FIELD OF THE INVENTION
The present invention provides a process for producing pH independent cellulase and a product thereof. More specifically, the invention relates to a process for producing a pH independent cellulase by thermo tolerant fungi Rhizopus oryzae NS5 strain, using pea pod.

BACKGROUND OF THE INVENTION
Cellulases are the important industrial enzymes which have wide application in various fields such as detergent, beverage and biofuel industries. In the biofuels production process, cellulases play a crucial role in the hydrolysis of cellulosic biomass into the fermentable sugars. For the enzymatic hydrolysis of biomass, cellulase system should have three main enzyme components namely, ß-1,4- endoglucanase (EC 3.2.1.4), ß-1,4-exoglucanase (EC 3.2.1.91), and ß-Dglucosidase (EC 3.2.1.21). Endoglucanase (EG) involves in breaking-down the crystalline structure of cellulose microfibrils to release separate polysaccharide chains whereas exoglucanase [cellobiohydrolases (CBH)], gradually alters longchain cellulose into the cellodextrins; and glucosidase (BGL), converts cellodextrins into distinct glucose molecules. Thus, synergic actions of all these enzyme components are required for the effective hydrolysis of cellulosic biomass. The synergic actions of cellulases depend on various factors such as its type, pH, temperature, substrate type, and concentration. pH is one of the most important factors for applicability of cellulases in pulp bleaching process as well as in bioconversion of biomass into biofuels. Therefore, one of the important characteristics of the enzyme, cellulase, its pH range for optimal activity, is likely to have an enormous effect on its applications.
EP2784156 relates to a method of producing cellulase by membrane separation and concentration of a cellulase component derived from filamentaous fungi wherein the pH condition at the time of filtration through an ultrafiltration membrane is preferred, to adjust the pH of an aqueous solution of cellulase derived from filamentaous fungi to pH 2.6 to 5.4 or pH 8.6 to 9.4. It is to be noted that, in the present invention, if the pH of the aqueous solution of cellulase derived from filamentaous fungi already is within a range of a pH of 2.6 to 5.4 or a pH of 8.6 to 9.4, the pH thereof is considered to have adjusted to such a range. It is to be noted that the permeability of an enzyme or protein through an ultrafiltration membrane is known to change by a pH condition.
Almost all of the industrial processes (bioconversion of cellulosic biomass/pulp-paper industries/detergent industries/beverages industries) involving cellulase are dependent on its optimal pH range. Enzyme cost is one of the most expensive steps in bioethanol production from lignocellulosic biomass as it accounts for about 40% of the total cost during ethanol production from cellulosic biomass.
On a commercial scale, cellulases are generally produced using purified cellulose like avicel and solka floc, which are expensive substrates. Therefore, it is important to produce large quantities of cellulase enzyme using abundantly available low cost substrates, like cellulosic biomass for large scale and economically feasible bioconversions. Fungi are the better producers of cellulases enzyme system than bacteria due to high penetration ability and fair production efficient cellulase enzymes.
The present invention takes into account the drawbacks of the prior art and provides a method, which does not require maintenance of pH, for producing cellulase which has a broad range of optimal pH for activity.

OBJECT OF THE INVENTION
The main object of the invention is to provide a solid state fermentation process for producing cellulase by Rhizopus oryzae NS5 strain using pea pod waste-based substrate which does not require maintenance of pH and product thereof.
Yet another object of the invention is to provide a substrate for solid state fermentation enabling cellulase production.

Yet another object of the invention is to provide a crude cellulase enzyme preparation which is highly economical due to its substrate cost.
Yet another object of the invention is to provide a method for producing cellulase which has a broad range of optimal pH for activity.
Yet another object of the invention is to provide cellulase which has broad range of industrial applications owing to its broad range of optimal pH for activity.
Yet another object of the invention is to provide a fermentation media for producing cellulase which does not require maintenance of pH which is a time consuming step and is uneconomical.
Yet another object of the invention is to provide a method for producing cellulase by solid state fermentation by thermo tolerant fungi Rhizopus oryzae NS5 strain.
Yet another object of the invention is to provide a method for isolation of crude extract of cellulase from the solid state fermentation media.
Yet another object of the invention is to provide a cellulase which has a broad range of optimal pH (1.0 to 14 pH) and hence useful for several industrial applications.

SUMMARY OF THE INVENTION
In the main embodiment of the present invention, a solid state fermentation process for producing cellulase by thermo tolerant Rhizopus oryzae NS5 strain is provided, using pea pod waste-based substrate which does not require maintenance of pH and further provides a method for producing cellulase which has a broad range of optimal pH for activity and require 37°C of temperature.
In another embodiment of the present invention is provided, a composition of a media which is used for the fermentation process comprising of 7 gms of a carbon substrate preferably, pea pod waste of 4 mm particle size per 100 ml of a basic media maintaining 70% moisture, wherein the basic media such as Mandel and Weber media comprising of KH2PO4 (1.5 gL-1), MgSO4.7H2O (0.3 gL-1), CaCl2.2H2O (0.4 gL-1), (NH4)2SO4(1.3 gL-1), FeSO4.7H2O (6.0 mgL-1), MnSO4.H2O (1.6 mgL-1), ZnSO4.7H2O (1.3 mgL-1), CoCl2.6H2O (2.0 mgL-1), 0.5% RGO (reduced graphene oxide) and Peptone (0.8 gL-1) and the substrate is achieved by drying, grinding and meshing to particle size of 4mm.
In yet another embodiment of the present invention, the enzyme activity of cellulase is 40 IU/gds (international units of enzyme activity per gram of dried solid) where one unit (U) of the enzyme activity is defined as “the amount of enzyme releasing 1 µmol reducing sugar per min” using glucose as a standard.
In yet another embodiment of the present invention, a method for producing cellulase using thermo tolerant Rhizopus oryzae NS5 strain by solid state fermentation and extracting crude cellulase preparation from the media. Cellulase is produced by adding the fungal mycelia disc of size 5 mm to a 250 ml Erlenmeyer flask containing fermentation media and incubating at 37°C ± 5 for around 72 hrs. The crude extract cellulase enzyme is prepared by adding 50 ml of sodium citrate buffer after 72 hrs to the fermentation media and shaking it at room temperature for 30 mins followed by filtering it using cotton to separate the liquid containing the cellulase and the solid particles. Prior to using the fungal mycelia for fermentation process, the growth of the fungi is maintained on Potato Dextrose Agar media.
Therefore, the present invention provides a method for producing cellulase in a highly cost effective way using easily available substrates such as pea pod waste, and additionally provides cellulase which has a wide range of applicability owing to its unique properties.

DETAILED DESCRIPTION OF THE INVENTION
The present invention will now be described more fully hereinafter. This invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough, and will fully convey the scope of the invention to those skilled in the art.
The present invention relates to a method for producing cellulase by solid state fermentation process using pea pod-waste-based substrate by thermo tolerant Rhizopus oryzae NS5strain.
In the preferred embodiment, the present invention provides a media for fermentation process which does not require maintenance of pH comprising of 7 gms of carbon substrate preferably, pea pod waste of 4 mm particle size per 100 ml of a basic media maintaining 70% moisture, wherein the basic media such as Mandel and Weber media comprising of KH2PO4 (1.5 gL-1), MgSO4.7H2O (0.3 gL-1), CaCl2.2H2O (0.4 gL-1),(NH4)2SO4 (1.3 gL-1), FeSO4.7H2O (6.0 mgL-1), MnSO4.H2O (1.6 mgL-1), ZnSO4.7H2O (1.3 mgL-1), CoCl2.6H2O (2.0 mgL-1), 0.5% RGO (reduced graphene oxide) and Peptone (0.8 gL-1).
In another preferred embodiment, pea pod waste substrate is prepared by completely drying the pea pod waste followed by grinding it to particles of 4 mm size and bringing the moisture content to 70% using Mandel and Weber media. Generally, fermentation processes have a pre-requisite of maintaining pH of the fermentation media for production of enzyme or for any kind other kind of enzymatic process mainly because : growth of the organism used for fermentation process, activity of the organism in the fermentation media, or activity of enzyme produced are highly sensitive to pH of the media. The optimal pH range of the enzyme produced by any fermentation process also largely depends on the factors affecting its production. Maintenance of pH is a major step in most of the enzyme-dependent processes. However, the fermentation media of 7 gms of pea pod waste per 100 ml of basic media providing 70% moisture eliminates the requirement of adjustment of pH of the media making the process easy, less time consuming and highly economical.
In yet another preferred embodiment, the present invention provides a method for producing cellulase using thermo tolerant Rhizopus oryzae NS5 strain by solid state fermentation and extracting crude cellulase preparation from the fermentation media wherein the Rhizopus oryzae NS5 strain is isolated, cultured and maintained on Potato Dextrose Agar media while the cellulase is produced by adding the fungal mycelia disc of size 5 mm to a 250 ml Erlenmeyer flask containing fermentation media and incubating at 37°C ± 5 for around 72 hrs. The crude extract cellulase enzyme is prepared by adding 50 ml of sodium citrate buffer after 72 hrs to the fermentation media and shaking it at room temperature for 30 mins followed by filtering it using cotton to separate the liquid containing the cellulase and the solid particles.
In yet another preferred embodiment, the enzyme activity of cellulase is 40 IU/gds (international units of enzyme activity per gram of dried solid) where one unit (U) of the enzyme activity is defined as “the amount of enzyme releasing 1 µmol reducing sugar per min” using glucose as a standard.
In yet another preferred embodiment, the present invention also relates to producing cellulase enzyme which has a broad range of optimal pH for activity ranging from 1.0 – 14 pH. Generally cellulases produced by bacteria work at acidic/highly acidic pH (~ pH 3.0 maximum), while cellulases produced by fungi work best at pH range of 4.5-7.0. Fungal cellulases with highly acidic and alkaline pH range are rare. The present invention provides cellulases produced by Rhizopus oryzae NS5 strain which have broad range of activity ranging from highly acidic to highly alkaline conditions, which is unconventional for fungal cellulases.
Although the invention has been illustrated and described with respect to one or more implementations, equivalent alterations and modifications will occur to others skilled in the art upon the reading and understanding of this specification and the annexed drawings. In addition, while a particular feature of the invention may have been disclosed with respect to only one of several implementations, such feature may be combined with one or more other features of the other implementations as may be desired and advantageous for any given or particular application. The names are purely indicative and are provided just to best describe the nature of the invention.
Therefore, the foregoing is considered as illustrative only of the principles of the invention. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation shown and described, and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the invention.

We claim:

1. A solid state fermentation process for producing a cellulase enzyme by thermo tolerant fungi strain, said process comprising steps of:
a) isolating the fungi strain;
b) culturing fungi strain;
c) maintaining fungi strain on Potato Dextrose Agar media;
d) adding the fungal mycelia disc of size 5 mm to a 250 ml Erlenmeyer flask containing a fermentation media and incubating at 37°C ± 5 for around 72 hrs;
e) adding 50 ml of sodium citrate buffer after 72 hrs to the fermentation media and shaking it at room temperature for 30 mins; and
f) filtering it using cotton to separate the liquid containing the cellulase and solid particles;
wherein,
the fungi strain is Rhizopus oryzae NS5 strain;
the fungi strain is cultured on a carbon substrate preferably, pea pod waste and a basic media to maintain 70% moisture and therefore obviating requirement of maintenance of pH and thus having a broad range of optimal pH for activity at 37°C ;
the basic media is preferably Mandel and Weber media comprising of KH2PO4 (1.5 gL-1), MgSO4.7H2O (0.3 gL-1), CaCl2.2H2O (0.4 gL-1),(NH4)2SO4 (1.3 gL-1), FeSO4.7H2O (6.0 mgL-1), MnSO4.H2O (1.6 mgL-1), ZnSO4.7H2O (1.3 mgL-1), CoCl2.6H2O (2.0 mgL-1), 0.5% reduced graphene oxide and Peptone (0.8 gL-1); and
the fermentation media comprises the carbon substrate preferably pea pod waste and the basic media.

2. The process as claimed in claim 1, wherein the carbon substrate is preferably pea pod waste with particle size of 4mm achieved by drying, grinding and meshing.

3. The process as claimed in claim 1, wherein the enzyme activity of cellulase produced is 40 IU/gds.

4. A cellulase enzyme obtained from fungi Rhizopus oryzae NS5 strain having enzymatic activity that is pH variation independent.

5. The cellulase enzyme as claimed in claim 4 having wide industrial applications including but not limited to biofuel industries for enzymatic hydrolysis of cellulosic biomass into fermentable sugars, paper and pulp industries as cleansing agent for clear paper whitening, detergent industries as a cleaner, juice and beverages industries for purification of juice or liquid.