|Title of Invention||
"AN IMPROVED PROCESS FOR THE PREPARATION OF CELLULASE."
|Abstract||This invention relates to an improved process for the preparation of cellulase which comprises culturing the strain of Trichoderma ressei having characteristics as herein described employing weeds selected from Parthenium hysterophourus, Eichornia crassipes, Ipomea fistulosa, Lantana camara at a pH of 4.5 at room temperature in presence of mild mutagen for a period of 10 hours, separating and recovering the cellulase by known methods.|
|Full Text||This invention relates to an improved process for preparation of cellulase. The cellulase prepared by the process of the invention is useful to hydrolyse cellulose to glucose which can be fermented to various value added compounds like ethanol 2,3-butanediol, acetone-butanol etc. The central role of energy in economic and social development has long since been recognized and a great deal of effort has been devoted to developing technologies for the extraction production and use of all type sof energy. The aim has always been to reduce costs, make systems more efficient and provide access to previously untapped energy sources. Presently, fossil fuels (coal, oil & gas) account for nearly 90% of the worlds commercial energy production. With the present rate of consumption, the world resources of fosssil fuels are likely to be exhausted by 2020 AD according to reliable estimates.
The possibility of achieving total replacement of the petrochemicals used for ethanol, acetone, butanol and 2,3-butanediol manufacture is the ultimate objective of this approach which certainly points to a promising future of lignocellulosic substrates as sources of chemicals and biochemicals. Propylene, which is one of the principal petrochemical building blocks, is required for butanol manufacture. Acetone, on the other hand, is manufactured by dehydrogenation of isopropanol while ethanol is manufactured from ethylene which is also an important petrochemical building block. 2,3-butanediol, too, is currently obtained from petroleum resources. All these chemicals can be
obtained by fermentation of glucose which, in turn, can be derived through saccharification of cellulose.
Lignocelluiose is considered to be the most abundant revewable source of fuel and organic feedstock which is greatly underutilized. Cellulose saccharification through enzymatic hydrolysis to obtain sugars which can be fermented to produce value added chemicals has received global attention ever since the realization of
the limited nature of fossil fuel reserves which is being depleted at an alarming rate.
. The promising species for this purpose is Trichoderma reesei which possesses
battery of cellulase degrading enzymes and strong secretory ability. Cellulose is produced in millions of tons annually through photosynthesis. The lignocellulosic biomass available in India includes agricultural residues, forestery wastes, jute wastes, coconut wastes. However, the availability of these wastes for the production of fuels and chemicals will determine whether it will play a significant role in the future - there are competing demands for their utilization. In particular, crop residues and bagasse are often used as fodder for livestock or in the paper industry. In this context, nuisance weeds and aquatic macrophytes such as Parthenium hysterophorus (congress grass), Eichornia crassipess (water hyacinth), Ipomea fistulosa, Lantana camara which are non-competing, no-cost and abundantly available throughout the country acquire significance as potential substrates for the production of sugars which can be fermented to various chemicals. The use of such an abundantly available and no-cost substrate could
help in achieving the twin objectives of environmental management, viz., resolving the problem of nuisance weed eradication and taking a step forward in the identification of alternate resources for meeting the growing demands for petrochemicals based products to fulfill our industrial and demostic requirements. Adoption of biotechniloical route to produce petrochemical based product from cellulose saccharification and fermentation enables rerouting of petrochemical building blocks for other uses leading to efficient management of non-renewable resources.
Despite the diversity of the microorganisms and their metabolites, the metabolic pathways for the biosynthesis of various chemicals originate from glucose. Glucose is metabolized to pyruvate through the EMP or ED pathway. Thereafter, the formation of different products is regulated by the ability of the microorganisms to produce energy, the utilization or disposal of excess electron and the presence of active key metabolic enzymes.
The rate limiting step in the conversion of cellulose to chemicals is the hydrolysis of cellulose to glucose. Cellulose is a chemically stable polymer, and its association with hemicellulose and lignin in plants makes it even more inaccessible to the hydrolytic agent - acid or cellulolytic enzymes. Enzymatic hydrolysis is preferred
today since it has the potential to overcome many of the inherent drawbacks of acid hydrolysis, viz., corrosion, byproduct formation and high capital costs.
Cellulase hydrolyses cellulose to glucose and cellobiose (a dimer of glucose). To date, the most extensively studied fungal strain exploited for commercial production of cellulase is Trichoderma reesei since it produces the jcomplete cellulase complex capable of hydrolyzing crystallin cellulose. However its production is subject to catabolite repression and its action is subject to and product inhibition. Moreover, cellulase production is expensive contributing as much as 50 percent to the overall costs of saccharification/fermentation processes. Out of this, the substrate used for enzyme production accounts for 30-40% of the expenses. So far, highly purified cellulose substrates have been used for commercial cellulase production.
Various mutation programmes at various laboratories have utilized different chemicals (NTG, kabicidin, diethyl sulphate, nitrosoethyl area etc.), alone or in combination with uv-light, gamma-irradiation or high energy electron bombardment. The reported enhancement in cellulase production by mutagenesis of T.reesei QM9414 (a mutant derived from T.reesei QM6a) is 1.2 to 1.5 times using harsh mutagens like kabicidin, NTG and UV light. The main object of the present invention is to provide an improved process
for the preparation of cellulase. Another object of the present invention is to provide an improved process for the preparation of cellulase employing the weeds which are abundantly available. In the process of the present invention, a relatively mild mutagen, sodium nitrite, in combination with UV light was utilized and a mutant producing 1.5 to 1.7 times more enzymes as compared to the parent strain (T.reesei QM9414) was isolated. The invention has been designed on the basis of our finding that the weeds available abundantly can be used for the preparation of cellulase.
Accordingly, the present invention provides an improved process for the preparation of cellulase which comprises culturing the strain of Trichoderma ressei having characteristics as herein described employing weeds selected from Parthenium hysterophorus , Eichornia crassipess , Ipomea fistulosa, Lantana camara at a pH of 4.5 at room temperature in presence of mild mutagen for a period of 10 hours, separating and recovering the cellulase by known methods.
In an embodiment of an invention the culturing is done at pH 4.5 and temperature of 30+2°C.
This mutant has a faster rate of sporulation and is less sensitive to inhibition by glucose and cellobiose. The mutant has been successfully grown on dried and size reduced congress grass both in solid as well as liquid culture. The improvisation in this process is in the utilization of pest weeds like congress grass for the production of cellulase and the use of hyperproducing catabolite repression resistant mutant developed by the use of a mutagen which has not been used earlier for the production of cellulase. This enzymes is used to hydrolyse cellulose to glucose which can be fermented to various value-added compounds like ethanol, 2,3-butanediol, acetone-butanol.
In another embodiment of the invention mild mutagen is selected from sodium nitrite in combination of UV light.
We claim :
1. An improved process for the preparation of cellulase which comprises
culturing the strain of Trichoderma ressei having characteristics as herein
described employing weeds selected from Parthenium hysterophorus ,
Eichornia crassipes, Ipomea fistulosa , Lantana camara at a pH of 4.5 at
room temperature in presence of mild mutagen for a period of 10 hours,
separating and recovering the cellulase by known methods.
2. An improved process as claimed in claim 1 wherein the mild mutagen
used is sodium nitrite in combination with U V light.
3. An improved process for the preparation of cellulase substantially as
herein described with reference to the examples.
|Indian Patent Application Number||1264/DEL/1994|
|PG Journal Number||31/2009|
|Date of Filing||15-Oct-1994|
|Name of Patentee||COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH|
|Applicant Address||RAFI MARG, NEW DELHI-110 001, INDIA.|
|PCT International Classification Number||C12N 9/42|
|PCT International Application Number||N/A|
|PCT International Filing date|