Description:FIELD OF THE INVENTION
[0001] The present disclosure relates to the field of production of biofuels. In particular, the present disclosure relates to a composition and process composition and process for depolymerization of cellulosic biomass, particularly lignocellulosic biomass. More particularly, the present disclosure relates to a secretome composition for hydrolysing cellulosic/lignocellulosic biomass and the process of obtaining the same.

BACKGROUND OF THE INVENTION
[0002] Agriculture sector generates surplus lignocellulosic biomass including corn stover, sugarcane bagasse, straws or feedstock, etc. To minimize the use of fossil fuel and thus to reduce carbon dioxide emission into the atmosphere, which in turn contributes adversely to climate change, alternative sources of fuel such as second-generation biofuel have been identified by scientists. Ethanol produced from biomass is considered as a second-generation biofuel. Specifically, lignocellulosic biomass is abundantly available carbon source which is majorly composed of cellulose, hemicelluloses, and lignin. In general, plants combine water and carbon dioxide, in presence of light and chlorophyll, and produce sugar building blocks. Between 0.1 and 1% of the available light is stored as chemical energy in plants. These sugar building blocks are the starting point of cellulose, hemicelluloses, and lignin found in almost all plants. The carbohydrate fractions (e.g., starch, cellulose, and hemicellulose) of biomass can be hydrolyzed into monosaccharides and converted into ethanol.
[0003] Promising methods of energy conversion are biologically mediated processes, in particular for the conversion of biomass into fuels. Biomass processing schemes involving enzymatic or microbial hydrolysis generally include 1) the production of cellulolytic enzymes such as cellulases, hemicellulases, xylanases and some other auxiliary enzymes; 2) the hydrolysis of cellulose and hemicellulose components of pre-treated biomass in to monomeric sugars such as glucose, xylose, arabinose and mannose; 3) the fermentation of sugars such as glucose, xylose, arabinose and mannose into ethanol.
[0004] Enzymatic activities required for native cellulose degradation are endoglucanases that cut at random in the cellulose polysaccharide chain of amorphous cellulose, generating oligosaccharides of varying lengths and consequently new chain ends; exoglucanases, including cellodextrinases and cellobiohydrolases, that hydrolyze cellulose from the chain end and generate cellodextrins and cellobiose; and lastly ß-glucosidases that hydrolyze soluble cellodextrins and cellobiose to glucose units.
[0005] The use of biomass for the conversion into fuels is limited by the inherent structure of lignocellulose, which is problematic in nature, leading to the requirement of potent lignocellulosic enzymes for the disruption of such lignocellulosic biomass. The conversion of these complex polymeric sugars like cellulose, hemicellulose and lignin into their corresponding mono-saccharides is the first step in the conversion of biomass to biofuels or other high value metabolites.
[0006] Because of its varied composition, organisms used as biocatalysts must produce digestive enzymes that can accommodate an assortment of substrates, in a wide range of conformations, and in a diverse of reaction environments. Present systems, for efficient usage of lignocellulosic substrates requires the addition of external enzymes at high levels and externally added enzymes are costly. Therefore, there is a long-standing need in the art of a composition and process for conversion of lignocellulosic biomass to biofuel that is economical. The present disclosure fulfils the existing needs, at least in part, and provides an improved composition for conversion of lignocellulosic biomass. Thus, it would be beneficial to isolate cellulases from cellulolytic organisms with high specific activity and high expression levels in host organisms in order to achieve efficient biomass saccharification.
[0007] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

OBJECTS OF THE INVENTION
[0008] An object of the present disclosure is to provide a composition and process for conversion of lignocellulosic biomass to biofuel that is economical.
[0009] An object of the present disclosure is to provide a secretome composition for hydrolysing lignocellulosic biomass.
[00010] An object of the present disclosure is to provide a secretome composition for hydrolysing lignocellulosic biomass obtained from a fungal species of Penicillium.
[00011] An object of the present disclosure is to provide a secretome composition for hydrolysing lignocellulosic biomass obtained from Penicillium marneffei.
[00012] An object of the present disclosure is to provide a process for producing a secretome composition for hydrolysing lignocellulosic biomass.
[00013] An object of the present disclosure is to provide a process for hydrolysing lignocellulosic biomass using a secretome composition.
[00014] An object of the present disclosure is to provide a secretome composition with high storage stability at ambient temperature.

SUMMARY OF THE INVENTION
[00015] The present invention relates to a composition and process for conversion of cellulosic/lignocellulosic biomass to biofuel.
[00016] In an aspect, the present disclosure relates to a composition for saccharification of cellulosic biomass particularly, lignocellulosic biomass.
[00017] In an aspect, the present disclosure relates to a secretome composition for hydrolysing cellulosic or lignocellulosic biomass.
[00018] In an embodiment, the secretome composition with cellulolytic activity comprises Carbohydrate-Active Enzymes or CAZymes.
[00019] In an embodiment, the secretome composition with cellulolytic activity comprises more than 25% of Carbohydrate-Active Enzymes or CAZymes.
[00020] In an embodiment, the secretome composition with cellulolytic activity comprises about 30 to 40% of Carbohydrate-Active Enzymes or CAZymes.
[00021] In an embodiment, the secretome composition with cellulolytic activity comprises 37% of Carbohydrate-Active Enzymes or CAZymes.
[00022] In an embodiment, the secretome composition with cellulolytic activity comprises about 201 proteins belonging to Carbohydrate-Active Enzymes or CAZymes class of enzymes.
[00023] In an embodiment, the secretome composition with cellulolytic activity comprises enzymes for carbohydrate metabolism, lipid metabolism, amino acid metabolism, and cell wall metabolism and proteases.
[00024] In an embodiment, the secretome composition with cellulolytic activity comprises Carbohydrate-Active Enzymes or CAZymes for starch degradation, pectin degradation, chitin degradation, lignin degradation, ß-1,3-glucan degradation, cellulose degradation, hemicellulose degradation and esters degradation.
[00025] The present disclosure relates to a secretome composition with cellulolytic activity comprising ß-Glucosidase, Endoglucanase, Xylanase, and Avicelase.
[00026] The present disclosure relates to a secretome composition for hydrolysing lignocellulosic biomass, wherein the secretome for hydrolysing lignocellulosic biomass is obtained from a fungal species of Penicillium.
[00027] The present disclosure relates to a secretome composition for degradation of substrates comprising Hemicellulose, Cellulose, ß-1,3-glucan, Lignin, Chitin, Pectin, and Starch.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
[00028] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
[00029] Figure 1A and 1B graphically illustrates the performance of the fungal isolates. Figure 1A graphically plots the average weight score of the 50 fungal isolates grown in Mandel’s media, calculated on the basis of enzymatic activity and Biomass hydrolyzing performance and Figure 1B graphically plots the relative enzyme activity and Biomass hydrolyzing potential on rescreening of top 6 strains grown on the standard enzyme production conditions.
[00030] Figure 2 illustrates graphically the saaccharification of pre-treated rice straw using Penicillium marneffei secretome composition grown on MM and MM-1 media along with commercial Advanced Enzyme (AE) used as a reference. Two secretome composition concentrations 5 FPU/gm and 10FPU/gm biomass are used with 15% solid biomass loading. a) Total released sugar obtained at different time intervals of saccharification with different secretome compositions. b) Percentage holocellulose conversion measured at each time point of saccharification.
[00031] Figure 3A and 3B graphically illustrates the Storage stability of the P. marneffei secretome composition stored at 4°C and 30°C for 60 days. Figure 3A illustrates the secretome composition activity (FPU) of stored enzyme at 4°C and 30°C for 60 days. Figure 3B illustrates the Biomass hydrolysis by stored secretome composition for 2 months with comparison to freshly prepared enzyme. Experimental condition is 10% biomass loading, 2.5 FPU/gm biomass in 5 ml reaction volume for 72hr.
[00032] Figure 4 graphically illustrates the effect of varying pH on various enzyme activity of the secretome composition obtained from P. maerneffei. Activity is measured using 50mM different pH buffers (pH: 2-4, Buffer: Glycine HCL; pH: 4.5-6.5, Buffer: Na-Citrate; and pH: 7, Buffer: Tris-HCL) and different substrate as per the enzyme activity. (a) Tested the beta-glucosidase activity using 4-Nitrophenyl ß-D-glucopyranoside as a substrate (b) endoglucanase using carboxymethyl cellulose as substrate, (c) Xylanase activity using birchwood xylan as substrate, (d), Avicelase activity using Avicel as substrate, (e), FPU using 50mg Whatman garde1 Filter paper as substrate, and (f) Biomass hydrolysis using acid treated rice straw with 5% biomass loading.
[00033] Figure 5 graphically illustrates the effect of temperature on various enzyme activities of the secretome composition from P. maerneffei. (a) FPU done using 50 mg Whatman Garde1 filter paper, (b) endoglucanase using carboxymethyl cellulose as a substrate, (c) beta-glucosidase using 4-Nitrophenyl ß-D-glucopyranoside as a substrate, and (d) Biomass hydrolysis using acid treated rice straw with 5% biomass loading.
[00034] Figure 6 graphically illustrates the secretome composition of P. marneffei grown on MM-1 media. (a) Functional and metabolic classification of the overall secreted secretome composition, (b) further classification of identified CAZymes by different glycoside hydrolases classes with respect to their catalytic properties, and (c) Distribution of secretome composition on the basis of substrate specificity of the different CAZymes.
[00035] Figure 7 graphically illustrates the distribution of molecular weights with respect to isoelectric point (pIs) of all the identified proteins and their secretory property.
[00036] Figure 8 graphically illustrates the abundance of the different CAZymes family in P. marneffei.

DETAILED DESCRIPTION OF THE INVENTION
[00037] The following is a detailed description of the embodiments of the present disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[00038] Unless the context requires otherwise, throughout the specification which follow, the word comprises and variations thereof, such as, comprises and comprising are to be construed in an open, inclusive sense that is as including, but not limited to.
[00039] Reference throughout this specification to one embodiment or an embodiment means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases in one embodiment or in an embodiment in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[00040] As used in the description herein and throughout the claims that follow, the meaning of “a”, “an”, and the includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of in includes in and on unless the context clearly dictates otherwise. The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[00041] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term about. Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.
[00042] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
[00043] All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., such as) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[00044] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[00045] Various terms are used herein. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[00046] The present invention discloses Penicillium marneffei, a eukaryotic cellular system which is systematically cultured P. marneffei P47 strain, P. marneffei P47-?CreA strain and P. marneffei P47-?CreA-CBHI strain, for secretion of a secretome composition as disclosed in the present invention and was deposited on 23rd August 2022 with ID MTCC accession numbers: MTCC 25572, MTCC 25573, and MTCC 25574.
[00047] The term “secretome” or “secretome composition” refers to the set of proteins expressed by an organism and secreted into the extracellular space expressed by a genome, typically represented at a given point in time.
[00048] The present invention relates to a secretome composition and process for the conversion of cellulosic and/or lignocellulosic biomass to biofuel.
[00049] In an aspect, the present disclosure relates to a secretome composition for saccharification of cellulosic biomass, particularly lignocellulosic biomass.
[00050] In an aspect, the present disclosure relates to a secretome composition for hydrolysing cellulosic and/or lignocellulosic biomass.
[00051] In an embodiment, the secretome composition with cellulolytic activity comprises Carbohydrate-Active Enzymes or CAZymes.
[00052] In an embodiment, the secretome composition with cellulolytic activity comprises more than 25% of Carbohydrate-Active Enzymes or CAZymes.
[00053] In an embodiment, the secretome composition with cellulolytic activity comprises about 30 to 40% of Carbohydrate-Active Enzymes or CAZymes.
[00054] In an embodiment, a secretome composition with cellulolytic activity comprises 37% of Carbohydrate-Active Enzymes or CAZymes.
[00055] In an embodiment, a secretome composition with cellulolytic activity comprises about 201 proteins belonging to Carbohydrate-Active Enzymes or CAZymes class of enzymes.
[00056] In an embodiment, a secretome composition with cellulolytic activity comprises enzymes for carbohydrate metabolism, lipid metabolism, amino acid metabolism, and cell wall metabolism and proteases.
[00057] In an embodiment, a secretome composition with cellulolytic activity comprises Carbohydrate-Active Enzymes or CAZymes for starch degradation, pectin degradation, chitin degradation, lignin degradation, ß-1,3-glucan degradation, cellulose degradation, hemicellulose degradation and esterases.
[00058] In an embodiment, a secretome composition is obtained from Penicillium marneffei cultivated in medium or substrate comprising 30-40%w/w of Hemicellulose, 10-15% w/w of Cellulose, 5-15% w/w of ß-1,3-glucan, 5-15% w/w of Lignin, 3-9% w/w of Chitin, and 3-9% w/w of Pectin.
[00059] In an embodiment, a secretome composition is obtained from Penicillium marneffei cultivated in a substrate or medium comprising 35% w/w of Hemicellulose, 13% w/w of Cellulose, 10% w/w/ of ß-1,3-glucan, 10% w/w of Lignin, 6% w/w of Chitin, and 5% w/w of Pectin.
[00060] The present disclosure relates to a secretome composition with cellulolytic activity comprising ß-Glucosidase, Endoglucanase, Xylanase, and Avicelase.
[00061] The present disclosure relates to a secretome composition for hydrolysing lignocellulosic biomass, wherein the secretome for hydrolysing lignocellulosic biomass is obtained from a fungal species of Penicillium.
[00062] The present disclosure relates to a secretome composition for degradation of substrates comprising Hemicellulose, Cellulose, ß-1,3-glucan, Lignin, Chitin, Pectin, and Starch.
[00063] In an embodiment, Penicillium species is an ascomycetous monophyletic fungi having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to Penicillium marneffei identified by sequencing of internal transcribed spacer.
[00064] In an embodiment, Penicillium marneffei or P. marneffei is a fungus having 99% similarity to internal transcribed spacer or ITS1-5.8S-ITS2 sequences from Penicillium sp. E/Rr/10/2, Penicillium sp. E/Rr/10/2, Penicillium sp. E/Rr/3/3, Talaromyces yunnanensis KUMCC 18-0208, Talaromyces sp. Cesf-10, and Talaromyces sp. OUCMDZ-5269 identified by internal transcribed spacer or ITS sequencing.
[00065] In an embodiment, Penicillium marneffei is identified by genome sequencing with maximum contig hits.
[00066] In an embodiment, the Penicillium marneffei is isolated from putrefying plant materials by a process comprising the steps of (a) isolating fungal strains from putrefying plant materials, (b) streaking the fungal strains in potato dextrose agar (PDA) to get colonies, (c) screening for cellulase enzymes in Mandel’s media (MM), (d) testing cellulolytic activity of the fungal strains, and (e) sequencing the fungal strains with highest cellulase enzymes activity.
[00067] In an embodiment, secretome composition may additionally be obtained from species such as but not limited to Myceliophthora thermophila, P. funiculosum, Fusarium graminearum and Talaromyces amestolkiae.
[00068] The potato dextrose agar (PDA) may comprise dehydrated Potato Infusion, Dextrose, and Agar.
[00069] The Mandel’s media (MM) may comprise (NH4)2SO4, KH2PO4, CaCl2·2H2O, MgSO4·7H2O, FeSO4·7H2O, MnSO4·H2O, ZnSO4·7H2O, CoCl2·6H2O, Peptone, and Tween-80.
[00070] In an embodiment, the cultivating media for enhanced secretome composition production in P. marneffei comprises bacto-peptone as organic nitrogen source in the range of 0.3-1% w/v; urea in the range of 0.01-0.1% w/v; Avicel as carbon source in the range of 0.8 -2% w/v and Ammonium sulphate as inorganic nitrogen source in the range of 0.05 – 0.2% w/v.
[00071] For optimization of media for enhanced secretome composition production in P. marneffei, 0.5% bacto-peptone used as an organic nitrogen source, 0.03% urea, 1% Avicel is used as carbon source, and 0.15% Ammonium sulphate is used as inorganic nitrogen source, respectively. Other inorganic salts, buffering agent and trace elements also added as part of the media.
[00072] In an embodiment, a method for hydrolysis of a cellulosic biomass for producing fermentable sugars comprises the steps of contacting the cellulosic biomass with a secretome composition from Penicillium marneffei at a temperature ranging from 40 °C to 85°C and at a pH ranging from 2 to 5 in presence of Avicel in the range of 0.8 -2% w/v for a time period ranging from about 48 to about 72hrs.
[00073] In an embodiment, the secretome composition comprises Endoglucanase in the range of 12-22 IU/ml, Beta-Glucosidase in the range of 2-10 IU/ml, Xylanase in the range of 110-220 IU/ml, and Avicelase in the range of 0.35-1.00 IU/ml.
[00074] In an embodiment, 1% wheat bran, which is a rich source of hemicellulose, cellulose, lignin, and t-glucan, is added to the Mandel’s media (MM) to obtain increased enzyme activity and protein concentration. It is observed that FPU from increased from 0.55 to 1.09 IU/ml, Endoglucanase from 9.02 to 19.93 IU/ml, Beta-Glucosidase 1.24 to 4.13 IU/ml, Xylanase 90.74 to 190.2 IU/ml, Avicelase 0.26 to 0.45 IU/ml, respectively. Protein concentration also increased significantly from 1.38 to 2.26 mg/ml.
[00075] The following table (Table 1) charts the relative correlation coefficients of cellulolytic enzyme with it’s hydrolytic potential to pre-treated biomass:
Table 1:
ARS/AWS Avicelase Endoglucanase ß-Glucosidase
ARSa 1.00 0.78 0.62 0.59
AWSb 1.00 0.71 0.50 0.53
aARS= acid treated rice straw. bAWS= acid treated wheat straw. Data are statistically significant with p<0.05.
[00076] Biomass hydrolysing potential and lignocellulolytic enzyme activities of the secretome composition from P. marneffei is evaluated on the MM and MM-1 media (Table 2) along with commercial enzyme “Advanced Enzymes (AE)” for reference. Acid-treated rice straw with 15% solid biomass loading at 50°C is evaluated with 5 FPU/gm and 10 FPU/gm biomass at different time intervals (24, 48 and 72 hrs.)
Table 2. Comparative enzyme activity and protein concentration of P. marneffei grown on MM and Mandel’s media with 1% wheat bran (MM-1).
FPU (IU/ml) Endoglucanase (IU/ml) Beta-Glucosidase (IU/ml) Xylanase (IU/ml) Avicelase (IU/ml) Protein concentration (mg/ml)
MM 0.55±0.04 9.02±0.01 1.24±0.03 90.74±0.68 0.25±0.01 1.38±0.03
MM-1 1.09±0.02 19.93±0.38 4.13±0.02 190.2±8.51 0.45±0.02 2.26±0.01
Enzyme activities are performed in duplicates and standard error are mentioned after ± sign. Enzyme activities are performed as per mentioned in the material and methods.
[00077] Released sugar (Glucose and Xylose) is evaluated. At 5 FPU with MM enzyme loading, total sugar released is 29, 37, 39 g/L with 36, 46, and 48% hydrolysis, however, at 10 FPU loading, total sugar is 50, 53, and 58 g/L with 61, 66, and 71% hydrolysis at 24, 48, and 72 hrs, respectively. Using MM-1 secretome composition at 5 FPU loading, total sugar released is 29, 38, and 38 g/L with 36, 47, and 47 % hydrolysis while at 10 FPU loading, total sugar released is 50, 56, and 58 g/L with 62, 69 and 71% hydrolysis at 24, 48, and 72 hrs, respectively. In reference commercial enzyme ‘AE’ using 5 FPU loading total sugar is 16, 23, and 25 g/L with 20, 29 and 31% hydrolysis whereas using 10 FPU loading total sugar is 32, 36, and 39 g/L with 40, 44 and 49% hydrolysis at 24, 48, and 72 hrs, respectively (Figure 2a & 2b). Maximum and equal hydrolysis is observed in both MM and MM-1 with 10 FPU loading that is 58 g/L released sugar with 58% hydrolysis in 72 hrs while in 5 FPU loading only 38-39% hydrolysis is observed which is more than hydrolysis efficiency of advanced enzyme (AE).
[00078] In an embodiment, the secretome composition from P. marneffei may be stored for about 30 to about 60 days at a temperature from about 4°C to about 30°C, preferably at 30°C, and may retain more than 98% of enzyme activity and more than 96% biomass hydrolyzing efficiency.
[00079] To measure enzyme stability, the relative enzyme activity (FPU) is evaluated every 10 days and found that activity is almost consistent till 60 days and retained 98.03 and 98.8% activity at 4°C or 30°C, respectively in 60 days (Figure 3a). Biomass hydrolysis using 60 days-stored samples at 2.5FPU/gm biomass with 10% biomass solid loading and fresh secretome composition also taken as a control for evaluation of the activity. The released glucose is monitored and found that stored secretome composition retained 96.9% and 97.9% hydrolysis efficiency of 4°C or 30°C stored samples, respectively (Figure 3b).
[00080] In an embodiment, P. marneffei may be incubated at a pH of about 2 to about 5, preferably at a pH of about 4, for secretion of secretome composition for hydrolysing cellulosic/lignocellulosic biomass.
[00081] For evaluation of optimal enzyme activity at different pH biomass hydrolysis is performed using 1.5 FPU (2 mL) secretome composition with 5% solid biomass loading for 24 hrs in 5 mL reaction volume. Enzyme activities are converted to relative percent. The highest FPU activity is observed at pH 4 which is significant from pH 2 and 5 (Figure 4e). The observed Beta-Glucosidase activity is 100% at pH 4 that is, highly significant at pH 3 and 4.5 (Figure 4a). The endoglucanase activity is highest at pH 5 and highly significant from adjacent points (Figure 4b). Xylanase activity is highest at pH 5, which found significant from pH 4 and 5.5 (Figure 4c). Avicelase activity is very close between pH 4-5 and highest at pH 4.5, found significant from pH 3 and 5.5 (Figure 4d). Biomass hydrolysis is highest at pH 5, which is significant from pH 4 and 6 (Figure 4f).
[00082] In an embodiment, P. marneffei may be incubated at about 40 to 85°C temperature, preferably at about 60-70°C temperature for secretion of secretome composition for hydrolysing cellulosic/lignocellulosic biomass.
[00083] For evaluation of optimal enzyme activity at different temperatures, biomass hydrolysis is performed using 1.5 FPU enzyme with 5% solid biomass loading for 24 hrs in 5 mL reaction volume. Enzyme activities are converted to relative percent. The optimal FPU activity is 100% at 60°C, which is significantly higher from the adjacent points, while it is 61% at the standard enzyme assay temperature of 50°C (Figure 5a). The optimum beta-Glucosidase activity is found at 70°C which is significant from adjacent points and only 40% activity is observed at 50°C (Figure 5c). The highest Endoglucanase activity is at 65°C which is significant from 60°C and 75°C although just 60% activity is observed at 50°C (Figure 5b). The optimal biomass hydrolysis is found at 55°C that is closest to 50°C, and significant difference is observed from 45°C and 60°C (Figure 5d).
[00084] In an embodiment, the process for preparing a secretome composition for hydrolysis of cellulosic biomass comprises the steps of,
i) cultivating Penicillium marneffei in Mandel’s media (MM) comprising bacto-peptone as an organic nitrogen source in the range of 0.3-1% w/v, urea in the range of 0.01-0.1% w/v, Avicel as a carbon source in the range of 0.8 -2% w/v and Ammonium sulphate as an inorganic nitrogen source in the range of 0.05 – 0.2% w/v;
ii) maintaining pH at about 2 to about 5;
iii) maintaining temperature of about 40 °C to 85°C; and
iv) extracting the secretome composition comprising Carbohydrate-Active Enzymes (CAZymes) comprising Endoglucanase, Beta-Glucosidase, Xylanase and Avicelase.
[00085] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.
EXAMPLE 1
Isolation of Fungus
[00086] Putrefying plant materials are collected from the forest and soil of Bangalore area, India and 49 strains of fungi are isolated. Trichoderma ressei NCIM1186 is procured from National Collection of Industrial Microorganisms (NCIM) as a reference. Cultures are plated for 6 days at 28°C in potato dextrose agar (PDA) (Himedia, India) and subcultures are taken in every 15 days. Secretome composition from each fungal strain is produced by inoculating PDA-grown mycelia in 50 mL Mandel’s media (MM) using 250 mL flask for screening. The liquid medium or the Mandel’s media (MM) with the inoculation is incubated at 28°C, 120 rpm for 6 days. This is harvested by centrifugation at 10,000 rpm for 10 min after 6 days. Endoglucanase, Avicelase, ß-Glucosidase activity and biomass hydrolysis is tested using the supernatant obtained after centrifugation for the identification of cellulolytic potential of the strains. Top six strains from the screening are rescreened on the basis of cumulative enzyme activity and biomass hydrolytic potential using standard enzyme production condition. 1 million spores are inoculated and cultured in 50 mL PD broth at 28°C, 120 rpm for 48 hrs as primary culture. Thereafter 10% of the primary culture is inoculated in a production media and incubated at 28°C, 120 rpm for 5 days.
[00087] The molecular identification of the most outperforming fungal strain is carried out by alignment of its ITS1–5.8S-ITS2 region with other available sequences in NCBI public databases. DNA is extracted from PD broth and characterized using Quick-DNA Fungal/Bacterial Kits (ZYMO RESEARCH). ITS region is amplified using ITS1 (5'-CCGTAGGTGAACCTTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') primers with DNA template. After running gel electrophoresis, the sequence is aligned with nonredundant nucleotide collection (nr/nt) (NCBI) using BLASTn tools. For accurate identification of species genome sequencing is carried out.
The fungal cultures grown for six days wherein the secretome composition is tested for the Avicelase, endoglucanase, and ß- glucosidase activity as well as biomass hydrolysing capacity using acid-treated rice straw and wheat straw. The respective enzyme performance is correlated and the weightage of each stain is estimated. Positive correlation is observed between biomass hydrolysis and the enzyme activity. The highest correlation is detected with Avicelase activity followed by endoglucanase and ß-glucosidase activity as can be noted from Table 1 above, thus highest correlation for the biomass hydrolysis is noted to be with crystalline substrate (Avicel) utilization rather than amorphous substrate (Carboxymethyl cellulase and 4-Nitrophenyl-ß-D- glucopyranoside or pNPG) utilization.
Evaluation of the strain performance
[00088] Weighted sum model (WSM) approach is applied to profile enzymes from each strain to evaluate the cumulative performance of strains. The WSM, is a “multicriteria decision analysis tool”, reflects a set of alternatives M and related criteria N and recognizes the most suitable alternative using given algorithm:

Where (AiWSM-score) = the WSM score of the suitable alternative, n = the number of criteria, aij = the real value of the ith alternative in relations to the jth criterion, and wj = the weight of the rank of the jth criterion. The Avicelase, Endoglucanase, and Beta-glucosidase activity data is taken as base criteria (N) for estimation of weighted sum score for acid treated rice and wheat straw. A relative weight (w) is allocated to the individual measure based on the Pearson Correlation Coefficients between the measures. The weight one is assigned for biomass hydrolysis, because it defines the overall saccharification capacity whereas other enzyme activities assigned the weight of their respective correlation coefficient. The ranking on average weight scores of the strains are determined based on independent biomasses (acid pre-treated rice and wheat straw). All enzyme performance data given as input and cumulative Pearson Correlation Coefficient is evaluated for each enzyme activity with respect to both acid treated rice straw and acid treated wheat straw to estimate the potential of each strain. Further, a weighted sum score (AiWSM-score) is created for each strain using enzyme normalized enzyme activity data as well as correlation coefficient values and evaluated the cumulative performance of strains. Weight one is assigned for biomass hydrolysis representing the overall saccharification capacity whereas weight of the other enzyme activities is assigned as their respective correlation coefficient (Table 1). Furthermore, independently evaluated the weight score of both biomasses (acid pre-treated rice and wheat straw) and define the ranking on average weight scores of the strains. Weight score obtained from WSM model is analysed and found that strain 59 achieved the highest score that is 22.82 (Figure 1A). Highest six weight scored strains 45, 47, 59, 79, 80, and 82 is re-screened using standard enzyme production condition. In rescreening, strain 45 showed 100% FPU, Avicelase and Endoglucanase activity while poor in biomass hydrolysis (36% and 21% in ARS and AWS). However, strain 47 appeared best in terms of biomass hydrolysis (100%) and second highest FPU (95%), Avicelase (81%), Endoglucanase (79%) activity. However, ß-Glucosidase activity is quite less (51%) in strain 47 while 100% in strain 59 (Figure 1B). Biomass hydrolysis is performed using 5% solid biomass loading in 5 ml reaction volume with equal enzyme volume loading. Biomass hydrolysis potential is given preference during screening for potential lignocellulolytic strain over other enzyme activities.
Determination of cellulase enzyme activities
[00089] Filter paper assay method (FPase/FPU) is used to evaluate the total cellulase activity by using NREL (National Renewable Energy Laboratory) protocol with some modification. 50 mg of Whatman filter paper (Grade I) is taken and incubated with 0.5 mL of diluted secretome composition and 1mL Citrate buffer having pH 4.5 at 50°C for 60 min and reaction is stopped using 3 mL DNSA (3,5-Dinitrosalicylic acid) and boiled at 98°C for 10 min, absorbance taken at 540 nm. Glucose release is monitored using calorimetric assay and 1 FPU classify the certain quantity of enzyme required to release 2 mg of glucose from the 50 mg of filter paper in 60 min of reaction. However, other enzyme assays such as for Avicelase (Cellobiohydrolase), Endoglucanase and Xylanase activity is performed at standard volume condition and glucose release is monitored using DNSA method and reaction is performed at 50°C.
[00090] Avicelase activity is performed by incubating 150µl diluted enzyme with 150µl 1% Avicel® PH-101 (Sigma) for 240 min. Endoglucanase and Xylanase activity assay is performed using 0.5mL 2% CMC (Sigma), 2% beechwood xylan (HiMedia), respectively with 0.5mL appropriate enzyme dilution and incubated for 30 min and stopped the reaction with respective volume of DNSA and boiled as mentioned above. One unit of Avicelase, Endoglucanase, and xylanase activity is defined as “amount of enzyme releasing 1 µmol of reducing sugar per min”. The ß-glucosidase activity is performed using 1mM p-nitrophenyl-ß-d-glucopyranoside (Sigma) at final concentration and reaction is carried out for 30 min and stopped with 1% sodium carbonate (pH 11.5). 4-nitrophenol is measured at 400 nm using a 4- nitrophenol standard curve. The one unit of ß-glucosidase activities is defined as the amount of given enzyme released 1 µmol of p-nitrophenol (pNP) per min.
Experimental design for the saccharification potential of the strains
[00091] Acid treated rice straw and Acid treated wheat straw at different volume is used for biomass hydrolysis experiments. Biomass hydrolysis is performed in 1.2 mL capacity 96-wells deep well plates with 250µl working volume for screening fungal isolates. Reaction is carried out in 50mM citrate buffer pH5 with 5% solid biomass loading and equal volume of protein loading. Hydrolysis is carried out by orbital shaking at 150 rpm at 50°C for 72 hrs. Soluble sugars present in alone biomass is also measured in similar manner. The concentration of hydrolyzed glucose is measured using D-Glucose Assay Kit (Megazyme, Ireland) and glucose (Sigma) taken as standard.
[00092] Secretome composition from most performing fungal strain P. marneffei is compared to a commercial enzyme (Advanced enzyme formulation) (AE) for determination of saccharification potential. Hydrolysis is carried out by different concentrations of enzyme loading for different time periods. Different protein concentration of 5 FPU/g and 10 FPU/g biomass loading with 15% biomass loading is used to test the performance of the secretome composition. The 15-20% dry biomass with 10 FPU/g protein loading is considered as industrially feasible biofuel solution. Saccharification is conducted using 100 mL flask with 20 mL working volume which is placed in the incubator shaker at 50°C and 200 rpm for 24-72 hrs. Control tests are also set up as mentioned above to detect the background free sugars in the biomass. On the completion of hydrolysis after each time period, hydrolysate is centrifuged at 10000 rpm for 10 min and collected the supernatant. Released monomeric sugars (glucose and xylose) are examined with high-performance liquid chromatography (HPLC) which is ran in isocratic mode with rate of 0.3 mL/min and temperatures of RI detector maintained at 35°C. 4 mM H2SO4 is used as mobile phase. The glucose and xylose yield are calculated with reference to prepared standard curves. The theoretical yield of monomeric sugars from hemicellulose and cellulose are estimated as per NREL’s protocol. Percent hydrolysis in the reaction is calculated with reference to theoretical sugar present in the biomass as well as gained sugars in biomass hydrolysis as described in the given formulas:

Wherein, [Glucose] = glucose concentration (g/L), [Xylose] = xylose concentration (g/L) gained after hydrolysis.
The [Cellulose Pre-treated Biomass] = cellulose content identified in the pre-treated biomass, and [Hemicellulose Pre-treated Biomass] = total hemicellulose content identified in the pre-treated biomass.
Protein concentration and SDS-PAGE Analysis of P. marneffei secretome composition
[00093] Samples of secretome composition stored at -20°C are analysed for determination of protein quality or any visual degradation using SDS-PAGE. The crude sample (30µl) is mixed with 6µl of 6X sample buffer comprising 10% glycerol, 10% SDS, 375 mM Tris-HCL pH 6.8, 0.03% Bromophenol blue, 60mM DTT and 5% Mercaptoethanol, and boiled for 10 min and subjected to the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).
[00094] Proteomics or large-scale study of the secretome composition is done in Orbitrap Fusion Lumos Tribrid Mass Spectrometer equipped with nano-LC Easy nLC 1200. The Mass spectroscopy is matched with database of P. marneffei draft genome sequence with 1% FDR using proteome DiscovererTM (Version 2.4.1.15, Thermo FisherTM Scientific Inc). 549 proteins are identified in the secretome composition from P. marneffei grown on MM-1 media which is higher than reported data on various fungal species such as P. echinulatum, P. funiculosum, Aspergillus fumigatus, P. janthinellum and T. reesei RUT-C30 which are 165, 195, 128, 85 and 65, respectively.
[00095] Of the 549 identified proteins which are identified and assigned functions using Blast2GO tool, 201 proteins belong to CAZymes which accounts for 37% of the overall secreted proteins as seen in Figure 6. The data disclosed in Figure 6 also identified functions of the other fraction of the secretome composition predicted as carbohydrate metabolism, lipid metabolism, amino acid metabolism, proteases or proteolytic activity, cell wall component and other catalytic activity share approximately 9%, 3%, 7%, 8%, 1%, and 35%, respectively. The molecular weights and isoelectric point (pI) of all the identified proteins are in the range of 8.2–175 kDa and 3.78– 9.77, respectively. However, carbohydrate active enzymes (CAZymes) mostly in the acidic range pI 4–6 and MW in the range of 20-120 kDa whereas Chitinase and 4-alpha-glucanotransferase are in a higher range than 170 kDa. This has been demonstrated graphically in Figure 7. A plot for molecular weight against the isoelectric point (pI) of the identified proteins (a), distribution of all the CAZymes along with other proteins represented with different colors as mentioned in the figure legends. Distribution of secreted proteins having secretory and non-secretory signal (b).
Distribution of CAZyme in P. marneffei
[00096] The distribution of CAZymes in different glycoside hydrolases classes are represented in Figure 8. From this graph in Figure 8 it can be seen that the it comprises 54% glycoside hydrolases-GH (422 protein in 72 families), 16% Glycosyl Transferase- GT (122 proteins with 34 families), 2% Polysaccharide Lyase- PL (13 proteins with 6 families), 3% Carbohydrate-Binding Module- CBM (27 proteins with 10 families), 4% hybrid glycoside hydrolases + carbohydrate-Binding Module- GH+CBM (29 proteins with 10 families), 6% Carbohydrate Esterase- CE (46 proteins with 9 families), and 15% Auxiliary Activities family enzymes- AAs (118 proteins with 12 families). Among the 201 CAZymes identified, 76% is GH (152 protein in 67 families), 2% PL (5 proteins with 3 families), 1% CBM (3 proteins with 2 families), 1% GH+CBM (3 proteins with 2 families), 7% CE (14 proteins with 7 families), and 12% AAs (24 proteins with 12 families). Over the secreted CAZyme, 69% proteins detected N-terminal secretory signals and 31% have no secretory signals, among the secretory signal’s proteins, 111, 12, 9, 5, 1, and 1 are belong to GH, AAs, CE, PL, CBM and GH+CBM family respectively. CAZyme in the secretome composition with non-secretory signals are 41, 12, 5, 2, and 2 belong to GH, AAs, CE, CBM and GH+CBM family respectively, without secretory signal PL is not detected. The GH is a large family of the CAZymes in comparison to others therefore predominance of GH and AAs in the secreted CAZymes correspond to 75.6% and 11.9%, respectively (Figure 8b). The relative abundance of the predicted CAZymes class in the genome in comparison to secretome composition (a); distribution of secreted CAZymes class having secretory and non-secretory N terminal signal (b); and proportional protein abundance of the top 10 secreted CAZymes on the basis of spectrum percent abundance (c) has been graphically presented in Figure 8.
[00097] Enzymes of other class are also obtained in the present secretome composition in higher fractions such as about 1.8% w/w a-L- arabinofuranosidase, 1.6% w/w of ß-xylosidase (GH3), about 1.1% w/w of Fn3_like ß-glucosidase (GH3), about 1% w/w of ß-glucosidase (GH3), about 0.9% w/w of Endoglucanase (GH74), about 0.8% w/w of Endo-1,3-ß- glucanase (GH17) and about 0.8% w/w of Endo-ß-1,4-mannanase F (GH5) (Figure 8c). Other enzymes present in smaller abundance but important for the lignocellulosic biomass deconstruction are Glycosidase (GH16), a/ß- glucosidase (GH31), a-galactosidase (GH27), Catalase-peroxidase (AA2), Glycoside hydrolase (GH3), 1,3-ß-glucanosyltransferase (GH72), Pectin lyase 1 (PL1), and lytic polysaccharide monooxygenase (AA9). Some of the enzyme have similar functions with different family of enzymes such as Glucanase (GH6 and GH7), a-L-arabinofuranosidase (GH62, GH51, GH54), Endoglucanase (GH74, GH17, GH12, GH30), ß-xylanase (GH10, GH11), Glycoside hydrolase (GH43, GH12) and ß-galactosidase (GH35, GH30). Thus, the P. marneffei in ideal environment produces a synergistic secretome composition of CBHI, CBHII, endoglucanase I, xylanases, ß-xylosidase, a- arabinofuranosidase, a-glucuronidase, Auxiliary enzymes and many other enzymes for hydrolysing lignocellulosic biomass.
[00098] In an embodiment, the secretome composition for hydrolysis of cellulosic biomass obtained from Penicillium marneffei, comprises glycoside hydrolases (GH), Glycosyl Transferase (GT), Polysaccharide Lyase (PL), Carbohydrate Esterase (CE), and Auxiliary Activities family enzymes (AA).
[00099] In an embodiment, the secretome composition for hydrolysis of cellulosic biomass obtained from Penicillium marneffei comprises a-L- arabinofuranosidase, ß-xylosidase (GH3), Fn3_like ß-glucosidase (GH3), ß-glucosidase (GH3), Endoglucanase (GH74), Endo-1,3-ß- glucanase (GH17) and Endo-ß-1,4-mannanase F.
[000100] In an embodiment, the secretome composition for hydrolysis of cellulosic biomass obtained from Penicillium marneffei comprises other enzymes such as Glycosidase (GH16), a/ß- glucosidase (GH31), a-galactosidase (GH27), Catalase-peroxidase (AA2), Glycoside hydrolase (GH3), 1,3-ß-glucanosyltransferase (GH72), Pectin lyase 1 (PL1), and lytic polysaccharide monooxygenase (AA9).
[000101] While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.

ADVANTAGES OF THE PRESENT INVENTION
[000102] The present disclosure provides a composition and process for conversion of cellulosic/lignocellulosic biomass to biofuel.
[000103] The present disclosure provides a secretome composition for hydrolysing cellulosic/lignocellulosic biomass.
[000104] The present disclosure provides a secretome composition for hydrolysing cellulosic/lignocellulosic biomass obtained from a fungal species of Penicillium.
[000105] The present disclosure provides a secretome composition for hydrolysing cellulosic/lignocellulosic biomass obtained from Penicillium marneffei.
[000106] The present disclosure provides a process for producing a secretome composition for hydrolysing cellulosic/lignocellulosic biomass.
[000107] The present disclosure provides a process for hydrolysing cellulosic/lignocellulosic biomass using a secretome composition.
[000108] The present disclosure provides a secretome composition with high storage stability at ambient temperature.
, Claims:1. A secretome composition for hydrolysis of cellulosic biomass obtained from Penicillium marneffei, wherein the secretome composition comprises Carbohydrate-Active Enzymes (CAZymes) comprising Endoglucanase, Beta-Glucosidase, Xylanase and Avicelase.
2. The secretome composition as claimed in claim 1, wherein the secretome composition comprises more than 25% of Carbohydrate-Active Enzymes.
3. The secretome composition as claimed in claim 1, wherein the secretome composition comprises about 30 to 40% of Carbohydrate-Active Enzymes.
4. The secretome composition as claimed in claim 1, wherein the secretome composition comprises Endoglucanase in the range of 12-22 IU/ml, Beta-Glucosidase in the range of 2-10 IU/ml, Xylanase in the range of 110-220 IU/ml, and Avicelase in the range of 0.35-1.00 IU/ml.
5. The secretome composition as claimed in claim 1, wherein the secretome composition comprises glycoside hydrolases (GH), Glycosyl Transferase (GT), Polysaccharide Lyase (PL), Carbohydrate Esterase (CE), and Auxiliary Activities family enzymes (AA).
6. A process for preparing a secretome composition for hydrolysis of cellulosic biomass comprises the steps of,
v) cultivating Penicillium marneffei in modified Mandel’s media (MM) comprising bacto-peptone in the range of 0.3-1% w/v, urea in the range of 0.01-0.1% w/v, Avicel in the range of 0.8 -2% w/v and Ammonium sulphate in the range of 0.05 – 0.2% w/v;
vi) maintaining pH at about 2 to about 5;
vii) maintaining temperature of about 40 °C to 85°C; and
viii) extracting the secretome composition comprising Carbohydrate-Active Enzymes (CAZymes) comprising Endoglucanase, Beta-Glucosidase, Xylanase and Avicelase.
7. The process for preparing a secretome composition as claimed in claim 5, wherein the pH is maintained at about 4.
8. The process for preparing a secretome composition as claimed in claim 5, wherein the temperature is maintained at about 60-70°C.
9. The process for preparing a secretome composition as claimed in claim 5, wherein the Penicillium marneffei is cultivated in media comprising 30-40%w/w of Hemicellulose, 10-15% w/w of Cellulose, 5-15% w/w of ß-1,3-glucan, 5-15% w/w of Lignin, 3-9% w/w of Chitin, and 3-9% w/w of Pectin.
10. A method for hydrolysis of a cellulosic biomass for producing fermentable sugars comprises the steps of
i) contacting the cellulosic biomass with a secretome composition from Penicillium marneffei at a temperature ranging from 40 °C to 85°C and at a pH ranging from 2 to 5 in presence of Avicel in the range of 0.8 -2% w/v for a time period ranging from about 48 to about 72hrs.