FIELD OF THE INVENTION:
The invention relates to a process for production of cellulase.
BACKGROUND OF THE INVENTION:
Alcohol is produced in grain-based distilleries by fermenting different grains like sorghum, millets, maize, barley, rice, wheat or any other grain rich in starch. Once the fermentation is complete, the fermented mash is distilled to separate alcohol from slurry. The whole stillage, remaining after distillation is centrifuged to separate wet cake and thin stillage. Wet cake is dried to make Distiller's Dried Grain (DDG) and the thin stillage is concentrated by vacuum evaporation to make thick slurry that is termed as Distiller's solubles. Distiller's Dried grain (DDG) and Distiller's solubles are mixed and such mixture is subjected to another step of drying to make Distiller's Dried Grain and Solubles (DDGS) (as shown in fig 1). The entire process of making DDGS is highly energy intensive and hence, extremely costly. Separate utilisation of DDG and solubles will lead to marked reduction in the energy input for drying. Exploring the value-added applications of these by-products is a priority for the grain based alcohol industry to economise the process costs.
Brewer's spent grain is a by-product of brewery, obtained by filtering the fermented wort and drying the filter-cake.
Cellulase is a group of enzymes that degrade cellulose, which is present in wood, cloth, paper, cotton, plant material and any other lignocellulosic material or other products derived from such materials. Cellulose is composed of P-1,4 linked glucose units. The glucose chain length may be as large as 8,000-12,000 units.

Cellulase breaks these bonds to release glucose or other oligo-saccharides or causes changes in the physical structure of the materials containing cellulose.
Cellulase hydrolyses 0-1, 4 glycosidic bonds to produce monomers, dimers and oligomers of glucose. There are primarily three enzymes that constitute the cellulase complex. These are endoglucanase, cellobiohydrolase andβ-glucosidase. Cellulase is extensively used in poultry feed for reduction of the fibre content and in textile for treatment of denim fabric and polishing of cotton textiles. This enzyme finds application in chemical process as for treatment of paper pulp. It also has potential in other industries such as production of glucose from cellulose, which can in-turn be used for production of ethanol, glycerol, acetic acid, high fructose syrup etc.
Being the most abundant polymer in the biosphere there is immense interest in utilising cellulose for production of energy and chemicals. Though processes are available for hydrolysis of cellulose efforts are continuously being made to reduce the cost of cellulose hydrolysis. Enzymatic hydrolysis process using cellulase for breakdown of cellulose is preferred industrial process. Thus reduction hi the cost of cellulase is an active area of research.
There are a wide variety of substrates that are used for production of cellulase. This includes lignocellulosic substrates e.g. cellulose from different sources, wheat bran, rice bran, sulfite liquor, grain husks, filter paper etc. In the prior art, production of cellulase has been reported from a variety of materials as mentioned above. The most widely used substrates are wheat bran, rice bran and Micro-crystalline Cellulose Powder (MCCP). The related art describes use of different strains and mutants of Trichoderma reesei for production of cellulase. Most of the commercially available preparations of cellulase are derived from this fungus.

DDGS, a by-product of grain distillery is currently used for the animal feed and thus fetches low value. Prior art reveals that DDGS can be utilised as animal feed (US Patent No. 5,260,089). In another alternative application of DDGS, it is used as a non-burning tobacco substitute (US Patent No. 4,807,648). The material has also been patented for its application as additive to the bakery products (US Patent No. 5,225,228).
In this invention DDG and distiller's solubles were produced separately from whole stillage instead of making DDGS. Since, DDG is easily available as a waste product, the applicants felt that it could be used as a substrate for production of cellulase.
Accordingly, the applicants conducted a study on the production of cellulase using by-product of grain distillery. The initial experiments were conducted with the fungus, T. reesei, which incidentally is a good cellulase producer. The Applicants observed to their surprise that the fungus not only grows well but also secretes a large amount of enzyme in the broth. The activity of the enzyme obtained using DDG as substrate was found to be comparable with those achieved using the commercially employed materials such as wheat bran and Micro-crystalline Cellulose Powder (MCCP).
OBJECTS OF THE INVENTION:
The main object of the invention is to provide an efficient and cost effective process for preparation of cellulase enzymes using the by-product of gram distillery and brewery as substrate.
Another object of the invention is to provide a process for production of cellulase in high yields.

SUMMARY OF THE INVENTION:
The invention provides a novel process for production of cellulase using distiller's dried grain as a substrate, said process comprising the steps of inoculating the substrate with micro-organisms capable of degrading cellulose, and incubation at set temperature between 24-40°C for 7 days and separation of the biomass by conventional methods to obtain cellulase.
STATEMENT OF THE INVENTION:
Accordingly, the present invention provides a process for production of cellulase and the said process comprising the steps:
a) inoculating a medium comprises by-products of grain distillery such as
herein described optionally along with Natick's salt medium, glycerol,
peptone, urea and assimilable carbon and nitrogen sources as substrate with
micro-organisms capable of producing cellulase such as herein described,
b) incubating the medium of step (a) at a temperature in the range of 24 to
40°C at a pH in the range of 4 to 7 with constant shaking at 200 to 300 rpm
for 5 to 10 days to obtain a fermented broth,
c) separating the biomass from the fermented broth and recovering the
supernatant by conventional methods, and
d) concentrating and obtaining cellulase from the supernatant by a
conventional method.
Yet in another embodiment of the present invention the by-products of grain distillery are selected from a group consisting of distiller's dried grain, distiller's dried grain soluble and brewer's spent grain or a combination thereof.
Further, in another embodiment of the present invention the distiller's dried grain and brewer's spent grain are obtained from grains selected from a group comprising sorghum, barley, corn, wheat and millets.

Still in another embodiment of the present invention the particle size of distillers dried grain and brewer's spent grain ranges from 100 to 1000 microns.
Yet in another embodiment of the present invention the carbon to nitrogen ratio of the medium is 10 to 20.
Still in another embodiment of the present invention the micro-organisms are selected from Trichoderma sp. Cellulomonas sp., Aspergillus sp. Bacillus sp., Clostridium sp. and Thermomonospora sp.
Further in another embodiment of the present invention the microorganism is Trichoderma reesei.
In still another embodiment, the biomass is separated by conventional methods selected from filtration and centrifugation.
In one more embodiment of the present invention the sterile air at a rate of 0.5 wm is passed over the medium.
Still in another embodiment of the present invention the biomass is separated from the culture of step (d) by conventional methods selected from filtration and centrifugation.
Further in another embodiment of the present invention the extraction of cellulase is effected by methods selected from batch, fed batch, continuous culture, submerged culture and solid-state culture.

Yet in another embodiment of the present invention the cellulase enzyme produced is used for applications in poultry feeds, reduction in fiber content of feed/ feed ingredients, cellulose hydrolysis, textile processing, food processing, pulp/ paper processing or any other such application.
Still in another embodiment of the present invention the Natick's medium comprises Peptone 1.0 g/1, KH2PO4 2.0 g/1, (NH4)2SO4 1.4 g/1, Urea 0.3 g/1, MgS04.7H2O 0.3 g/1, CaCl20.3 g/1, FeS04.7H2O 5.0 mg/1, MnSO4.H2O 1.6 mg/1, ZnSO4.7H2O 1.4 mg/1, CoCl2 2.0 mg/1.
In yet another embodiment, the by-product is recovered by methods such as solvent precipitation, ultra filtration, chromatography and vacuum evaporation.
A process for the production of cellulase substantially as herein described with reference to the examples and drawings.
Distilleries producing potable alcohol from grain also produce a by-product called Distiller's dried grain and solubles (DDGS) in large quantities. DDGS is produced by drying the distiller's wet grain to make distiller's dried grain (DDG), to which, Distiller's solubles are mixed followed by drying (as shown in fig 1). DDGS is conventionally used as cattle feed. In this invention the drying process was altered to produce DDG and Distiller's solubles separately.
DDG was used for cellulase production. DDGS has also been used for

production of cellulase in this invention, but the use of DDG is preferable both in terms of the final activity achieved as well in terms of the reduction in the step for production of DDGS.
The Applicants observed that the production of cellulase employing distiller's dried grain is more than other conventional substrates. The productivity of enzyme produced using DDG is compared with that achieved in the conventional media. The enzyme manufactured thereof and concentrated by the process described herein can be used for the various industrial applications e.g. poultry feed, textile processing, production of ethanol, detergents etc.
The invention is described in detail hereinbelow providing illustrative examples which should not be construed as limitations on the inventive concept embodied in the present invention.
Brief description of the accompanying drawings:
Fig 1 represents the conventional method for production of distiller's dried grain. The process of the invention involved growing the micro-organism, Trichoderma reesei (NCIM 1186; QM 9414) in medium containing cellulose, peptone, salts and glycerol. This inoculum was added to medium containing DDG and allowed to ferment and secrete cellulase. The process for cellulase production on DDG was compared with the other routinely employed substrates for the industrial production of the enzyme. The process under investigation was also studied for the optimal concentration of DDG required for the maximum enzyme yield, for requirement of different supplements, for the effect of scale-up etc. The final broth obtained therein was centrifuged to remove the biomass and the enzyme concentrated by ammonium sulphate precipitation. The enzyme preparation thus obtained was studied for the application.
The strain of Trichoderma reesei QM 9414 was obtained from NCIM and is known to produce cellulase extra-cellularly. The cellulase production was examined on

Congo red Carboxy Methyl Cellulose agar medium. The clearance zone surrounding the colonies indicates production of cellulase. Also the organism produced large amount of cellulase when grown for 7 days at 30°C in the medium containing MCCP, a source of nitrogen and salts. The first step of the process involved growth of cell mass. To achieve this, the cell mass of the organism was grown in the medium containing cellulose source, glycerol (0.5%), a nitrogen source and salts.
Trichoderma reesei grown on MCCP agar slant incubated at 30°C for 7days was used to inoculate the flask containing Natick's medium. The preinoculum was grown for 24 hours and was used to seed the flask containing 5% DDG. The culture not only grew well but also produced high level of cellulase enzyme.
The examples hereinbelow describe the method for the production of cellulase. The experiments have been carried out using different substrates such as distiller's dried grain as substrate, solubles and brewer's spent grain. It may bee added that the inventive concept is to primarily enable all sorts of substrates, as mentioned hereinabove and scope of the invention should not be deemed to be confined to the substrates herein illustrated as any person skilled in the art can easily substitute DDGS for DGG or Brewer's spent grain and attain the same results.
Example 1:
Trichoderma reesei (NCIM 1886; QM 9414) was grown on the MCCP agar slants for 7 days. The temperature was maintained at 30° C for initial two days and then incubating at 25 °C for subsequent five days. Fresh slants were used to inoculate the preculture medium for the growth of cell mass. The medium contained MCCP 10 g/1 and also contained the following ingredients: Peptone 1.0 g/1, KH2PO4 2.0 g/1, (NH4)2SO4 1.4 g/1, Urea 0.3 g/1, MgSO4.7H2O 0.3 g/1, CaCl2 0.3 g/1, FeSO4.7H2O 5.0 mg/1, MnSO4.H2O 1.6 mg/1, ZnSO4.7H2O 1.4 mg/1, CoCl2 2.0 mg/1. These salts are commonly referred to as "Natick's Medium". These salts were

not added in medium with DDG however DDG was used at concentration of 5% and MCCP at the concentration of 1%. The particle size of the DDG was reduced to below 200 microns. The concentration of wheat bran was 5% w/v. The pH of the medium was adjusted to 5.3. The flasks were incubated at 30°C for 24 h at 250 rpm. The fully-grown inoculum was used to seed the flasks containing Natick's medium, DDG, and wheat bran. The flasks were incubated at 30°C for 7 days at 250 rpm. The samples, 5 ml each, were periodically removed for analysis of enzyme activity. The mycelia and undegraded mass was removed by centrifugation at 6000 rpm for 15 min. The activity of the enzyme was determined in terms of the filter paper units (FPU). The enzyme activity determined in media containing different substrates is given hi table 1.
Table 1: Cellulase activity using different substrates
(Table Removed)

Similar results were obtained when the experiment was conducted employing distiller's dried gram solubles.
Example 2:
In another set of experiment cellulase production from T. reesei was studied using DDG prepared from corn after ethanol fermentation. The fermentation and the subsequent process used for producing corn DDG was identical to that used for producing DDG from sorghum used for cellulase production in Example 1. The other parameters of the process for production of cellulase were same as hi Example 1. The cellulase activity achieved using DDG from corn was 57 u/dl while the cellulase activity obtained using DDG from sorghum as substrate was 64 u/dl.
Example 3:

In another set of experiment cellulase production from T. reesei was studied using brewer's grain obtained after ethanol fermentation using barley as raw material. The fermentation and the subsequent process used for producing corn DDG was identical to that used for producing DDG used for cellulase production in Example 1. The other parameters of the process for production of cellulase were same as in Example 1. The cellulase activity achieved using brewer's gram was 59 u/dl while the cellulase activity obtained using DDG from sorghum as substrate was 64 u/dl.
Example 4:
In another set of experiments, similar set-up as hi example 1 was adopted to study the effect of different concentration of DDG in the medium. The different amounts of DDG suspended hi water with pH adjusted at 5.3 were sterilized in separate flasks. The activity was studied at the end of 7 days. The optimum concentration for production maximum FPU was found to be at the level of 5%.
Table 2: Effect of different concentration of DDG and activity of the components of cellulose
(Table Removed)

Example 5:
The process was scaled-up from the flask level to the 19 litre fermenter. Control fermentation was run with Natick's medium and the yields were compared with those obtained with DDG as substrate. During the fermentation the pH was adjusted by addition of alkali to 5.4. After the initial drop in the pH it was

maintained at 3.5. The dissolved oxygen was maintained at the level of 40%
saturation by the cascade control of airflow rate and agitation.
Table3: Fermenter studies on production of cellulase on DDG and MCCP.
(Table Removed)

Production of cellulase using DDG was also tried by solid state fermentation which also gave good results.
Example 6:
The broth obtained after the fermentation as explained in example 1 was centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant from the centrifugation was used for precipitation of the enzyme. The broth at 4°C was gradually subjected to increasing saturation level of ammonium sulphate and centrifuged at each stage. The supernatant at each step was subjected to the subsequent saturation level. The precipitate formed at each stage was dissolved in buffer.
Table 4: Concentration of cellulase by ammonium sulphate precipitation.
(Table Removed)
Example 7:
The concentrated enzyme obtained from the example 4 was used to study the reduction of fibre content in rice bran, which is an ingredient of animal feed. Rice bran was treated with the enzyme produced using DDG and the fibre content was determined. The fibre content of rice bran was reduced from 13.27% to 10.67%. This indicates that the enzyme can reduce the fibre content by about 20%. The fibre reduction in feed components is very important for poultry feed.

We Claim:
1. A process for the production of cellulase, said process comprising the steps:
a) inoculating a medium comprising by-products of grain distillery such as
herein described optionally along with Natick's salt medium, glycerol,
peptone, urea and assimilable carbon and nitrogen sources as substrate
with micro-organisms capable of producing cellulase,
b) incubating the medium of step (a) at a temperature in the range of 24 to
40°C at a pH in the range of 4 to 7 with constant shaking at 200 to 300 rpm
for 5 to 10 days to obtain a fermented broth,
c) separating the biomass from the fermented broth and recovering the
supernatant by conventional methods, and
d) concentrating and obtaining cellulase from the supernatant by a
conventional method.

2. A process as claimed in claim 1, wherein the by-products of grain distillery are
selected from a group consisting of distiller's dried grain, distiller's dried grain
soluble and brewer's spent grain or a combination thereof.
3. A process as claimed in claim 2, wherein the distiller's dried grain and
brewer's spent grain are obtained from grains selected from a group
comprising sorghum, barley, corn, wheat and millets.
4. A process as claimed in claim 2, wherein the particle size of distillers dried
grain and brewer's spent grain ranges from 100 to 1000 microns.
5. A process as claimed in claim 1, wherein the carbon to nitrogen ratio of the
medium is 10 to 20.
6. A process as claimed in claim 1, wherein the microorganisms are selected from
Trichoderma sp. Cellulomonas sp., Aspergillus sp. Bacillus sp., Clostridium sp. and
Thermomonospora sp.

7. A process as claimed in claim 6, wherein the microorganism is Trichoderma
reesei.
8. A process as claimed in claim 1, wherein sterile air at a rate of 0.5 vvm is
passed over the medium.
9. A process for the production of cellulase substantially as herein described with
reference to the examples and drawings.