CELLULOSE FREE NUTRIENT MEDIUM FOR ENHANCED PRODUCTIVITY AND ACTIVITY OF CELLULASE FREE ENZYMES
FIELD OF INVENTION
The present invention relates to soluble alkaline lignocellulosic extract (SALE) as an enriched nutrient medium for enhanced production of cellulase free enzymes, more particularly cellulase free xylanase and process thereof.
BACKGROUND OF INVENTION
Agro residues are primarily composed of lignocellulose, a complex fibrous structure of the sugar polymers, cellulose and hemicellulose, which is strongly intermeshed with the aromatic co-polymer lignin. Both the cellulose and hemi-cellulose fractions are potential source of fermentable sugars. For this purpose, the sugars must be made available from the complex fibrous biomass by means of physical/chemical pretreatments followed by enzymatic treatments. Agro residues have been utilized for microbial degradation process for production of high value products. Treated agro residues are primarily employed in solid state fermentation. Though, lignocellulosic residues have been used in submerged fermentation processes, the problems of low bulk density, mass and heat transfer besides control of parameters, are overwhelming.
Attempts have been made to use lignocellulose in solid state fermentation as well as submerged fermentation. US patent no 5,981,233 discloses use of thin rye stillage as a source of lignocellulose after some special pre treatment for the production of xylanase. However, increasing concentrations of rye stillage inhibited the growth and enzyme production using Trichoderma reesei. Other lignocellulosics viz. wheat straw, rice straw, rice husk and sugarcane baggase were reportedly utilized for the production of xylanase enzyme in solid state fermentation, where alkali treatment and subsequent washing of the residues led to the reduction in its enzymatic activity using Melanocarpus albomyces IIS 68 (1).
Lignocellulosic residues were also used in submerged fermentation process for the production of xylanase enzyme using Melanocarpus albomyces IIS 68 resulting in 170 IU/ml of enzyme activity in 122 h fermentation operation (2). Though, the presence of

lignocellulosic residues in submerged fermentation media requires repeated sterilization, longer time duration to attain optimum enzyme activity and poses limitations on their use at high concentrations due to their low bulk density, it also leads to poor control of environmental parameters, such as, pH, dissolved oxygen, and temperature in a bioreactor arising due to poor heat and mass transfer.
Lignocellulose provides nutrients to growing microbial cultures which either through bioconversion or metabolic synthesis convert it into value-added bio-products as outlined in Figure-1.
Hydrolytic enzymes, one of them xylanases, have tremendous application potentials in the pulp and paper industry in addition to existing uses. Xylanase enzymes have conventionally been produced by using enzymatic-hydrolysed or non-hydrolysed lignocellulosic residues as inducers of the enzyme. The presence of the lignocellulosic residues in fermentation media requires repeated pre-sterilization at elevated temperatures. Moreover, use of the insoluble residues in fermentation media poses limitation on its inclusion at high concentrations, due to its low bulk density. For cellulase-free xylanase production using fermentation, a cellulose-free nutrient medium is required, since most microorganisms used in such fermentation have inducible enzyme system for both xylanase and cellulase.
Xylanase is a hemicellulase enzyme which hydrolyzes xylans (also called "wood gums") to xylose and other sugars. The enzyme is also known as endo-1, 4-.beta.-D-xylanase or 1, 4-.beta.-D-xylan xylanohydrolase and belongs to the EC 3.2.1.8 category of enzyme.
Xylanase are polysaccharides formed from 1, 4- beta.-glycoside-linked D-xylopyranose with short side-chains of different composition, and may contain arabinose, glucose, galactose and/or glucuronic acid as well as acetyl and methyl groups in the molecule.
Xylans are components of many deciduous and coniferous trees, as well as cereals, bran, pectin, tragacanth, plant gums, etc. Xylans are structurally diverse. Therefore, complete degradation of branched, partially acetylated xylans requires the action of a variety of xylanases to hydrolyze the xylans to xylose and other sugars. The mode of action of

xylanases is complex and is realized in conjunction with other (to some extent synergistic) enzymes.
PRIOR ART OF THE INVENTION
US Patent No.4, 182,780 describes a process and an apparatus for alkali treatment of lignocellulosic material for increasing feed value of lignocellulosic materials. The objective of this invention is different from our present invention.
US Patent No.4, 048,341 refers to a process and an apparatus for increasing feed value of lignocellulosic material. Thus the objective of this invention and present invention are very different.
DE 10139949 describes solid state fermentation process
DE 4017522 refers to new cellulase free xylanase enzyme produced by culturing Thermomyces lanuginosus in a nutrient medium containing lignocellulose.
US 5,981,233 teach process for manufacturing a xylanase-rich enzyme complex by cultivating microorganism of the genus. Trichoderma in a nutrient medium containing pre-treated thin stillage of rye.
Anita Jain [3] reports the production of xylanase by thermophilic Melanocarpus albomyces IIS 68 by solid state fermentation and is different from the approach adopted in the present invention.
Saraswat et al.[4] describes the induction of maximum number of isoenzymes on wheat straw and in fact that xylanases act synergistically to bring about hydrolysis of xylans probably explain highest xylanase activity on the lignocellulosic substrate.
Proceedings of 6th International Conference on Pulp & Paper Industry [5] describes the use of xylanase enzyme as a pre-bleaching agent the objective was also to reduce or eliminate the use of elemental chlorine in bleach sequences to reduce pollution loads from bleaching. It also reveals the production and characterization of Xylanase enzyme produce from Melanocarpus albomyces. The strain was grown on medium containing wheat straw of mesh size below 250nm providing highest enzyme activity. However, it was observed that pH between 5.5 to 7.0 provided in a retained enzyme activity upto 60%, whereas that pH above 7 loss of enzyme activity was observed.

Use of lignocellulose residues such as wheat bran, rice bran, and wheat straw in fermentation medium, is associated with many problems due to its insoluble nature. Therefore it is necessary to develop a process wherein the polymer of pentose present in lignocellulosic materials to be available in soluble and unhydrolyzed form, which can be used in submerged fermentation avoiding the limitations of the prior art processes. Thus the present invention provided surprising results by using an alkali soluble lignocellulosic extract as one of the components of nutrient medium in the submerged fermentation process for the production of cellulase free enzymes, more particularly cellulase free xylanase with high yield and enzyme activity. The process of the invention also eliminates the tedious step of repeated sterilization in the fermentation process and accomplishes enhanced enzyme activity in a shorter duration of time.
OBJECTS OF THE INVENTION
Main objective of the invention is to provide nutrient medium free from cellulose for the enhanced production of cellulase free xylanase.
An objective of the invention is to provide alkali soluble portion of lignocellulose as carbon source and inducer for the production of cellulase free xylanase in submerged fermentation process.
Another object of the invention is to provide nutrient medium as a potential inducer for xylanase enzyme.
Yet another object of the invention is to provide nutrient medium comprising cellulose free carbon source.
Still another object of the invention is to provide nutrient medium which does not warrant repeated sterilization in the submerged fermentation process.
Still yet another object of the invention is to provide a fermentation process which is devoid of limitation of poor control of environmental parameters such as pH, dissolve oxygen and temperature arising in a bioreactor arising due to poor heat and mass transfer.
Further object of the invention is to provide an economical and improved submerged fermentation process.

SUMMARY OF THE INVENTION:
The present invention relates to soluble alkali lignocellulosic extract (SALE) as a cellulase free enriched nutrient medium as carbon source for induction of cellulase free enzymes, more particularly cellulase free xylanase enzyme and the invention also relates to use of said nutrient medium in a process for high production and enhanced enzyme activity of cellulase free xylanase in submerged fermentation process.
DETAILED DESCRIPTION OF INVENTION
Treatment of lignocelluloses, such as wheat straw, with alkali separates the hemicelluloses in soluble form from the insoluble lignocellulose, and thus the extracted portion can be employed successfully in the submerged fermentation process. In accordance with the present invention, high production and enhanced activities of thermophilic xylanase are accomplished using Melanocarpus albomyces IIS 68 in media containing SALE.
The present invention thus relates to increasing the utility value of lignocellulosic materials by alkali-treatment and using the said soluble portion as nutrient-rich form as an inducer and carbon source in submerged fermentation. SALE is found to contain no free reducing sugars and is free from cellulose. The reducing sugar is detected only after enzymatic hydrolysis by xylanases. Also, it is a potential inducer for xylanases but not cellulase, and thus, it can be employed on industrial scale as a cellulose-free carbon source. It could be used for induction of other enzymes as well, such as mannanase.
One of the preferred embodiments of the present invention provides cellulose free medium for enhanced production and activity of cellulase free enzyme from micro organism, the said medium comprising of:
W/w (%)
a) Soluble Alkaline Lignocellulosics Extract (SALE) 70.0 to 96.0;
b) Mineral salt 0.03 to 0.07;
c) KH2PO4 0.2 to 0.06;
d) K2HPO4 0.03 to 0.08; and
e) Nitrogenous source 0.12 to 0.20

and an acid to adjust the pH between 7.0 to 7.2.
The lignocellulosic material used is selected from the group consisting of wheat straw, rice straw, barley straw and corn cob. The mineral salt used is selected from the group consisting of magnesium sulphate and magnesium chloride.
The nitrogenous source is selected from the group consisting of urea, ammonium nitrate, ammonium phosphate and di-ammonium hydrogen phosphate
The microorganism used is selected from the group consisting of Melanocarpus albomyces, Aspergillus niger, Trichoderma resei, Schizophyllum radiatum and Thermomyces lanuginosus.
The acid used for adjusting the pH is selected from the group consisting of ortho-phosphoric acid, succinic acid, malic acid, acetic acid, sulphuric acid, citric acid and pyruvic acid
The cellulase free enzyme obtained is cellulase free xylanase.
One more embodiment of the invention provides a process for the preparation of cellulase free xylanase, said process comprising steps of:
(a) preparing soluble alkaline lignocellulosic extract (SALE) by treating lignocellulosic material with an aqueous alkali solution, filtering to obtain a clear filtrate,
(b) adjusting the pH of the filtrate of step (a) in the range of 7.0 to 7.2 with an acid,
(c) adding mineral salt, nitrogenous source, dipotassium hydrogen phosphate and potassium dihydrogen phosphate,
(d) autoclaving at a temperature of about 120°C for twenty minutes,
(e) innoculating with preculture seed and incubating at a temperature of about 45° C at a speed of 220 rpm for a time period of about 30 hrs., and
(f) obtaining cellulase free xylanase
In the following section, the invention will be described primarily with reference to wheat straw, but it should be noted that the invention, is in no way, limited to this but related

to the treatment of all types of lignocellulosic materials, such as rice straw, corn stalk, jute sticks and so on.
In the present invention the example described should not be construed to limit the scope of the invention. The present invention has been described in terms of its specific embodiments and certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of present invention.
EXAMPLES
EXAMPLE 1 Preparation of Soluble Alkaline Lignocellulosics Extract (SALE):
Size of wheat straw, received from thrasher unit, is 4.5 cm and less. It is further cut in a pulverizer to size 6 mm and less. 4 g wheat straw from pulverizer is washed with demineralized water once and the washed water is discarded. The residue is re-suspended in distilled water and 1.12 g KOH is added. The final volume of the suspension is adjusted to 100 ml with distilled water. The suspension is heated at 70°C for one hour.
Thereafter, on cooling, the suspension is filtered to remove suspended residues. The clear supernatant so obtained has hemicellulose in soluble and unhydrolyzed form. This filtrate is termed as soluble alkaline lignocellulosic extract (SALE).The pH of SALE is adjusted to 7.2 by 44 % H3PO4. The SALE could be supplemented with additional nutrients (eg. nitrogen source, Mg+2 sources etc.) for using it as fermentation medium for growth of microorganism or for induction of enzymes requiring hemicellulose as an inducer.
EXAMPLE 2
The fungus has optimum growth temperature of 45°C and pH of 7.2 and produces thermophilic xylanase, which finds application in paper and pulp industry. High xylanase activities are obtained by Melanocarpus albomyces IIS 68 using SALE as sole carbon source and inducer in shake flask. Melanocarpus albomyces IIS 68 was isolated from compost in 1985[6]
The pure and potent culture maintenance is carried on Sabouraud Dextrose Agar and the pure colonies are separated by further sub culturing. The culture is then grown on YPSS media slants; the composition of the same is given in Table-1. This is a conventional media.

The best growth is observed in 4-5 days. The Pre-Cultures (PC) are raised on media proposed in Table-2.
Since the fungal growth is characterized by mycelia pellets, a few neutral glass beads (4mm diameter) are included in the flask to prevent clumping of pellets. For PC-I stage, the culture from one slant is transferred into 25 ml of media taken in 100 ml flask. After incubation for 36-42 h at 45°C, 5 ml of PC-I culture is transferred to 95 ml of media containing media components for 100ml working volume in 500 ml flask for PC-II stage. After 24 hours, 25 ml of PC-II stage is transferred to 100 ml production media (Table-3) in 500 ml flask. The fermentation is carried out at 45°C for 24-30 hours. Thereafter, the broth is harvested and the mycelium is separated from the broth by filtration. The enzyme is present in aqueous phase (filtrate)
EXAMPLE 3:
Process for obtaining cellulase free xylanase. Steps adopted are enumerated below:
Pure and Potent Culture Maintenance:
a. Potent Culture maintenance
For the pure and potent culture maintenance of Melanocarpus albomyces IIS 68, the yeast phosphate soluble starch (YPSS) culture media was used. At times purity of culture was ensured using serial dilution technique and such pure culture was sub-cultured using slants of the same media, which were sub-cultured every month.
b. Serial Dilution Technique
1. 1 ml sample prepared from stored culture was suspended in 9 ml of sterile saline (Sodium Chloride 0.85 %) to make a microbial suspension.
2. Serial dilutions 10-2 10-3 & so on were prepared.
3. 0.1 ml of aliquot of various dilutions were used to inoculate sterile yeast
phosphate soluble starch (YPSS) media petri plates, which was uniformly spread
with help of sterile glass spreader.
4. Petri plates were incubated in an inverted position for 3-4 days at 45 °C, in a BOD incubator.

c. Media Composition and preparation
i. Yeast Phosphate Soluble Starch (YPSS) Media Composition: Slant Media Table I
(Table Removed)
ii. YPSS Media Preparation:
1. 'A', 'C and 'D' media components were dissolved in 100 ml distilled water and the pH was set to 7.0.
2. Component 'E' was added as a jellying agent and autoclaved at 15psi for 20 min.
3. The stock solutions of 'B' was autoclaved separately and added aseptically in the requisite amount at room temperature.
4. Approximately 7 ml of YPSS media was poured into 30 ml capacity test tubes after proper mixing of all the components.
5. Test tubes were kept in slanting position for solidification of the media.
d. Enzyme Production:
1. 100 ml SALE obtained by extracting wheat straw with alkali (was taken and autoclaved at 121°C for 20 min.
2. Other components were added from stock solution.
3. Each production flask was inoculated with one Pre culture (seed) flask (i.e. 25ml) and incubated at 45°C at 220 rpm.
Sampling was performed after required incubation time i.e. 18, 24, 30 hrs.

a. Media Composition and preparation I) Pre-culture media composition:
(Table Removed)
Pre-culture media preparation:
1. In 100 ml volumetric flask, 23.5 ml volume of distilled water was taken.
2. All other media components were autoclaved (15 psi and 121 °C) separately and added aseptically from their stock solutions.
3. Each seed media containing flask was inoculated with one slant, which was scrubbed gently at the upper surface with sterile loop after taking some of media (approximately 5 ml) into it and shaked carefully before transferring inoculum into respective flask.
4. The seed culture was incubated in orbital shaker having temperature and agitation set points at 45°C and 130 rpm respectively for 48 hours. Microscopic observations were made at various steps to check any contamination, especially before inoculation.
II) Production Media composition:
(Table Removed)
i. Preparation of Wheat Straw:
1. Primary sieving of wheat straw was performed using the sieve of mesh size
above 1000 micron.
2. Primary sieving was followed by clearing of all the hard pieces of straw, which makes grinding easy.
3. To reduce the moisture content the wheat straw was put in the aluminum tray, which was kept in the oven at 80°C for 2-3 h.
4. Further grinding of the wheat straw was done using domestic grinder giving 8-10 second operating pulse.
5. The wheat straw was sieved and the fractions between 50-63 micron mesh sizes were taken.
6. Wheat straw was collected and washed with de-mineralized (DM) water once and air dried on blotting paper for 30 min followed by oven drying at 80°C for 7-8 h.
ii. Preparation of wheat straw extract:
1. 4 % wheat straw was taken in 100 ml distilled water and 0.2M KOH was added.
2. The flask was incubated in hot water bath at 70 °C for 1h.
3. Cooling was done in running tap water for 2-3 min in shaking manner followed by cold water bath for l-2min.
4. The extract was filtered through muslin cloth and neutralized with 44% H3PO4 to pH 7.2 and the total volume was made to 100ml by adding distilled water.
EXAMPLE 4: Enzyme Activity Estimation:
A. Preparation of Sodium Phosphate Buffer (pH 6.0 and 0.05 M):
To 150 ml of 0.05 M Sodium di-hydrogen ortho-phosphate (NaH2PO4.2H2O), about 30 ml of 0.05 M di-Sodium hydrogen ortho phosphate (Na2HPO4) to obtain a buffer solution of 0.05M and pH 6.0
B. Preparation of Substrate:
1. 1.0 % oat spelt xylan was suspended in sodium phosphate buffer mentioned above.
2. Preheating was done at 70°C for 10 minutes, followed by magnetic stirring for 35-40 minutes at 25-35°C.
C. Standard Operating Procedure:
1. The enzyme sample was taken in an eppendroff tube and centrifuged at 10,000 rpm for 10 min. in a cooling centrifuge.
2. From the supernatant, at least, three suitable dilutions were made
3. Buffer blank (having 2 ml buffer), substrate blank (having 1.8ml substrate, and 0.2 ml buffer), enzyme blank (0.2 ml suitably diluted enzyme + 1.8 ml buffer), and combined enzyme plus substrate blank (having 1.8ml substrate only) were prepared.
4. Added 1.8 ml of substrate from the flask, while on magnetic stirrer, into each assay tube and kept the tubes in 70°C water bath for 10 minutes for temperature equilibration.
5. Added 0.2 ml of suitably diluted enzyme to the successive tubes in an order with 15 sec interval (phased manner). Each tube was taken out of 70°C water bath for this addition and placed back immediately into the water bath Enzyme should not be added to any of the blank tube.
6. Started beeper timer as soon as 0.2 ml enzyme was added to the first tube.
7. After 5 min. of incubation, with respect to the first test tube, the tubes were taken out from the water bath in the same order with an interval of 15 seconds (phased manner). As soon as a test tube was taken out of the water bath, it was put in ice bath followed by immediate addition of 3 ml DNS

reagent, to stop the hydrolysis reaction (phased manner). In case of combined enzyme plus substrate blank tube, 3ml DNS was added followed by 0.2 ml suitably diluted enzyme.
8. After cooling all the tubes were placed in 100°C water bath for 5 min.
9. The tubes were cooled in circulating water ice bath for lmin.
10. 20 ml distilled water was added at room temperature to each tube and shaked
properly.
11. Optical density of substrate blank and enzyme blank and combined blank was
checked with respect to buffer blank.
12. Optical density of the assay tubes was measured at 540 nm, using 1cm path
length cuvette, with respect to combined enzyme plus substrate blank.
13. Calculate mg xylose produced in the assay mixture using standard
curve from the measured optical density in step 12.
Calculating enzyme activity of the sample:
Calculation
mg xylose produced in assay tube = O.D / 0.683 (from standard curve)
(Equation Removed)
(Taking molecular weight of xylose=150; hydrolysis time = 5 min., and enzyme sample volume = 0.2 ml, D.F is dilution factor) Enzyme Activity (IU/ ml) = 250-300 mg xylose in assay tube * 6.66 * D.F
EXAMPLE 5: Enzyme activity accomplished in Solid State Fermentation:
The reported production of xylanase enzyme [4] using Melanocarpus albomyces IIS 68 and its enzyme activity is 170 IU/ml in 122 h fermentation operation.
EXAMPLE 6: Enzyme production using only SALE and no salts
Only SALE and buffer components without any mineral salts component resulted in only 25 % activity of the cellulase free xylanase as compared to SALE used in complete media as referred to in claim 1.

Main Advantages of Present Invention
(1) Avoids repeated sterilization in the fermentation process.
(2) High productivity of cellulose free enzymes.
(3) Considerable reduction in fermentation process time.
(4) Accomplishment of high enzyme activity.
(5) Cost effective fermentation process.
REFERENCES
1. Process Biochem., Vol.30, No. 8, p.705-709, 1995
2. Journal of Fermentation and Bioengineering, Vol. 83, No. 4, p. 352-357, 1997
3. Process Biochem, Vol.30, No. 8, pp.705-709, 1995.
4. Saraswat, V. and Bisaria V.S.,"Biosynthesis of xylanolytic and xylan de-branching
enzymes in Melanocarpus albomyces IIS 68", J. Ferment. Bioeng., Vol. 83, No.
4,p.352-357, 1997.
5. Mishra S., et al., "Melzyme LS- A novel enzyme formulation for Pre-bleaching of Kraft pulp," Proceedings of 6th International Conference on Pulp and Paper Industry, New Delhi, India, p.73-86, 2003.
6. Rao N.J., et al., "Xylanase pre-bleaching of agro-residue pulps," Proceedings of 6th International Conference on Pulp and Paper Industry, New Delhi, India, p.87- 104, 2003.
7. J. Gen. Microbiol., Vol.131, p. 3017-3027, 1985).

I/We claim:
1. Cellulose free medium for enhanced production and activity of cellulase free enzyme
from micro organism, the said medium comprising of:
w/w (%)
a) Soluble Alkaline Lignocellulosics Extract (SALE) 70.0 to 96.0
b) Mineral salt 0.03 to 0.07
c) KH2PO4 0.2 to 0.06
d) K2HPO4 0.03 to 0.08
e) Nitrogenous source 0.12 to 0.20
and an acid to adjust the pH between 7.0 to 7.2.
2. The medium of claim 1, wherein the lignocellulosic material used is selected from the group consisting of wheat straw, rice straw, barley straw and corn cob.
3. The medium of claim 1, wherein the mineral salt used is selected from the group consisting of magnesium sulphate and magnesium chloride.
4. The medium of claim 1, wherein the nitrogenous source is selected from the group consisting of urea, ammonium nitrate, ammonium phosphate and di-ammonium hydrogen phosphate
5. The medium of claim 1, wherein the microorganism used is selected from the group consisting of Melanocarpus albomyces, Aspergillus niger, Trichoderma resei, Schizophyllum radiatum and Thermomyces lanuginosus.
6. The medium of claim 1, wherein the acid used for adjusting the pH is selected from the group consisting of ortho-phosphoric acid, succinic acid, malic acid, acetic acid, sulphuric acid, citric acid and pyruvic acid
7. The medium of claiml, wherein the cellulase free enzyme obtained is cellulase free xylanase.
8. A process for the preparation of cellulase free xylanase, said process comprising steps of:

(g) preparing soluble alkaline lignocellulosic extract (SALE) by treating lignocellulosic material with an aqueous alkali solution, filtering to obtain a clear filtrate,
(h) adjusting the pH of the filtrate of step (a) in the range of 7.0 to 7.2 with an acid,
(i) adding mineral salt, nitrogenous source, dipotassium hydrogen phosphate and potassium dihydrogen phosphate,
(j) autoclaving at a temperature of about 120°C for twenty minutes,
(k) innoculating with preculture seed and incubating at a temperature of about 45° C at a speed of 220 rpm for a time period of about 30 hrs., and
(l) obtaining cellulase free xylanase