccharide to be produced, may be a monosaccharide from lignocellulose. Specifically, glucose, xylose, arabinose, fructose, mannose, galactose, and the like.
[0111]
[Bleaching process of the pulp]
　bleaching process of pulp according to the present invention, the a variant xylanase, in which comprises contacting the pulp.
　Bleaching process of pulp according to the present invention, even in deactivated easily under enzymes, for using the variant xylanase acting stably for a long period of time, be done generally with a quenching easily under enzymes can be, it can be bleached efficiently pulp.
[0112]
　The pulp used in the step of contacting the mutant xylanase and pulp, and wood pulp and non-wood pulp. The wood pulp, include, for example, those such as softwood and hardwood as a raw material. As the non-wood pulp, such as bagasse and a pomace sugarcane, straw, hemp, those cotton, etc. as a raw material. In addition, also include waste paper pulp and waste paper, such as newspapers and magazines as a raw material.
　These pulps and mechanical pulps extracted fibers by a physical force from the raw material, it is chemically treated to roughly classified into chemical pulp removed fibers. The mechanical pulp, for example, groundwood pulp, refiner ground pulp, thermomechanical pulp, include chemithermomechanical pulp. As the chemical pulp, e.g., kraft pulp, alkaline pulp, the sulfite pulp and the like.
[0113]
　In the step of contacting the mutant xylanase and pulp, in addition to said mutant xylanases may be used in combination with other hemicellulases and lignin-degrading enzymes. Thus, it is possible to enhance the bleaching of pulp.
　Origin of hemicellulase and lignin degrading enzyme is not limited, filamentous fungi, basidiomycetes, bacteria and the like.
[0114]
　Pulp bleaching method according to the present invention, in addition to the step of contacting the mutant xylanase pulp preferably further comprises a delignification step and a bleaching step.
　As used herein, delignification step, as long as for positively removing the lignin from the pulp may be in any way, it is possible to use a method that is implemented conventionally. For example, a method as described in JP-A-2004-263310.
[0115]
　Encompassed in the present specification, the bleaching step may be a step of performing a bleaching process to the pulp, for example, removal of the lignin remaining in the pulp, the overall process for the purpose of improvement of brightness of the pulp it is intended to. Further, the bleaching step is a step subsequent to the delignification step, it is possible to use a method that is implemented conventionally. For example, a method as described in JP-A-2010-1594.
[0116]
　The step of contacting the mutant xylanase pulp according to the present invention, when performing a combination of the delignification step and the bleaching step comprises the steps of contacting the mutant xylanase and pulp, the delignification from the processing step may be performed at any point of the process subsequent to the bleaching step. That is, the step of contacting the mutant xylanase and pulp may be carried out either before or after the front and rear and the bleaching step of the delignification step. The step of contacting the mutant xylanase and pulp can also be carried out concomitantly with said delignification step or the bleaching step.
　Step of contacting the mutant xylanase pulp according to the present invention is preferably used as part of the bleaching step. Among them, from the viewpoint of exhibiting the performance of the mutant xylanases of the present invention to its fullest extent, the use of lignin content less stages in the bleaching step is more preferable.
[0117]
　The step of contacting the mutant xylanase and pulp, of the bleaching step, for example, chlorine, chlorine dioxide, nitrogen dioxide, hypochlorite, oxygen, hydrogen peroxide, chemical bleaching using ozone It can also be used as part of the bleaching process.
[0118]
[Detergent]
　Detergent of the present invention contains the mutant xylanases may also contain other components as required.
　Detergent of the invention, even under severe conditions where the enzyme is deactivated easily by containing the mutant xylanase exhibits stable activity, can improve the performance of the detergent.
[0119]
　The detergent of the present invention, laundry detergent, various detergents such as detergents for automatic dishwashers are included. In addition, it can be used as a household and detergents for industrial use. Further, the detergent of the present invention can also be used as modifiers for clothing textiles.
　When using the detergent of the present invention as modifiers of apparel textiles, the clothing fibers, may be used cotton fibers, hemp fibers, and modifiers of cellulose-containing fibers such as rayon and Tencel.
[0120]
　Detergent of the invention, the in addition to mutant xylanase also other enzymes may be formulated according to the application. Other enzymes can be used those known in the art. For example, proteases, cellulases, amylases, and lipases. The origin of these other enzymes are not limited, filamentous fungi, basidiomycetes, bacteria and the like.
[0121]
　Detergents other than the other enzymes of the present invention, such as surfactants, cleaning aids, bleaching agents, the components used for ordinary detergent such as a fluorescent agent may be blended.
　Examples of the surfactant include anionic surfactants, nonionic surfactants, amphoteric surfactants, mention may be made of cationic surfactants. Preferably anionic surfactants, non-ionic surfactants.
　Examples of the anionic surfactant, for example, sodium fatty acid (soap), alpha-sulfonated fatty acid esters, sodium linear alkyl benzene sulfonate (LAS), sodium alkyl sulfate (AS), alkyl ether sulfate ester ( AES), alpha-olefin sodium sulfonate (AOS), include sodium alkyl sulfonate.
　The non-ionic surfactants such as polyoxyalkylene alkyl ether (AE), polyoxyethylene alkyl phenyl ether (APE), sucrose fatty acid salt esters, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene fatty esters, alkanol amides.
[0122]
　As the cleaning aid, alkaline buffers, divalent metal ion scavenger or the like can be mentioned anti-soil redeposition agents, specifically, tripolyphosphates, polyphosphates such as pyrophosphates, A-type zeolite aluminosilicates such as sodium carbonate, sodium sesquicarbonate, carbonates such as sodium hydrogen carbonate, polyethylene glycol, carboxylic acid polymer, polyvinyl alcohol, polyvinyl pyrrolidone, polymers such as polyglycidyl acid salt, cellulose derivatives such as carboxymethyl cellulose, and aminocarboxylic acid type polymers such as polyaspartic acid.
[0123]
　As the bleaching agent, for example, sodium hypochlorite, dichloroisocyanurate salt, sodium chlorite, hydrogen peroxide, sodium percarbonate, sodium perborate, peracetic acid, hydrosulfite, and dioxide thiourea acid .
　The fluorescent agent, such as bis (triazinylamino) stilbene disulfonic acid derivative or bis styryl biphenyl derivatives.
[0124]
　Detergent of the invention, the surfactant, the cleaning aid, the bleaching agent can be prepared in a conventional manner in combination with the fluorescent agent.
　Form of the detergent can be selected depending on the application, can for example, liquid, powder, granule, paste, be a solid or the like.
[0125]
[Animal Feed]
　animal feed of the present invention contains the mutant xylanase, other components may be contained if necessary.
　Animal feed of the present invention, even under severe conditions where the enzyme is deactivated easily, containing the mutant xylanase exhibits stable activity. Thus, the animal feed of the present invention, since the abundant plant fibers included in the animal feed is decomposed, thereby improving the absorption efficiency of the plant nutrients in animals ingesting animal feed, digestion in the stomach of the animal You can improve the performance.
　The content of the mutant xylanase in animal feed of the present invention is not particularly limited as long as it is an amount capable of improving the digestibility in the stomach of the animal animal feed.
[0126]
　Examples of the animal feed, for example, ready-made animal feed containing xylan, like cereals and the like. Incidentally, among other cereals, wheat, maize, rye, barley, oats, Torichikeru, such as rice and sorghum are preferred.
[0127]
　Wherein said variant xylanase in animal feed of the present invention can also be used in combination other and feed additives, and other enzymes.
　Examples of the other feed additives such as vitamins feed additives, mineral feed additives, amino acid feed additives, and the like penetrating protectant.
　Examples of other enzymes, e.g., cellulase and amylase, such as proteases and the like. The origin of these enzymes is not limited, it is possible to use a filamentous fungi, Basidiomycetes, the enzyme derived from bacteria or the like.
[0128]
　Animal feed of the present invention can be used in a wide variety of animals. Preferably, chickens, turkeys, ducks, poultry such as geese, cows, horses, ruminants sheep, etc., boar such as pigs, and rodents and fish rabbit like.
[0129]
　Animal feed of the present invention, as long as the variant xylanase is added may be one produced by any method, the production method of the animal feed is not particularly limited. The addition of the the variant xylanase of animal feed, for example, feed production before, may be carried out in any final stage of the feed production or manufacturing such phase. Moreover, the variant xylanase can also be added directly to the ready-made animal feed formed into pellets or mash form. Incidentally, the variant xylanase, by adding directly to the drinking water, can also be included in animal feed.
[0130]
[Bread modifier]
　bread modifier of the present invention contains the mutant xylanases may also contain other components as required.
　Bread modifier of the present invention, even under severe conditions where the enzyme is deactivated easily, containing the mutant xylanase exhibits stable activity. Thus, bread modifier according to the present invention, under conditions of 35 ° C. ~ 40 ° C., to indicate the stable activity during the fermentation process during bread that one to two hours, the hemicellulose portion of the wheat flour can be decomposed, it is possible to modify the nature of the bread.
[0131]
　Bread modifier of the present invention, in addition to the variant xylanase, may include other bakery modifier. Examples of other bread modifier, for example, monoglycerides, organic acid monoglycerides, glycerin fatty acid esters, propylene glycol fatty acid esters, sorbitan fatty acid esters, phospholipids, ascorbic acid and derivatives thereof, organic acids, amino acids, salts, etc. and the like.
[0132]
　The types of bread for which the addition of bread modifier of the present invention, by mixing the bread material, triturated, fermented, as long as it is manufactured by performing firing, etc., bread, etc. on the other, special bread, cooking bread, pastry, it included steamed bread, hot cakes, and donuts.
　The material of the pan, flour, water, such as water or milk, yeast, sugar, salt, fats and oils (shortening, lard, margarine, butter, liquid oil, etc.) and the like. If necessary, eggs, seasoning (glutamine acids, nucleic acids, etc.), swelling agents, can also be added perfumes. Also, if the flour is the main raw material, it is also possible to use rye flour, and rice flour to wheat flour. Incidentally, the dough in this specification, by mixing the bread material, means that kneading.
[0133]
　Method for producing bread is not particularly limited as long as it is a method usually used as long as it contains the fermentation step, for example, a straight method, the sponge method, a liquid type method can be used.
　Fermentation process, can be used a method commonly used, it is possible to shorten the fermentation time by setting the fermentation temperature to be higher than room temperature, it is preferably carried out for 1-2 hours at 35 ℃ ~ 40 ℃.
[0134]
　Bread modifier of the present invention, for example, may be mixed with powder raw materials wheat flour, or use pre-dissolved in water, also be added during the process in the form of powdered or liquid good.
　Having described embodiments of the present invention, which is illustrative of the present invention, it is also possible to adopt various other configurations.
Example
[0135]
　Further illustrate the present invention by the following examples, but the present invention should not be construed by the following examples limitation all. Also shows the component contents in the composition of Example "%" are by weight unless otherwise specified.
[0136]
EXAMPLE 1 Measurement method (Standard assay) of xylanase activity
　hydrolyzed DNS method the amount of reducing sugars xylan (Bailey et al., 1992) was measured by, were measured xylanase activity.
　Substrates used for the evaluation, 1% 100mM sodium citrate buffer (pH 4.5) (w / w) Birchwood xylan (manufactured by Sigma-Aldrich) were mixed vigorously, and centrifuged for 15 minutes at 5000 × g after, using the supernatant.
　The xylanase against substrate solution were mixed so as to be 0.1% (w / w), and reacted with stirring at 45 ° C. 30 minutes, measuring the amount of reducing sugars in the reaction mixture obtained, xylanase the activity was measured.
[0137]
Example 2 Site-Directed Mutagenesis Preparation and characterization of xylanase mutants by
(1) the expression vector YEp-GAPDHp-GAs-TVX, YEp-GAPDHp-GAs-ACX construction of
　(a) obtaining a promoter sequence: Saccharomyces genomic DNA sequences cerevisiae as a template, a promoter sequence of glyceraldehyde-3-phosphate dehydrogenase (hereinafter abbreviated as GAPDH) (GenBank Accession Number: A35397.1 ) was obtained by the PCR method. Primers used for the PCR were SEQ ID NO: 55 and 56 shown in Table 4.
[0138]
　(B) obtaining the signal sequence: the genomic DNA sequence of Rhizopus oryzae as a template, glucoamylase gene signal sequence (GenBank Accession Number: D00049.1) was obtained by the PCR method. Primers used for the PCR were SEQ ID NO: 57 and 58 shown in Table 4.
[0139]
　(C) ligation of the promoter sequence and signal sequence: The PCR amplified the DNA sequence, was purified by phenol / chloroform solution, it was collected by ethanol precipitation. After digesting the purified promoter sequence and signal sequence with restriction enzyme BglII, purified excised fragment containing the target DNA by the respectively agarose electrophoresis. Such fragments thus obtained, was ligated with DNA ligase (Takara Shuzo Co., Ltd.) (hereinafter, abbreviated as GAPDHp-GAs).
[0140]
　(D) T. viride amplification of xylanase II gene: T. The genomic DNA sequence of viride as a template, T. The total length of viride xylanase II gene (nucleotide sequence and secretion signal sequence encoding a xylanase gene) was obtained by PCR. Primers used for the PCR were SEQ ID NO: 59 and 60 shown in Table 4. The sequence obtained is shown in SEQ ID NO: 74 of the Sequence Listing.
[0141]
　(E) A. cellulolyticus of xylanase gene amplification: A. Genomic DNA sequences of cellulolyticus as a template, A. The total length of cellulolyticus xylanase gene (nucleotide sequence and secretion signal sequence encoding a xylanase gene) was obtained by PCR. Primers used for the PCR, SEQ ID NO: 61 and 62 described in Table 4. The sequence obtained is shown in SEQ ID NO: 75 of the Sequence Listing.
[0142]
　(F) a promoter sequence, the signal sequence, xylanase gene linked in: T. obtained in (d) The viride xylanase II as a template, a nucleotide sequence encoding a xylanase gene except the signal sequence was obtained by PCR. Primers used for the PCR were SEQ ID NO: 63 and 64 shown in Table 4. Then purify the resulting fragment was digested with the restriction enzymes SacI, and ligated with the GAPDHp-GAs fragment (hereinafter, abbreviated as GAPDHp-GAs-TVX).
　In addition, A. obtained in (e) Similarly for cellulolyticus xylanase I, the gene for secreted protein portion was obtained by PCR, after purification was digested with restriction enzymes SacI, the was GAPDHp-GAs fragment and ligated (hereinafter, abbreviated as GAPDHp-GAs-ACX). Note that primers used for PCR are described in SEQ ID NO: 65 and 66 shown in Table 4.
　Incidentally, the method of purification and ligation of DNA fragments shown here is the same as (c).
[0143]
　Introduction into (g) expression vector: digesting the GAPDHp-GAs-TVX fragment and budding yeast multi-copy vector YEp24 (ATCC 7769) with restriction enzymes XmaI and BamHI (the former about 1.3 kbp, the latter about 7.4 kbp) , after purification, ligated, T. viride was obtained xylanase II variant fabrication plasmid (hereinafter referred to as YEp-GAPDHp-GAs-TVX) . Similarly, GAPDHp-GAs-ACX and YEp24 was digested with restriction enzymes XmaI and BamHI restriction enzymes (former about 1.5 kbp, the latter about 7.4 kbp), after purification, ligated, A. cellulolyticus was obtained xylanase I variant creation expression vector (hereinafter referred to as YEp-GAPDHp-GAs-ACX) .
　Incidentally, the method of purification and ligation of DNA fragments shown here is the same as (c). In addition, YEp24 can be obtained from the American Type Culture Collection is a cell-microbial bank.
[0144]
[Table 4]

[0145]
(2) site-directed mutagenesis
　mutant used in the embodiment of the present invention, a template an expression vector constructed in (1), mutated by the "LA PCR in vitro mutagenesis Kit" of Takara Shuzo Co., Ltd. did.
　Primers, using synthetic oligonucleotides.
　The expression vector YEp-GAPDHp-GAs-TVX as template, following the SEQ ID NO: 5 and SEQ ID NO: 6 in Table 5, and SEQ ID NO: 7 and SEQ ID NO: 8, and SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: PCR was carried out using a 12 as primers to obtain a mutant xylanase expression vector YEp-GAPDHp-GAs-TVX01.
　The expression vector YEp-GAPDHp-GAs-ACX as a template, the following sequence of SEQ ID NO: 21 and SEQ ID NO: 22 in Table 5 as primers, substituted 154th leucine residue is a methionine residue of SEQ ID NO: 2 to obtain a YEp-GAPDHp-GAs-L154M having substituted amino acid residues.
　The expression vector YEp-GAPDHp-GAs-ACX as a template, the following SEQ ID NO: 13 and SEQ ID NO: 14 in Table 5, and SEQ ID NO: 15 and SEQ ID NO: 16, and SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, and SEQ ID NO: 23 and SEQ ID NO: 24, and SEQ ID NO: 25 and SEQ ID NO: 26, and SEQ ID NO: 27 and SEQ ID NO: 28, the sequence of SEQ ID NO: 29 and SEQ ID NO: 30 as primers, was similarly obtained the YEp-GAPDHp-GAs-ACX01.
[0146]
　The expression vector YEp-GAPDHp-GAs-ACX as a template, and SEQ ID NO: 15 and 16 below Table 5, and SEQ ID NO: 21 and SEQ ID NO: 22, and SEQ ID NO: 31 and SEQ ID NO: 32, and SEQ ID NO: 33 and SEQ ID NO: 34 the sequences of the primers were similarly obtained YEp-GAPDHp-GAs-ACX02.
[0147]
　The expression vector YEp-GAPDHp-GAs-ACX as a template, the following SEQ ID NO: 21 and SEQ ID NO: 22 in Table 5, and SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38 If, as a primer the sequence of SEQ ID NO: 39 and SEQ ID NO: 40, was similarly obtained YEp-GAPDHp-GAs-ACX03.
　The plasmid containing the mutant xylanase was transformed into competent cells of E. coli HB101 (manufactured by Toyobo Co., Ltd.) to obtain transformants.
　Plasmids were prepared by alkaline SDS extraction from the cells, to determine the nucleotide sequence of the xylanase gene portion in DNA sequencer, a mold YEp-GAPDHp-GAs-TVX, xylanase gene of YEp-GAPDHp-GAs-ACX the region encoding, it was confirmed that amino acid substitution has been introduced.
[0148]
[table 5]

[0149]
(3) Transformation into yeast production mutant xylanase
　expression vector having the mutant xylanases made by (2), competent cells of Saccharomyces cerevisiae BY4741 using Fast-Yeast Transformation Kit (G- Biosciences) transformed into, SD-Ura agar medium (0.67% Yeast-nitrogen base without amino acids ( Difco), 2% glucose, 0.5% casamino acid, 0.077% -Ura DO Supplement (Clontech) , 2% agar, 30 ° C. with deionized water) above were cultured for 48 hours. The results obtained variants in Table 6.
[0150]
[Table 6]

[0151]
(4) Evaluation of stability of mutant xylanases
　obtained colonies SD-Ura liquid medium (4% glucose, without agar, except SD-Ura liquid medium are the same as defined above medium composition) planted, 24 hours at 30 ° C. after pre-culture, inoculation 2% and by 48 hours the culture, after centrifugation the supernatant containing the mutant xylanase, was heated at 50 ° C. ~ 55 ° C., the residual by measurement method described in example 1 activity was assessed.
[0152]

　Example 2 0-72 hours after the heat treatment the mutant xylanases prepared by the method noted in (2) at 50 ° C., the residual activity was measured by the measuring method of Example 1. The results are shown in Table 7. WT is the wild type, F, L, S, R, in the amino acid sequence of SEQ ID NO: 1 in the sequence listing, the amino acid residue corresponding to the 27th tyrosine residue phenylalanine, corresponding to the 29th asparagine residue amino acid residue is a leucine residue, amino acid residue is a serine residue corresponding to the 44th asparagine residue, the amino acid residue corresponding to the 58th lysine residue represents a replacement of the arginine residue. Incidentally, the amino acid residue corresponding to the 27th tyrosine residue in the production of the mutant xylanase was replaced by a phenylalanine residue (Table 7 (e)), and SEQ ID NO: 41 and SEQ ID NO: 42 in Table 8 using primers.
[0153]
[Table 7]

[0154]
[Table 8]

[0155]
　Substituted amino acid residue of the specific amino acid residues are believed to result in heat stabilization, shown in WO 2007/115391 pamphlet, WO 2001/27252 pamphlet, WO 2005/108565 pamphlet mutant xylanases with groups as shown in Table 7 (d), (f) and (h), was completely inactivated after 24 hours.
　Further, as shown in Table 7 (e) and (g), mutant xylanases or 44th asparagine residue serine residue having a substituted amino acid residues 27 th tyrosine residues is replaced with a phenylalanine residue for even mutant xylanases with substituted amino acid residue substituted with group I was completely inactivated after 24 hours.
[0156]
　However, 29 th mutant xylanases asparagine residue has a substituent amino acid residue leucine and 58 lysine residue is replaced with an arginine residue (Table 7 (d)), the 27 th tyrosine residue based on the improved residual activity by replacing the phenylalanine residue showed 50 percent or more residual activity after 72 hours (Table 7 (c)).
　Further by substituting the 44th asparagine residue of the variant xylanase serine residues, while maintaining the wild type (Table 7 (a)) moderate activity, leaving 70% of the activity after 72 hours was (Table 7 (b)).
　Incidentally, have many variants comparable nature as TVX01 with quadruple mutation point of the present invention, specifically, at the same time having a quadruple mutation point, the amino acid sequence of SEQ ID NO: 1 of Sequence Listing in, which 47th glycine residue is substituted with a cysteine residue, which 52nd glutamine residue has been substituted with a lysine residue, which 59th valine residue is substituted with an isoleucine residue, 67 th which asparagine residue has been substituted with an aspartic acid residue, which 69th asparagine residue is replaced with an isoleucine residue, which 80th serine residue is substituted with alanine residue, the 97th asparagine residue obtained by substituting the aspartic acid residue, which 105th leucine residue is substituted with a methionine residue, which 109th threonine residue is substituted with alanine residue Which 120th threonine residue is substituted with arginine residue, which 143rd threonine residue is replaced with an isoleucine residue, which 151 th asparagine residue has been substituted with serine residue, 161 serine residue which group is substituted with leucine residue, 186th serine residue like those substituted with a threonine residue.
[0157]

　with an expression vector comprising a nucleic acid represented by the nucleotide sequence encoding the amino acid sequence of the mutant xylanases prepared by the described in Example 2- (2) Method, according to the procedure described in Example 2- (3) yeast were transformed, mutant xylanase production yeast liquid culture, after 24 hours heat treatment at a supernatant of the culture solution 50 ° C., the residual activity was measured by a measuring method shown in example 1.
　The residual activity of the mutant xylanase was 50%. Further, towards the said variant xylanase was as high as 1.13 with respect to the wild-type 1 also initial reaction rate before the heat treatment.
[0158]

　with an expression vector comprising a nucleic acid represented by the nucleotide sequence encoding the amino acid sequence of the mutant xylanases prepared according to the procedure described in Example 2- (2), Example 2- (3) yeast transformed with noted technique, mutant xylanase production yeast liquid culture, after 0-48 hours heat treatment at supernatant 50 ° C. of the culture solution, the residual activity was measured by a measuring method shown in example 1. The results are shown in Table 9. In the table, WT indicates the wild type.
　The liquid medium used herein is a SD (not including Ura) medium containing 4% glucose.
[0159]
[Table 9]

[0160]
　Wild-type A. cellulolyticus xylanase after heat treatment of 16 hours, was completely inactivated (Table 9 (i)). On the other hand, the residual activity of the mutant xylanases is increased, it showed more than 50% residual activity after 48 hours (Table 9 (j), (k) or (l)). ACX02, ACX03 (Table 9 (k) and (l)) is approximately equal to the wild-type well for the reaction initial rate before the heat treatment, is led to ACX01 showed nearly twice the activity of the wild-type (Table 9 ( j)).
　Note that illustrates many of the variants comparable nature as ACX01 with all mutation point of ACX01, specifically, in addition to mutation point having the ACX01, in SEQ ID NO: 3 amino acid sequence described in the sequence listing, 133 th serine residue of the asparagine residue, position 176 glutamine residues like those substituted with an arginine residue.
　Similarly, the ACX02, its many variants with all mutation point showed comparable properties and ACX02, specifically, in addition to mutation point having the ACX02, of SEQ ID NO: 3 in the Sequence Table acids in sequence, the 90th threonine residue is a serine residue, the 132 th glutamine residue is an arginine residue, the 133rd serine asparagine residues, 174th serine residue is threonine residue, 195 the proline residue is a histidine residue, an arginine residue at position 176 glutamine residues, 197th serine residue is the aspartic acid residue, the 217th glycine residue and those substituted with a glutamic acid residue and the like.
　Further, shows a number of variants comparable nature as ACX03 with all mutation point of ACX03, specifically, in addition to mutation point having the ACX03, in SEQ ID NO: 3 amino acid sequence described in the sequence listing, 176 th glutamine residue of like those substituted with an arginine residue.
[0161]
COMPARATIVE EXAMPLE
　When improving the heat resistance of the enzyme by mutagenesis, typically initial rate of reaction is known to result in significantly reduced or completely inactivated. Also in this application, although the improved heat resistance, initial reaction rate is lowered mutants could be obtained was almost. One example is shown in Table 10. WT is the wild type, MT represents a mutant.
　Note that these mutants were prepared by the method described in Example 2- (2).
　MT1 is the plasmid YEp-GAPDHp-GAs-TVX a template, and SEQ ID NO: 43 and SEQ ID NO: 44 below Table 11, and SEQ ID NO: 45 and SEQ ID NO: 46, a primer sequence of SEQ ID NO: 47 and SEQ ID NO: 48 did.
　MT2 is a plasmid YEp-GAPDHp-GAs-TVX as template, and SEQ ID NO: 7 and SEQ ID NO: 8 below Table 11, and SEQ ID NO: 47 and SEQ ID NO: 48, a primer sequence of SEQ ID NO: 49 and SEQ ID NO: 50 did.
　MT3 is a plasmid YEp-GAPDHp-GAs-ACX as a template, the following SEQ ID NO: 51 and SEQ ID NO: 52 in Table 11, were as primers the sequence of SEQ ID NO: 53 and SEQ ID NO: 54.
[0162]
　The mutation point of MT1 in Table 10, in SEQ ID NO: 1 amino acid sequence as set forth in the Sequence Listing, 13 th tyrosine residue phenylalanine residue (Tyr13Phe), 47 th glycine residue is a cysteine residue (Gly47Cys), indicating that the 151 th asparagine residue is substituted with serine residue (Asn151Ser).
　Further, the mutation point of MT2, in SEQ ID NO: 1 amino acid sequence as set forth in the Sequence Listing, 46th valine residue isoleucine residue (Val46Ile), 58 lysine residues arginine residue (Lys58Arg), 151 It indicates that the asparagine residue is replaced with a serine residue (Asn151Ser).
[0163]
　The mutation point of MT3, in SEQ ID NO: 2 amino acid sequence described in the sequence listing, the 100th serine residue cysteine ​​residue (Ser100Cys), 144 th aspartic acid residue is replaced with cysteine ​​residues (Asn144Cys) indicating that.
[0164]
[Table 10]

[0165]
[Table 11]

[0166]
Example 3 T. mass production of TVX01 and ACX02 the viride was the host
(1) T. mass production of TVX01 with viride
(a) Construction of plasmid TVX01-pCB1
　a nucleotide sequence encoding a variant xylanase TVX01 obtained in Example 2 as a template, was obtained by PCR The DNA sequence of the xylanase portion.
　Primers used for the PCR were SEQ ID NO: 67 and SEQ ID NO: 68 in Table 12.
　T. obtained in Example 2- (1) (d) The total length of viride xylanase gene as a template, the DNA sequence of the signal portion obtained by the PCR method. Primers used for the PCR were SEQ ID NO: 69 and SEQ ID NO: 70 in Table 12.
　Wherein the DNA sequence of the DNA sequence and the xylanase portion of the signal sequence portion linked by the PCR method, the StuI to sequence upstream of the initiation codon of the signal sequence portion was obtained an array containing the XhoI downstream of the stop codon.
　Primers used for the PCR were SEQ ID NO: 71 and SEQ ID NO: 72 in Table 12. The amplified DNA fragment of 0.7 kbp, the TOPO TA cloning kit (Invitrogen) and inserted into pCR2.1-TOPO expression vector according to the attached protocol, yielding plasmid TOPO-TVX01.
[0167]
[Table 12]

[0168]
　Plasmids "TOPO-TVX01" was cleaved with StuI and XhoI, to obtain a gene fragment of about 0.7kbp "TVX01-N". On the other hand, pCB1-Eg3X-hphless a (WO 2011/021616 pamphlet) was digested with StuI and XhoI, to recover a fragment of about 7 kbp. Recovered fragment of about 0.7kbp gene fragment "TVX01-N" was ligated with DNA ligase (Takara Shuzo Co., Ltd.), to prepare plasmid "TVX01-pCB1". In accordance with the conditions attached to the kit's instructions for the reaction conditions, such as an enzyme. Plasmid "TVX01-pCB1", the host of the T. At the viride, it was constructed to express the TVX01 using its own initiation codon.
[0169]
(B) T. due to the plasmid "TVX01-pCB1" Preparation Example transformants viride 3- (1) - (a ) by the plasmid "TVX01-pCB1" obtained in T. Transformation of viride is obtained according to the process described in WO 2011/021616 pamphlet, was performed. Uracil biosynthetic gene (pyr4) is deficient strain T. The viride strain 2 strain as a host, transformation was carried out by coating lance formation method using the pyr4 gene Neurospora crassa (Neurospora crassa) as a selection marker. In addition, T. viride strain 2 strain can be obtained according to the method described in paragraph [0102] of Japanese Patent No. 4644603. That is, as described in paragraph [0102] of the specification Patent No. 4,644,603, 10 Trichoderma viride MC300-1 strain (FERM BP-6047) 9 with a spore suspension of the order of CFU / ml, UV lamp 2 lamps under lighting at a height of 30 cm, and irradiated with gentle mixing. Applying a spore suspension after UV irradiation to the selection medium and cultured for 7 days at 28 ° C.. Were selected grown strains, is a uracil-requiring strain of Trichoderma viride T. The viride strain2 shares were acquired. The composition of the selection medium, dihydrogen 1 Potassium minimal medium [0.2% phosphoric acid, 0.4% ammonium sulfate, 0.03% urea, 0.03% magnesium sulfate heptahydrate, 0.03% calcium chloride, 0.5% glucose, 2.5% agar, 0.01% trace elements (5mg iron sulfate heptahydrate, 1.56 mg manganese sulfate heptahydrate, 1.4 mg of zinc sulfate heptahydrate, 2 mg of cobalt chloride those dissolved in water 1L)] in is obtained by adding 5-fluoroorotic acid uridine and 1 mg / mL of 10 [mu] g / mL.
　T. The viride strain 2 strain was suspended in protoplast enzyme solution (1 mg / mL beta-glucuronidase, 0.3 mg / mL chitinase, 0.3 mg / mL Zaimoriesu, 0.5 mol / L sucrose), and the mycelium was protoplasted. The suspension was filtered and centrifuged, SUTC buffer and washed with (0.5 mol / L sucrose, 10 mmol / L calcium chloride, 10 mmol / L Tris-HCl (pH 7.5)).
[0170]
　The protoplasts were suspended in SUTC buffer 100 [mu] L, there a DNA solution 10μL containing the 10μg amount of plasmid "TVX01-pCB1" is entered DNA solution 10μL and pyr4 gene was added and allowed to stand on ice for 5 minutes . Then 400μL of PEG solution (60% PEG4000,10mmol / L calcium chloride, 10 mmol / L Tris-HCl (pH 7.5)) was added, and the mixture was allowed to stand for 20 minutes in ice, the SUTC buffer 10mL was added, centrifuged separated. The collected protoplasts were suspended in SUTC buffer 1 mL, overlaid with soft agar on a minimum medium containing 0.5 mol / L sucrose by 200 [mu] L, after 5 days of culture at 28 ° C., transplanted again minimal medium grown colonies and it was here formed colonies as a transformant.
[0171]
(C) culturing and identification of transformants "TVX01-pCB1"
　to select a strain grown in the introduction to minimal medium plasmid "TVX01-pCB1", and cultured according to the method described in WO 98/11239 pamphlet . Centrifuge the obtained culture solution, after separating the culture supernatant and cells, the culture supernatant filter (pore size: 0.2 [mu] m) to prepare a culture supernatant by filtered sterilization through. The culture supernatant was electrophoresed separated using 12% Gel SDS-PAGE mini (Tefco Co.), were selected culture supernatants band TVX01 is satisfactorily detected. The culture supernatant, which was selected, was "a large amount prepared TVX01".
[0172]
(2) T. mass production of ACX02 using Viride
(A) T. modification of ACX02 gene codon suitable for expression in Viride
　ACX02 gene T. In viride, in order to highly express the activity the protein, A. the signal sequence and ACX02 gene cellulolyticus created a DNA related to changes in total 24 points of a base. In Table 13, "position of the base" sequence name "ACX02" wild type A. represented by SEQ ID NO: 4 coincides with the position of the base cellulolyticus xylanase gene. Moreover, "the position of the base" sequence name "A.cellulolyticus signal sequence" denotes a position of a base represented by SEQ ID NO: 73.
[0173]
[Table 13]

[0174]
　This modification ACX02 gene, T. One in which was designed in consideration of the use frequency distribution of codons in viride. This modified ACX02 gene, in the Corporation Jean design, was artificially synthesized. Note when the artificial synthesis, EcoRI and StuI to sequences upstream of the start codon, were designed to contain a XhoI and HindIII downstream of the termination codon. To pUC19 of the EcoRI / HindIII, resulting in plasmid codon modified ACX02 gene has been inserted "pACX02".
[0175]
(B) Plasmid ACX02-pCB1 Construction
Plasmids "pACX02" was cut with StuI and XhoI, to obtain a gene fragment of about 850bp "ACX02-N". On the other hand, pCB1-Eg3X-hphless a (WO 2011/021616 pamphlet) was digested with StuI and XhoI, to recover a fragment of about 7 kbp. Recovered fragment about 850bp gene fragment "ACX02-N" was ligated with DNA ligase (Takara Shuzo Co., Ltd.), to prepare plasmid "ACX02-pCB1". In accordance with the conditions attached to the kit's instructions for the reaction conditions, such as an enzyme. Plasmid "ACX02-pCB1", at the host Trichoderma viride, was constructed to express ACX02 using its own initiation codon.
[0176]
(C) Preparation of transformants Trichoderma viride by plasmid "ACX02-pCB1"
　Example 3- (2) - Transformation of Trichoderma viride by plasmid "ACX02-pCB1" obtained in (b) is WO according to the method described in 2011/021616 pamphlet, it was carried out. Trichoderma viride strain2 strain is uracil biosynthesis gene (pyr4) deficient strain as a host, transformation was carried out by coating lance formation method using the pyr4 gene Neurospora crassa (Neurospora crassa) as a selection marker. The Trichoderma viride strain 2 strain was suspended in protoplast enzyme solution (1 mg / mL beta-glucuronidase, 0.3 mg / mL chitinase, 0.3 mg / mL Zaimoriesu, 0.5 mol / L sucrose), and the mycelium was protoplasted. The suspension is filtered, centrifuged, SUTC buffer and washed with (0.5 mol / L sucrose, 10 mmol / L calcium chloride, 10 mmol / L Tris-HCl (pH 7.5)).
　The protoplasts were suspended in SUTC buffer 100 [mu] L, there a 10μg amount of plasmid "ACX02-pCB1" is entered DNA solution 10μL and pyr4 gene containing DNA solution 10μL was added and allowed to stand on ice for 5 minutes. Then 400μL of PEG solution (60% PEG4000,10mmol / L calcium chloride, 10 mmol / L Tris-HCl (pH 7.5)) was added, and the mixture was allowed to stand for 20 minutes in ice, the SUTC buffer 10mL was added, centrifuged separated. The collected protoplasts were suspended in SUTC buffer 1 mL, overlaid with soft agar on a minimum medium containing 0.5 mol / L sucrose by 200 [mu] L, after 5 days of culture at 28 ° C., transplanted again minimal medium grown colonies and it was here formed colonies as a transformant.
[0177]
(D) culturing and identification of transformants "ACX02-pCB1"
　to select a strain grown in introducing plasmid "ACX02-pCB1" minimal medium and cultured according to WO 98/11239 pamphlet.
　Centrifuge the obtained culture solution, after separating the culture supernatant and cells, the culture supernatant filter (pore size: 0.2 [mu] m) to prepare a culture supernatant by filtered sterilization through. Shoshi the culture supernatant to SDS-PAGE, and selected the culture supernatant bands ACX02 is satisfactorily detected. The culture supernatant, which was selected, was "a large amount prepared ACX02".
[0178]
EXAMPLE 4 Temperature and stability of transition of TVX01 with changes in pH
　by using a TVX01 large quantities have been prepared by the method described in Example 3, experiments were carried out described below. 200mM of various buffers and xylanase (below) were mixed in a 1: 1, after a certain time processing at various temperatures, was allowed to stand for 5 minutes the ice bath, the residual activity was measured by the method shown in Example 1. Here, the buffer used was sodium buffer citrate (pH 4.5), Tris-HCl buffer (pH 8 ~ pH 9), and glycine sodium buffer solution (pH 10).
　TVX01, in PH4.5,50 ° C., even after 72 hours heat treatment had a 86% activity. Further, in yet 70 ° C. at a high temperature, pH 5.5 (pH of the mutant xylanase stock), also after heat treatment of 5 minutes had 68% activity. In any conditions, the wild-type is a conditional loss of fully active, the variant residual activity at acidic and high temperature was improved.
　Further, after heat treatment of 60 minutes at pH8 ~ pH10,50 ℃, wild-type residual activity is 28% at pH 8, was 8% pH 9 with respect to completely deactivated in pH 10, the variant , 83% in pH 8, pH 9 at 60%, had 56% of activity in pH 10. In addition, wild-type completely lose activity 60 ° C., even after heat treatment of 60 minutes, the xylanase is 30% pH 8, pH 9 at 17%, has a 10% activity at pH 10, a base sex and even residual activity at a high temperature was improved.
[0179]
　As described above, TVX01 of the present invention, pH 4.5, under conditions in which the wild-type enzyme such as pH 8 ~ pH 10, and 50 ° C. ~ 70 ° C. is significantly deactivated, showed improvement in significant residual activity.
[0180]
Example 5 Temperature and transition stability ACX02 with changes in pH
　by using a ACX02 large quantities have been prepared by the method described in Example 3, it was subjected to the same experiment as in Example 4.
　Mass prepared ACX02, in PH4.5,50 ° C., had activity of 84% after 72 hours heat treatment. Similarly treated wild-type xylanase, it has reduced activity up to 45%, wherein said variant xylanase than the wild type, the residual activity at acidic and high temperature was improved.
　Further, after heat treatment of 60 minutes at pH8 ~ pH10,50 ℃, whereas lost wild-type fully active, ACX02 is 34% pH 8, pH 9 5%, have a 2% activity at pH10 and it has also remained active in basic and high temperatures was improved.
　As described above, ACX02 of the present invention, pH 4.5, under conditions in which wild-type such as pH 8 ~ pH 10, and 50 ° C. is significantly deactivated, showed improvement in significant residual activity.
[0181]
Example 6 saccharification of lignocellulosic feedstock (1)
　was placed a conical flask in dry weight 2g of hardwood kraft pulp (LBKP), was prepared in large quantity by the method of Example 3 ACX02, TVX01, T. As an experimental control wild-type xylanase and A. from viride added to each 52mg equivalent by the Erlenmeyer flask with protein weight of cellulase solution containing a wild-type xylanase from cellulolyticus, the reaction solution of 20g was prepared by injecting 20mM sodium citrate buffer (pH 4.5), silicone stopper in was sealed. This was followed by gentle stirring to saccharification reaction at 50 ° C.. The results are shown in Table 14. WT represents the wild type xylanase.
[0182]
[Table 14]

[0183]
　After 72 hours, A. cellulolyticus derived and T. Whereas residual activity of the wild-type xylanase from viride was decreased to around 30% (Table 14 (m), (o)), mass prepared ACX02 and mass prepared TVX01 90% or more residual activity It showed (Table 14 (n), (p)).
[0184]
Saccharification reaction of Example 7 lignocellulosic feedstock
(2) (1) saccharification reaction
　was placed a separable flask in dry weight 40g hardwood kraft pulp (LBKP), was prepared in large quantity by the method of Example 3 TVX01, ACX02 in addition to the corresponding portions the separable flask 347mg cellulase solution with protein weight each containing, to prepare a reaction solution 400g injected 20mM sodium citrate buffer (pH 4.5). Thereafter, gently stirred to saccharification reaction at 50 ° C., the product monosaccharides amount after 72 hours the reaction was analyzed by HPLC.

analytical instrument: JASCO HPLC
Column: ULTRON PS-80H (300 × 8mm; Shinwa Kako Co., Ltd.)
Analysis Temperature: 50 ° C.
Mobile phase: aqueous perchloric acid pH2.1
[0185]
(2) recovery of the enzyme
　the saccharification reaction (72 hours reaction) was collected precipitate was centrifuged at 7000 × g. One of the centrifugal supernatant commercial UF membrane (trade name: microza UF pencil module AIP-0013D, manufactured by Asahi Kasei Chemicals Corporation) was treated with, to obtain a concentrated fraction.
[0186]
(3) re-saccharification reaction using the recovered enzyme
　into a separable flask was charged with 7000 × g centrifugation of the saccharification reaction, and the enriched fraction obtained by the UF membrane treatment, hardwood kraft pulp ( adding LBKP) as a 40g as final solid content, subjected to gentle stirring to saccharification reaction at 50 ° C., the product monosaccharides amount after 72 hours the reaction was analyzed by HPLC.
[0187]
(4) saccharification reaction results
　after the first reaction started (enzyme recycling 0 time) 72 hours, the concentration of accumulated glucose and xylose in saccharification reaction solution containing the TVX01 was 79.1 g / L. Among them, the concentration of accumulated glucose was 65.1 g / L. On the other hand, the wild-type T. concentration of accumulated glucose and xylose in saccharification reaction containing viride xylanase is 78.0 g / L, the concentration of accumulated glucose was 64.7 g / L. Similarly, in the saccharification reaction solution containing ACX02, accumulation concentration of glucose and xylose 61.3 g / L, the concentration of accumulated glucose was 49.9 g / L. The enzyme in these glycation reaction solution was recycled to the saccharification reaction by the method.
[0188]
　Concentration of accumulated reuse first glucose and xylose, 70.7 g / L in saccharification reactions containing TVX01, wild-type T. was 53.1g / L in the saccharification reaction solution containing 53.3g / L, ACX02 saccharification reaction solution containing viride xylanase.
　Concentration of accumulated reuse first glucose, 58.7 g / L in saccharification reactions containing TVX01, wild-type T. was 43.5g / L in the saccharification reaction solution containing 45.0g / L, ACX02 saccharification reaction solution containing viride xylanase.
[0189]
　Concentration of accumulated reuse second glucose and xylose, 63.6 g / L in saccharification reactions containing TVX01, wild-type T. was 42.6g / L in the saccharification reaction solution containing 42.3g / L, ACX02 saccharification reaction solution containing viride xylanase.
　Concentration of accumulated reuse second glucose, 53.0 g / L in saccharification reactions containing TVX01, wild-type T. was 34.6g / L in the saccharification reaction solution containing 35.8g / L, ACX02 saccharification reaction solution containing viride xylanase.
　Sugar concentration of accumulated reuse 0-th results obtained are expressed as a relative value is 100%. The results are shown in Table 15 and Table 16. It should be noted, WT represents the wild type xylanase.
[0190]
[Table 15]

[0191]
[Table 16]

[0192]
　As shown in Table 15, in recycling the saccharification reaction the enzyme, mutant xylanases TVX01 and ACX02 of the present invention that can be suitably used revealed.
　Further, as shown in Table 16, the use of the mutant xylanases of the present invention, not only the productivity of xylose, glucose productivity was also revealed that it is possible to improve.
　In addition, even with TVX01 and ACX02 of the present invention to softwood-derived pulp (NBKP), the same effect was obtained.
[0193]
Example 8 bleaching process of the pulp
(1) xylanase treatment of pulp
　as a pulp, using a commercially available milk carton. Raw milk carton is an end member such as out when they are thinned wood and lumber softwood, a virgin pulp containing lignin is coloring component.
　The well-scrubbed milk cartons cut to about 5cm angle, between about 2 to 5 days, immersed in water. Thereafter, peel the polyethylene film surface.
　Heating the water soaked before paper to 50 ° C., is added a mutant xylanases TVX01 and mutant xylanase ACX02 of the present invention. Further, for comparison, the procedure is as in the xylanase of the respective wild-type. Xylanase used herein are all intended derived from a filamentous fungus. Amount of xylanase, consider to be the optimum mixing ratio. In addition, to consider so that the processing time is also the best. In addition also prepared which do not xylanase treatment.
[0194]
　Then added a commercially available chlorine bleach, in pH 7 ~ pH 10, to half a day to stand the pieces of paper. Well after washing with water, finely torn, pieces of paper in a mixer for home at the same time as the appropriate amount of water and stir until no longer visible.
　The fibers processed into paper-like using a commercially available papermaking apparatus, after removal of water, and dried.
[0195]
(2) Whiteness measurements
　in the ultraviolet-visible spectrophotometer whiteness meter, measuring whiteness of paper was created (JIS Z 8715) above.
(3) in the case of adding the mutant xylanase TVX01 and mutant xylanase ACX02 of the present invention can bleach the pulp even under conditions of pH 7 ~ pH 10.
[0196]
Example 9 Detergent
(1) fluff cleaning cloth
　to the subject washing, using old fabrics fluffed. As the detergent, a commercial detergent, using a material obtained by adding mutated xylanase TVX01 or mutant xylanase ACX02 of the present invention. Further, for comparison, the procedure is as in the xylanase of the respective wild-type. Xylanase used herein are all intended derived from a filamentous fungus. In addition also prepared which do not xylanase treatment. Amount of xylanase, consider to be the optimum mixing ratio. Adding timing is set to be at the same time as turning on the detergent.
[0197]
　Put water 800ml into a separable flask of 1L, adding the detergent and xylanase, while rotating at 60 rpm, at pH7 ~ pH10,50 ℃, to clean the 1 hour. Then, to dry naturally.
[0198]
(2) fuzz removal of the measurement
　to observe the removal degree of fuzz in the stereomicroscope. Also, the fabric fuzz removal rate after washing is measured using a spectral colorimeter.
(3) in the case of adding the mutant xylanase TVX01 and mutant xylanase ACX02 of the present invention, pH 7 ~ pH 10, suppressing fuzz under the conditions of temperature 50 ° C..
[0199]
Example 10 Animal Feed
(1) preparation of an animal feed
　in powder feed laboratory animals, the addition of mutant xylanases TVX01 or mutant xylanase ACX02 of the present invention. Further, for comparison, the procedure is as in the xylanase of the respective wild-type. Xylanase used herein are all intended derived from a filamentous fungus. In addition also prepared which do not xylanase treatment. Amount of xylanase considered to be the optimum blending ratio, the mixture is pelletized.
[0200]
(2) Measurement of cell wall degrading of the molding after the animal feed
　was allowed to stand overnight molded animal feed, sliced along the blade of commercially available razor performs Gram stain on the slide, staining intensity of cell walls under an optical microscope to observe. Also, the animal feed was allowed to stand overnight, mixed vigorously to 100mM sodium citrate buffer (pH 4.5), was centrifuged for 15 minutes at 5000 × g, was separated the supernatant, contained in the supernatant the amount of reducing sugar is measured by the DNS method (Bailey et al., 1992).
[0201]
Example 11 Bread modifier
(1) bread prepared
　by the straight method, to manufacture bread. Feed formulation is shown in Table 17 below. All use of the material for the home that are generally commercially available.
[0202]
[Table 17]

[0203]
　As bread modifier, adding mutant xylanases TVX01 or mutant xylanase ACX02 of the present invention. Further, for comparison, the procedure is as in the xylanase of the respective wild-type.
　Addition timing is at the same time as the mixing raw materials. Amount of xylanase, consider to be the optimum mixing ratio. Also, to prepare those that do not xylanase treatment.
　The resulting dough was fermented over about 1 hour to 2 hours until approximately two times larger at about 37 ° C.. After that, firing in a microwave oven.
[0204]
(2) grain structure observed pan
　bread was baked sliced along the blade of commercially available razor, observing the grain structure by stereomicroscope.
(3) Measurement of loaf volume
　after standing overnight the baked bread, by rapeseed displacement method, measuring the loaf volume of baked bread.
(4) in the case of adding the mutant xylanase TVX01 and mutant xylanase ACX02 of the present invention, under the conditions of 1 to 2 hours at 35 ° C. ~ 40 ° C. in the fermentation process, are those capable of stably reaction .
[0205]
　November 25, 2011 the disclosure of Japanese Patent Application No. 2011-257389 and April 2012, filed on the 24th Japanese Application No. 2012-099096 filed in herein incorporated by reference in its entirety It is.
　All documents described herein, patent applications, and technical standards, each individual publication, patent application, and that the technical specification is incorporated by reference to the same extent as if marked specifically and individually, It incorporated by reference herein.

WE claims

Hydrolyzate manufacturing method of lignocellulose comprising contacting the lignocellulosic material and the thermal stability xylanase.
[Requested item 2]
　The lignocellulosic feedstock is pulp hydrolyzate process according to claim 1.
[Requested item 3]
　Saccharification reaction containing claim 1 or saccharification of lignocellulose obtained by the saccharification product manufacturing method according to claim 2, and recovering the thermostable xylanase, and thermal stability xylanase recovered, ligno hydrolyzate manufacturing method comprising the method comprising producing a hydrolyzate contacting the cellulosic material, the.
[Requested item 4]
　The saccharification reaction liquid was solid-liquid separated by centrifugation or microfiltration membranes, by ultrafiltering separated liquid with an ultrafiltration membrane, and the heat stability xylanase and saccharification of lignocellulose separated and recovered , hydrolyzate method according to claim 3.
[Requested item 5]
　Producing said centrifugation or a microfiltration membrane in a solid-liquid thermostable xylanase recovered in the separated solid and ultrafiltration membranes, saccharified by contacting the lignocellulosic feedstock, saccharification of claim 4 Production method.
[Requested item 6]
　The thermostable xylanase, as compared to the corresponding wild-type xylanase, the initial rate of reaction is not less than 70%, compared to 50 ° C. and 24 hours of heat treatment after the xylanase activity before heat treatment xylanase activity 50% above, and the and saccharification material producing method according to claim 1 which is a mutant xylanase to any one of claims 5 having a substituted amino acid residue.
[Requested item 7]
　Wherein said variant xylanase, in SEQ ID NO: 1 amino acid sequence as set forth in the Sequence Listing, 29 th asparagine residue is replaced with a leucine residue, the 58th lysine residue is replaced by an arginine residue, 27 th tyrosine residue is replaced by other different amino acid residue and a tyrosine residue, and 44 th variants having at least a substituted amino acid residue substituted with other different amino acid residues asparagine residue and the asparagine residue hydrolyzate process according to claim 6 which is a xylanase.
[Requested item 8]
　The substituted and 27th tyrosine residue of the mutant xylanase is, different from the amino acid residue is a tyrosine residue is a phenylalanine residue, is and replaced with 44th asparagine residue, the asparagine residue hydrolyzate process according to claim 7 using the mutant xylanase is different from the amino acid residue is a serine residue in the hydrolyzate production.
[Requested item 9]
　Wherein said variant xylanase, in SEQ ID NO: 2 amino acid sequence described in the Sequence Listing, at least 154 amino acid residues, saccharification of claim 6 which is a variant xylanase has been replaced with another amino acid residue Production method.
[Requested item 10]
　The mutant xylanase, substituted amino acid residue 33 asparagine residue is replaced with an aspartic acid residue, the 36th substituted amino acid residue glycine residue is replaced with an arginine residue, 90 th threonine residue substituted amino acid residue group substituted by a serine residue, 132 th substituted amino acid residue glutamine residue is substituted with an arginine residue, 154th substituted amino acid residue leucine residue is replaced with a methionine residue group, 174th substituted amino acid residues serine residue is substituted with a threonine residue, 195th substituted amino acid residues proline residue is replaced with a histidine residue, 197th serine residue is asparagine residue the substituted amino acid residue substituted amino acid residue substituted, and the 217 th glycine residue is replaced with glutamic acid residues less The hydrolyzate production process according to claim 9 using a mutant xylanase also contains a saccharified production.
[Requested item 11]
　The mutant xylanases, 30 th substituted amino acid residue isoleucine residue is replaced with a valine residue, 33rd substituted amino acid residues asparagine residue is replaced with an aspartic acid residue, the 36th glycine residue claim a replacement-amino acid residue group is substituted with an arginine residue, and 154th leucine residue is a mutant xylanase comprising at least a substituted amino acid residue substituted with a methionine residue to the hydrolyzate produced 9 saccharification product manufacturing method according to.
[Requested item 12]
　Of the variant xylanase, 30th substituted amino acid residue isoleucine residue is replaced with a valine residue, the 59th substituted amino acid residue serine residue is substituted with a threonine residue, 154th leucine residue substituted amino acid residue but substituted with a methionine residue, the 239th substituted amino acid residue tyrosine residues is replaced with a histidine residue, and 242 th substituted amino acid residue in which the cysteine ​​residue is substituted with a serine residue hydrolyzate process according to claim 9 using a mutant xylanases that contains at least a group hydrolyzate production.
[Requested item 13]
　In SEQ ID NO: 1 amino acid sequence as set forth in the Sequence Listing, 29 th asparagine residue is replaced with a leucine residue, the 58th lysine residue is replaced by an arginine residue, 27th tyrosine residue of tyrosine residues other different is replaced with an amino acid residue, and 44 th mutant xylanases with at least a substituted amino acid residue substituted with other different amino acid residues asparagine residue and the asparagine residue and.
[Requested item 14]
　In SEQ ID NO: 1 amino acid sequence as set forth in the Sequence Listing, are replaced with 27th tyrosine residue, other different amino acid residue and a tyrosine residue is a phenylalanine residue, and the 44 th asparagine residue substituted is the mutant xylanase according to claim 13, which is a serine residue different from the amino acid residue is an asparagine residue.
[Requested item 15]
　In SEQ ID NO: 2 amino acid sequence described in the Sequence Listing, at least 154 th leucine residue, mutant xylanases has been replaced with another amino acid residue.
[Requested item 16]
　In SEQ ID NO: 2 amino acid sequence described in the sequence listing, substituted amino acid residue 33 asparagine residue is replaced with an aspartic acid residue, a substituted amino acid residue 36 th glycine residue is replaced with an arginine residue , substituted amino acid residues 90 th threonine residue is substituted with serine residue, 132 th substituted amino acid residue glutamine residue is substituted with an arginine residue, 154th leucine residue is methionine residue substituted substituted amino acid residue, a substituted amino acid residue 174 serine residue is replaced with a threonine residue, 195th substituted amino acid residues proline residue is replaced with a histidine residue, 197th serine location residue substituted amino acid residue substituted with asparagine residue, and the 217th glycine residue is replaced with glutamic acid residue Variant xylanase of claim 15 which contains at least the amino acid residues.
[Requested item 17]
　In SEQ ID NO: 2 amino acid sequence described in the sequence listing, substituted amino acid residue 30 isoleucine residue is replaced with a valine residue, 33rd substituted amino acid residues asparagine residue is replaced with an aspartic acid residue , 36th glycine residue of claim 15 substituted amino acid residue substituted with arginine residues and that the 154 th leucine residue contains at least a substituted amino acid residue substituted with a methionine residue mutant xylanase.
[Requested item 18]
　In SEQ ID NO: 2 amino acid sequence described in the Sequence Listing, 30 th substituted amino acid residue isoleucine residue is replaced with a valine residue, a substituted amino acid residue 59 serine residue is replaced with a threonine residue, substituted amino acid residues 154 th leucine residue is replaced with a methionine residue, the 239th substituted amino acid residue tyrosine residues is replaced with a histidine residue, and 242 th cysteine ​​residues in the serine residue variant xylanase of claim 15 comprising at least a substituted substituted amino acid residue.
[Requested item 19]
　The nucleic acid represented by the nucleotide sequence encoding the amino acid sequence of the mutant xylanase according to claims 13 to any one of claims 18.
[Requested item 20]
　An expression vector comprising the nucleic acid of claim 19.
[Requested item 21]
　Transformant containing the expression vector of claim 20.
[Requested item 22]
　E. coli transformant of claim 21, Bacillus subtilis, yeasts, actinomycetes or filamentous fungi cells from the host cell.
[Requested item 23]
　The filamentous fungus, Trichoderma spp., Acremonium spp, transformant according to claim 22 belonging to Humicola genus or Aspergillus genus.
[Requested item 24]
　The filamentous fungus, Trichoderma viride, Acremonium cellulolyticus, transformant according to claim 22 or claim 23 which is a Humicola insolens or Aspergillus niger.
[Requested item 25]
　From at least one of either culturing the transformant according to one paragraph, the culture of the transformant and the transformant was cultured according to claim 24 claim 21, claim claim 13 method for producing a mutant xylanases comprising recovering the mutant xylanase according to 18 any one of.
[Requested item 26]
　Manufactured by the method of claim 25 mutant type xylanase.
[Requested item 27]
　Compositions containing mutant xylanases according to any one of claims 18 and claim 21 claim 13.
[Requested item 28]
　A mutant xylanase according to any one of claims 13 to claim 18 and claim 21, contacting a pulp
bleaching process of pulp containing.
[Requested item 29]
　Detergent comprising a variant xylanase according to claims 13 to any one of claims 18 and claim 21.
[Requested item 30]
　Animal feed comprising the variant xylanase according to claims 13 to any one of claims 18 and claim 21.
[Requested item 31]
　Pan modifier made comprising the variant xylanase according to any one of claims 18 and claim 21 claim 13.