FIELD OF THE INVENTION:
This invention relates to a method for enzymatic delignification of lignocellulosic raw materials.
BACKGROUND OF THE INVENTION:
The use of lignocellulosic biomass for the production of bioethanol is progressively gaining importance due to the gradual depletion of fossil fuel and several advantages of lignocellulosic substrates. The raw material costs could be reduced by utilization of low value crops with high turnover which do not serve any function as food ingredients. Bottleneck for the utilization of lignocellulosic biomass is the presence of lignin, which consists of an asymmetrical and heterogeneous arrangement of polyprenoid residues that resist the chemical and enzymatic digestion of cellulose and hemicellulose polymer. To make this cellulose and hemicellulosemore accessible for hydrolysis, pretreatment, i.e. the disintegration of lignin is essential. Over the years several numbers of different lignin degradation strategies have been reported [Dagnino EP, Chamorro ER, Romano SD, Felissia FE, Area MC. Optimization of the acid pretreatment of rice hulls to obtain fermentable sugars for bioethanol production. Ind Crop Prod 2013; 42:363-368; Haykir NI, Bahcegul E, Bicak N, Bakir U. Pretreatment of cotton stalk with ionic liquids including 2-hydroxy ethyl ammonium formate to enhance biomass digestibility. Ind Crop

Prod 2013; 41:430-436; Binod P, Satyanagalakshmi K, Sindhu R, Janu KU, Sukumaran RK, Pandey A. Short duration microwave assisted pretreatment enhances the enzymatic saccharification and fermentable sugar yield from sugarcane bagasse. Renew Energ 2012; 37:109-116; Zhu JY, Verrill SP, Liu H, Herian VL, Pan X, Rockwood DL. On polydispersity of plant biomass recalcitrance and its effects on pretreatment optimization for sugar production. Bioenergy Res 2011; 4:201-210; Banerjee S, Sen R, Mudliar S, Pandey RA, Chakrabarti T, Satpute D. Alkaline peroxide assisted wet air oxidation pretreatment approach to enhance enzymatic convertibility of rice husk Biotechnol Prog 2011; 27:691-697; Saritha M, Arora A, Lata. Biological pretreatment of lignocellulosic substrates for enhanced delignification and enzymatic digestibility. Indian J Microbiol 2011; 1-9;]. Among them enzymatic process offers major advantages over conventional processes [Mtui GYS. Recent advances in pretreatment of lignocellulosic wastes and production of value added products. Afr J Biotechnol 2009; 8:1398-1415].
US Patent No. 4008120 entitled 'Process for delignification and bleaching a lignocellulosic product' to carles et al (1977) discloses a process where the delignification and bleaching of lignocellulosic products is effects by mixing an aqueous suspension of the

lignocellulosic product with a chlorine-containing substance and an oxygen-containing substance, simultaneously or sequentially, and they, by reaction in situ, give rise to gases in the nascent state. The chlorine and/or oxygen are in the so-called "singlet" state, or in a state of higher energy.
US Patent No. 4409032 for 'Organosolv delignification and saccharification process for lignocellulosic plant materials' [Paszner L, Chang P, (1983)], discloses a method for rapid hydrolysis of cellulose containing material by cooking under pressure at 180°C to 220°C, with acetone-water solvent mixture carrying from 0.05 to 0.25 weight percent of phosphoric, sulfuric or hydrochloric acids. US Patent No. 5023097 for "Delignification of non woody biomass to Tyson, George J, (1991) discloses a method in which a non-woody biomass is delignified through extrusion technology, utilizing hydrogen peroxide and an alkali agent, to break down complex biomass materials. The process is useful in forming a highly absorbant fiber material for use as a dietary fiber or an absorbant fiber. Alternatively, the process is useful for preparing dietary feeds for ruminant animals, as well as produces a broad range of alcohols or polymers from the non-woody lignocellulosic substrate.

US Patent No. 511746 for 'Cellulase-free endo-xylanase enzyme of use in pulp delignification' to Bernier et al., (1992), teaches a method of treating lignocellulosic material with a cellulase-free endo-xylanase for delignification, brightening and viscosity improvement. The endo-xylanase is obtained by the overexpression of the xylanase gene using a cellulase-negative recombinant microorganism of the genus Streptomyces.
Laccase mediated delignification of lignocellulosics biomass are the major area of interest of different researchers. Till date, few patents have been made in this particular area. In a patent 5385641 an efficient processes for delignification of cellulosic biomass was reported which is a three-stage process for the delignification of cellulose-containing raw materials, pulping is initially performed with a solution of concentrated aqueous acetic acid at an elevated temperature and under an elevated pressure. In US Patent 5525195 a process for high consistency delignification by alkali pretreatment. The invention relates to processes having improved efficiency for enzymatic delignification of unbleached pulp of cellulosic feedstocks. In another US Patent 5705383 a method was described for the use of certain white rot fungi in the reduction of the pitch and/or lignin content of cellulosic materials. In another patent 0406617, lignin peroxide mediated pretreatment of

Hardwood Kraft Pulp has been reported. The invention was directed to an improved process for the enzymatic delignification of lignocellulosic materials for use in the pulp and paper industry.
Delignification of lignocellulosics by enzymatic method is one of the prime research areas in order to generate clean forms of fuels from lignocellulosic. But there are several problems associated to these methods. Use of chemical and physical methods for lignin degradation serves various problems for subsequent processes for bioethanol production. Also, prolonged incubation period for desired level of delignification results higher cost of enzymes.
The following Table (Table-I) highlights the disadvantages of the various methods available for pretreatment (Table I).



OBJECTS OF THE INVENTION;
It is therefore an object of this invention to propose a method for enzymatic delignification of lignocellulosic raw materials, which is simple and easy to perform.
It is a further object of this invention to propose a method for enzymatic delignification of lignocellulosic raw materials, which is fast and cost-effective.
These and other objects of the invention will be apparent from the ensuing description. At the outset of the description which follows, it

is to be understood that the ensuing description only illustrates a particular form of this invention. However, such a particular form is only an exemplary embodiment and the teachings of the invention is not intended to be taken restrictively.
BRIEF DESCRIPTION OF THE INVENTION:
According to this invention is provided a method for enzymatic delignification of lignocellulosic raw materials.
In accordance with this invention is disclosed yellow laccase mediated pretreatment of different lignicellulosic materials for efficient degradation of lignin present in the substrates. The process involves yellow laccase mediated pretreatment/ delignification of lignocellulosic having the lignin content between 17%-24%, such as for example Bambusa bamboo, rice straw, Ricinus communis and Lantana camara.
The fungal strain used for the enzyme production is a novel strain belonging to Lentinus squarrosulus (MR 13) isolated from local soil of IIT-Kharagpur campus and [20,251 U/gds]. The yellow laccase enzyme produced form Lentinus squarrosulus (MR 13) was used for delignification of these substrates. This is the first report of the yellow laccase production from Lentinus squarrosulus (MR 13)

Response Surface Methodology (RSM) based three level Central Composite Design (CCD) was employed for optimization of enzymatic pretreatment of Bambusa bambos, rice straw, Ricinus communis and Lantana camara.
The delignification is performed at a pH of 6.5 to 7.5, preferably 6.3 to 7.0 and temperature in the range of 35 to 45°C, preferably 35 to 41°C. The solid to liquid ratio is maintained at 2-6mL/g, preferably 2.2 to 3 mL/g and the incubation time ranges from 4 to 8 hours, preferably from 6 to 8 hours. The enzyme concentration is 400 to 600 1U/ml.
The invention will now be explained in greater detail of the help of the following non-limiting example.
Materials and methods
Raw material
Bambusa bamboo, rice straw, Ricinus communis and Lantana camara was collected from nearest field of IIT (Indian Institute of Technology) Kharagpur, India. It was then air dried, milled and passed through 0.2 mm sieve, and stored at room temperature for subsequent use.
Enzyme production
Yellow laccase was produced from newly isolated strain of Lentinus squarrosulus (MR 13) in solid state fermentation. The fungal

myceliums were separated from crude enzyme extracts by centrifugation at 5, 000 rpm for 5 minutes at 4°C. The clear supernatant of yellow laccase was used for enzymatic pretreatment of both the substrates.
Enzyme assay
Yellow laccase assay was carried out by using ABTS as substrate.
Enzymatic pretreatment of lignucellulosic biomass
Enzymatic pretreatment of Bambusa bamboo, rice straw, Ricinus communis and Lantana camara was carried out by using O.lmol/L of phosphate buffer (pH 6.5 to 7.5) and required volume of laccase enzyme. For the substrate resultant solid residue was washed with distilled water. Samples were withdrawn periodically, and the solid residue was used for lignin estimation. After delignification the solid residue was washed with distilled water. Then it was air dried overnight at 60 °C and subsequently used for further studies.
Laccase mediated delignification of Bambusa bamboo results 90.51% lignin degradation after 8 h of incubation at 41 °C, for rice straw 85.23% lignin was degraded after 6.67 h of incubation at 39 °C, for Lantana camara 89.2% lignin degradation was observed after 7.5 h of incubation at 35 °C and for Ricinus communis 85.725 % lignin was degraded after 6.2 h and 40.6 °C. Enzymatic delignification of substrates resulted higher lignin degradation in short incubation time.

The bioethanol production and consumption in India and the projected demand for bioethanol requirements in India is show in Table-2 and Table-3 respectively.

Therefore, the commercial importance of the invention is apparent. The invention will be very useful for the renewable energy sector and possible end-users will be oil-industries and agricultural industries.

WE CLAIM:
1. A process for enzymatic delignification of lignocellulosic raw
materials comprising subjecting the raw material to the steps of
drying, milling and sieving,
subjecting the raw material to treatment with laccase enzyme in
phosphate buffer,
incubating the same for 4 to 8 hours,
periodically withdrawing samples of the solid residue for lignin
estimation, and after complete delignification, washing the solid
residue and drying.
2. The process as claimed in claim 1, wherein yellow laccase is
produced from Lentinus squarrosulus (MR 13) by solid state
fermentation, followed by separating the fungal mycelium from crude
enzyme by centrifugation to obtain a clear supernatant of the yellow
laccase.
3. The process as claimed in claim 1, wherein the pH of the
medium is in a range of 6.5 to 7.5, preferably, 6.5 to 7.0.
4. The process as claimed in claim 1, wherein the temperature is
in the range of 35 to 45°C, preferably 35 to 41°C.

5. The process as claimed in claim 1, wherein the liquid to solid ratio is 2-6 mL/g, preferably 2.2 to 3 mL/g.
6. The process as claimed in claim 1, wherein the enzyme concentration is in the range of 400 to 600 1U/ml.
7. The process as claimed in claim 1, wherein the raw material is selected from Bambusa bamboo, rice straw, Lantana camara, Ricinus communis.