Enzyme assisted juice extraction from sugar cane

Abstract

The present invention provides a improved process for enzyme-assisted sugar cane juice extraction.

Description

[0001] ENZYME ASSISTED JUICE EXTRACTION FROM SUGAR CANE

[0002] REFERENCE TO A SEQUENCE LISTING

[0003] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] The invention relates to sugar cane processing and more particularly to a process for improving sugar cane juice yield.

[0006] BACKGROUND OF THE INVENTION

[0007] Sugar cane is composed of two parts: stalk and trash. The stalk is stout, jointed, fibrous, and rich in sugars, particularly sucrose, and is the portion normally associated with sugar cane (cleaned cane), i.e., the part of the cane plant between plantation level and the end node of the upper stalk. Sugar cane trash is divided into three principal components: green leaves, dry leaves, and tops (Neto, 2005, Characterization of sugarcane trash and bagasse, In: Hassuani, S.J.; Leal, M.L.R.V.; Macedo, I.C. Biomass Power Generation. Sugarcane Bagasse and Trash, Published by PNUD and CTC, 1st edition, Piracicaba, Brazil, p. 24). The green leaves are green and yellow leaves, tops are the part of the cane plant between the top end and the last stalk node, and dry leaves are leaves that have already dried and are normally brownish in color.

[0008] Sucrose accumulation is greater at the base of the stalk, and the amount of reducing sugars and cellulose is superior in the tops (Triana et al., 1990, Atlas of sugarcane bagasse, Published by Geplacea and Icidca, Mexico, p. 139). The length of the stalk varies and depends on the variety of the plant and the cultural management provided. An adult stalk may be less than two meters to over six meters in length, which affects the number and length of internodes. The diameter of the stalk also varies, oscillating in its middle part from 250 to 350 mm. The color depends on the chlorophyll content, anthocyanin content, and aspects of agronomy (Triana et al., 1990, supra).

[0009] The stalks are prepared to obtain a cane juice broth, which is subsequently used for Plantation white sugar, refined sugar, brown sugar or ethanol production. The residual fraction of the stalk after processing is known as bagasse. Besides bagasse, sugar cane trash is another residue of sugar and ethanol production. The amount of trash from sugar cane harvesting depends on several factors such as harvesting system (burnt or unburned cane), topping, height, cane variety, age of crop (stage of cut), climate, and soil.

[0010] Bagasse and trash are lignocellulosic materials and are composed of cellulose, hemicelluloses, and lignin. Cellulose and hemicellulose compose the carbohydrate fraction of material. Cellulose is a homopolysaccharide composed of D-glucose units joined together by P(1 — >4) glucosidic linkages. Hemicellulose is a heteropolysaccharide composed of D-glucose, D- galactose, D-mannose, D-xylose, L-arabinose, acetic acid, D-glucuronic acid, and 4-O-methyl-D- glucuronic acid units. Lignin is a complex aromatic macromolecule formed by radical polymerization of three phenyl-propane alcohols, namely p-coumarilic alcohol, coniferilic alcohol, and synapilic alcohol. In the plant cell wall, lignin and hemicelluloses involve the cellulose elementary fibrils, providing protection against chemical and / or biological degradation (Kuhad et al., 1997, Microorganisms and their enzymes involved in the degradation of plant fiber cell walls, Advances Biochemical Engineering Biotechnology, vol. 57, pp. 45-125,). Quantitatively, cellulose, hemicelluloses, and lignin are present in bagasse in a proportion of 42-55.7%, 20-28.8% and 22.2-31.4%, respectively. Non-structural components of biomass, namely, extractives (2-10%) and ash (1-2.4%) are the other substances that are part of the chemical composition of sugar cane bagasse.

[0011] Globally, sugarcane milling process involves mechanical unit operations, extensive use of energy and water to extract sugarcane juice and / or sugar. Even with the extensive use of energy and water, 0.5-0.7% of sugar which has substantial value goes into low value bagasse. In particular, the sugarcane milling process involves mechanical, high energy and high-water usage to extract sugarcane juice / sugar. Even after use of high energy and water, approximately 0.6 % sugar goes as a waste along with bagasse.

[0012] Further improvements in sugarcane processing are desired, where the efficiency of the enzymatic process to extract the more juice and sugar from bagasse by using enzymatic technology to increase the yield of sugarcane juice and sugar in the sugar manufacturing process by reducing the residual sugar or POL% Bagasse and moisture in bagasse.

[0013] SUMMARY OF THE INVENTION

[0014] In a first aspect the present invention relates to a process for improving yield of sugar cane juice comprising steps of: a. contacting a substrate derived from sugarcane with an enzyme composition comprising one or more hydrolytic enzymes in presence of imbibition water; b. separating the mixture of sugar cane juice and bagasse; c. clarifying the separated sugar cane juice; and d. recovering the sugar from sugar cane juice.

[0015] Brief Description of the Figures

[0016] Figure 1 shows the process of extraction of sugar cane juice.

[0017] DEFINITION

[0018] Alpha-amylase: Alpha-amylases (EC 3.2.1.1) are a group of enzymes which catalyze the hydrolysis of starch and other linear and branched 1 ,4 glucosidic oligo- and polysaccharides. The skilled person will know how to determine alpha-amylase activity. It may be determined, e.g., by measuring residual activity after stressing the sample at pH 4.0 using a commercial alphaamylase activity assay kit, such as kits containing G7-pNP substrate and alpha-Glucosidase, e.g., manufactured by Roche / Hitachi (cat. No.11876473) or Sigma-Aldrich (Catalog number MAK009).

[0019] Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase” means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing alpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzyme acts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1 ,3)- and / or (1 ,5)-linkages, arabinoxylans, and arabinogalactans. Alpha-L-arabinofuranosidase is also known as arabinosidase, alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase, polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranoside hydrolase, L- arabinosidase, or alpha-L-arabinanase. For purposes of the present invention, alpha-L- arabinofuranosidase activity is determined using 5 mg of medium viscosity wheat arabinoxylan (Megazyme International Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100 mM sodium acetate pH 5 in a total volume of 200 pl for 30 minutes at 40°C followed by arabinose analysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

[0020] Arabinoxylan-containing material: The term “Arabinoxylan-containing material” means any material containing arabinoxylan. Arabinoxylan is a hemicellulose found in both the primary and secondary cell walls of plants, including woods and cereal grains, consisting of copolymers of two pentose sugars, arabinose and xylose. The arabinoxylan chain contains a large number of 1 ,4-linked xylose units. Many xylose units are substituted with 2-, 3- or 2,3-substituted arabinose residues.

[0021] Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucoside glucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminal non-reducing beta-D- glucose residues with the release of beta-D-glucose. Beta-glucosidase activity can be determined using p-nitrophenyl-beta-D-glucopyranoside as substrate according to the procedure of Venturi et al., 2002, J. Basic Microbiol. 42: 55-66. One unit of beta-glucosidase is defined as 1.0 pmole of p-nitrophenolate anion produced per minute at 25°C, pH 4.8 from 1 mM p-nitrophenyl-beta-D- glucopyranoside as substrate in 50 mM sodium citrate containing 0.01% TWEEN® 20.

[0022] Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of short beta (1— >4)-xylooligosaccharides to remove successive D-xylose residues from non-reducing termini. Beta-xylosidase activity can be determined using 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citrate containing 0.01 % TWEEN® 20 at pH 5, 40°C. One unit of beta-xylosidase is defined as 1 .0 pmole of p-nitrophenolate anion produced per minute at 40°C, pH 5 from 1 mM p-nitrophenyl-beta-D- xyloside in 100 mM sodium citrate containing 0.01% TWEEN® 20. Cellobiohydrolase: The term “cellobiohydrolase” means a 1 ,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91) that catalyzes the hydrolysis of 1 ,4-beta-D-glucosidic linkages in cellulose, cellooligosaccharides, or any beta-1 , 4-linked glucose containing polymer, releasing cellobiose from the reducing or non-reducing ends of the chain (Teeri, 1997, Crystalline cellulose degradation: New insight into the function of cellobiohydrolases, Trends in Biotechnology 15: ISO- 167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: why so efficient on crystalline cellulose?, Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity is determined according to the procedures described by Lever et al., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS Letters, 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. In the present invention, the Tomme et al. method can be used to determine cellobiohydrolase activity.

[0023] Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or “cellulase” means one or more (e.g., several) enzymes that hydrolyze a cellulosic material. Such enzymes include endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. The two basic approaches for measuring cellulolytic activity include: (1) measuring the total cellulolytic activity, and (2) measuring the individual cellulolytic activities (endoglucanases, cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al., Outlook for cellulase improvement: Screening and selection strategies, 2006, Biotechnology Advances 24: 452-481. Total cellulolytic activity is usually measured using insoluble substrates, including Whatman N°1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc. The most common total cellulolytic activity assay is the filter paper assay using Whatman N°1 filter paper as the substrate. The assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

[0024] For purposes of the present invention, cellulolytic enzyme activity is determined by measuring the increase in hydrolysis of a cellulosic material by cellulolytic enzyme(s) under the following conditions: 1-50 mg of cellulolytic enzyme protein / g of cellulose in PCS (or other pretreated cellulosic material) for 3-7 days at a suitable temperature, e.g., 50°C, 55°C, or 60°C, compared to a control hydrolysis without addition of cellulolytic enzyme protein. Typical conditions are 1 ml reactions, washed or unwashed PCS, 5% insoluble solids, 50 mM sodium acetate pH 5, 1 mM MnSO4, 50°C, 55°C, or 60°C, 72 hours, sugar analysis by AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

[0025] Cellulosic material: The term “cellulosic material” means any material containing cellulose. Cellulose is a homopolymer of anyhdrocellobiose and thus a linear beta-(1 -4)-D-glucan, while hemicelluloses include a variety of compounds, such as xylans, xyloglucans, arabinoxylans, and mannans in complex branched structures with a spectrum of substituents. Although generally polymorphous, cellulose is found in plant tissue primarily as an insoluble crystalline matrix of parallel glucan chains. Hemicelluloses usually hydrogen bond to cellulose, as well as to other hemicelluloses, which help stabilize the cell wall matrix.

[0026] Endoglucanase: The term “endoglucanase” means a 4-(1 ,3;1 ,4)-beta-D-glucan 4- glucanohydrolase (E.C. 3.2.1.4) that catalyzes endohydrolysis of 1 ,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, beta-1 ,4 bonds in mixed beta-1 ,3-1 ,4 glucans such as cereal beta-D-glucans or xyloglucans, and other plant material containing cellulosic components. Endoglucanase activity can be determined by measuring reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452-481). Endoglucanase activity can also be determined using carboxymethyl cellulose (CMC) as substrate according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH 5, 40°C.

[0027] Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase” or “Family GH61” or “GH61” means a polypeptide falling into the glycoside hydrolase Family 61 according to Henrissat B., 1991 , A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316: 695-696. The enzymes in this family were originally classified as a glycoside hydrolase family based on measurement of very weak endo-1 ,4-beta-D-glucanase activity in one family member. The structure and mode of action of these enzymes are non-canonical and they cannot be considered as bona fide glycosidases. However, they are kept in the CAZy classification on the basis of their capacity to enhance the breakdown of lignocellulose when used in conjunction with a cellulase or a mixture of cellulases.

[0028] GH5 polypeptide: refers to a polypeptide with enzyme activity, the polypeptide being classified as member of the Glycoside hydrolase family 5 in the database of Carbohydrate-Active EnZymes (CAZymes) (http: / / www.cazv.orq / ).

[0029] GH5_21 xylanase: “GH5_21 xylanase” is an abbreviation for Glycoside Hydrolase Family 5 subfamily 21 endo-beta-1 ,4-xylanases that possess a three-dimensional structure characterized by a (P / a) 8 barrel and use a glutamine residue as a catalytic nucleophile / base.

[0030] GH5_35 xylanase: “GH5_35 xylanase” is an abbreviation for Glycoside Hydrolase Family 5 subfamily 35 endo-beta-1 ,4-xylanases that possess a three-dimensional structure characterized by a (P / a) 8 barrel and use a glutamine residue as a catalytic nucleophile / base.

[0031] GH8 polypeptide: refers to a polypeptide with enzyme activity, the polypeptide being classified as member of the Glycoside hydrolase family 5 in the database of Carbohydrate-Active EnZymes (CAZymes) (http: / / www.cazy.orq / ). GH30 polypeptide: refers to a polypeptide with enzyme activity, the polypeptide being classified as member of the Glycoside hydrolase family 30 in the database of Carbohydrate-Active EnZymes (CAZymes) (http: / / www.cazy.org / ).

[0032] GH10 polypeptide: refers to a polypeptide with enzyme activity, the polypeptide being classified as member of the Glycoside hydrolase family 10 in the database of Carbohydrate-Active EnZymes (CAZymes) available at http: / / www.cazv.org / . (Lombard, V.; Golaconda Ramulu, H.; Drula, E.; Coutinho, P. M.; Henrissat, B. (21 November 2013). "The carbohydrate-active enzymes database (CAZy) in 2013". Nucleic Acids Research. 42 (D1): D490-D495’ Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (January 2009). "The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics". Nucleic Acids Res. 37 (Database issue): D233-8).

[0033] GH11 polypeptide refers to a polypeptide with enzyme activity, the polypeptide being classified as member of the Glycoside hydrolase family 11 in the database of Carbohydrate-Active EnZymes (CAZymes).

[0034] GH62 polypeptide: refers to a polypeptide with enzyme activity, the polypeptide being classified as member of the Glycoside hydrolase family 62 in the database of Carbohydrate-Active EnZymes (CAZymes).

[0035] GH43 polypeptide: refers to a polypeptide with enzyme activity, the polypeptide being classified as member of the Glycoside hydrolase family 43 in the database of Carbohydrate-Active EnZymes (CAZymes).

[0036] GH51 polypeptide: refers to a polypeptide with enzyme activity, the polypeptide being classified as member of the Glycoside hydrolase family 51 in the database of Carbohydrate-Active EnZymes (CAZymes).

[0037] Polypeptide having cellulolytic enhancing activity: The term “polypeptide having cellulolytic enhancing activity” means a GH61 polypeptide that catalyzes the enhancement of the hydrolysis of a cellulosic material by enzyme having cellulolytic activity. For purposes of the present invention, cellulolytic enhancing activity is determined by measuring the increase in reducing sugars or the increase of the total of cellobiose and glucose from the hydrolysis of a cellulosic material by cellulolytic enzyme under the following conditions: 1-50 mg of total protein / g of cellulose in PCS, wherein total protein is comprised of 50-99.5% w / w cellulolytic enzyme protein and 0.5-50% w / w protein of a GH61 polypeptide having cellulolytic enhancing activity for 1-7 days at a suitable temperature, e.g., 50°C, 55°C, or 60°C, and pH, e.g., 5.0 or 5.5, compared to a control hydrolysis with equal total protein loading without cellulolytic enhancing activity (1-50 mg of cellulolytic protein / g of cellulose in PCS). In a preferred aspect, a mixture of CELLUCLAST® 1.5L (Novozymes A / S, Bagsvaerd, Denmark) in the presence of 2-3% of total protein weight Aspergillus oryzae beta-glucosidase (recombinantly produced in Aspergillus oryzae according to WO 02 / 095014) or 2-3% of total protein weight Aspergillus fumigatus beta-glucosidase (recombinantly produced in Aspergillus oryzae as described in WO 2002 / 095014) of cellulase protein loading is used as the source of the cellulolytic activity.

[0038] The GH61 polypeptides having cellulolytic enhancing activity enhance the hydrolysis of a cellulosic material catalyzed by enzyme having cellulolytic activity by reducing the amount of cellulolytic enzyme required to reach the same degree of hydrolysis preferably at least 1 .01 -fold, e.g., at least 1 .05-fold, at least 1 .10-fold, at least 1 .25-fold, at least 1 .5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20-fold.

[0039] Protease: The term “proteolytic enzyme” or “protease” means one or more (e.g., several) enzymes that break down the amide bond of a protein by hydrolysis of the peptide bonds that link amino acids together in a polypeptide chain. A protease may include, e.g., a metalloprotease, a trypsin-like serine protease, a subtilisin-like serine protease, and aspartic protease.

[0040] Sugar cane: The term “sugar cane” means any tall perennial grass of the genus Saccharum (family Poaceae, tribe Andropogoneae). Native to warm temperatures in tropical regions, sugar cane has stout, jointed, fibrous stalks that are rich in sugar, and measure two to six meters (six to nineteen feet) tall. All sugar cane species interbreed, and the major commercial cultivars are complex hybrids.

[0041] Sugar cane bagasse: The term “sugar cane bagasse” means the fibrous residue remaining after sugar cane stalks are crushed to extract their juice.

[0042] Xylanases / polypeptide with xylanase activity: The term “xylanase” means a 1 ,4-beta- D-xylan-xylohydrolase (E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1 ,4-beta-D-xylosidic linkages in xylans. Xylanase activity can be determined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C. One unit of xylanase activity is defined as 1 .0 pmole of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6. Xylanases can be found in, e.g., the GH5, GH30, GH10, and GH11 families.

[0043] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

[0044] For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

[0045] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment) For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

[0046] (Identical Deoxyribonucleotides x 100) / (Length of Alignment - Total Number of Gaps in Alignment).

[0047] DETAILED DESCRIPTION OF THE INVENTION

[0048] The invention relates to the extraction of cane juice with a high yield of sugar from sugar cane, more particularly, with a process for the extraction of cane juice and thereby sugar from sugar cane and / or sugar cane bagasse

[0049] The present invention relates to a process for improving yield of sugar cane juice comprising steps of: a. contacting a substrate with an enzyme composition comprising one or more hydrolytic enzymes in presence of imbibition water; b. separating the mixture of sugar cane juice and bagasse; c. clarifying the separated sugar cane juice; and d. recovering the sugar from sugar cane juice.

[0050] The description will enable one skilled in the art to make and use the invention, and it describes several embodiments, adaptations, variations, alternatives, and uses of the invention. These and other embodiments, features, and advantages of the present invention will become more apparent to those skilled in the art when taken with reference to the following detailed description of the invention in conjunction with any accompanying drawings.

[0051] Sugar is produced commercially from sugar cane and sugar beet. Batch or continuous processes for the extraction of sugar from sugar cane and sugarbeet by diffusion are well known and have been successfully employed commercially for many years. The conventional method of extracting sugar juices from sugar cane is by the use of mills wherein the cane is crushed and its juices partly squeezed out leaving a form of bagasse from which a major portion of the juices have been extracted. These conventional methods do not generally make it possible to remove more than 80% of the moisture content from the sugar cane.

[0052] Sugarcane processing involves several stages that transform the raw sugarcane stalks into valuable products. The process typically begins with the harvesting of mature sugarcane, where the stalks are cut and collected in the fields. Once harvested, the sugarcane is transported to the sugar factory. At the sugar factory, the sugarcane stalks go through a series of steps to extract the juice. The first step is the preparation stage, where the cane is shredded or crushed using heavy machinery, typically a series of rotating knives or large rollers. This preparation helps in maximizing juice extraction and facilitates the subsequent processes.

[0053] Next comes the extraction stage, where the prepared sugarcane undergoes crushing or pressing to extract the juice. Traditionally, this was done using large rollers or mills that squeeze the sugarcane to release the juice. Modern milling facilities often employ more advanced machinery, such as crushers or mills powered by electricity or engines, to efficiently extract the juice from the sugarcane. The prepared cane passes through 3 to 7 individual mills connected in a series called “Milling Train” or “ Milling Tandem”. The juice collected from the first mill is called “primary juice”. The fibrous material from the first mill then undergoes subsequent mills in series and thereby till the last mill. Just before the last milling stage, the fibrous material goes through the washing step using 30% water (based on cane processed) maintained at 60 to 70°C which helps to further reduce the residual sugar in the fibres. The term water of %imbibition water is used interchangeably. This fibrous material after the last milling stage is called “Bagasse”.

[0054] In an embodiment of the invention, the substrate is sugar cane or shredded sugarcane or bagasse or milling intermediate or diffusion intermediate.

[0055] In an embodiment of the invention, the contacting the substrate with an enzyme composition is done before or during milling stage.

[0056] In an embodiment of the invention, the contacting substrate with an enzyme composition is done before or during diffusion stage.

[0057] In an embodiment of the invention, the contacting substrate with an enzyme composition is done before clarification step.

[0058] In an embodiment of the invention, the contacting substrate with an enzyme composition is done before, during and / or after shredding step.

[0059] In an embodiment of the invention, contacting the substrate with said one or more hydrolytic enzymes for a time period which is less than 72 hours.

[0060] In an embodiment of the invention, contacting the substrate with said one or more hydrolytic enzymes at a temperature which is within the range of 20°C -80°C.

[0061] In an embodiment of the invention, the imbibition water added to the process may be reduced in the range of 0.5-15% w / w to per / kg of sugarcane.

[0062] The juice from the last milling is pumped in a counter-current direction to the direction of solids as shown in the Figure 1 . The juice collected from milling stages 1 and 2 in a mixed juice tank and referred to as “mixed juice”.

[0063] The extraction stage can also be performed using the diffusion method. In this step, hot water is passed through the cane material, allowing it to dissolve and extract the sugar-containing juice. The resulting mixture, called "cane juice," is collected and separated from the residual solids, known as "bagasse”. The addition of an enzyme or mixture at any stage during the sugarcane extraction helps to loosen the cell walls of the sugarcane thereby releasing more juice during the extraction.

[0064] Once the juice is extracted, it undergoes clarification and filtration processes to remove impurities, such as suspended solids and debris. These processes help improve the quality of the juice and ensure that the end products are clean and pure.

[0065] In an embodiment of the invention, the recovered sugar is refined by conventional methods.

[0066] In an embodiment of the invention, the recovered sugar has more than 95% of sucrose.

[0067] Post clarification, the juice is typically subjected to heating and evaporation to concentrate the sucrose content. This is typically done in multiple stages using evaporators, which utilize heat to evaporate the water content and leave behind a concentrated juice, known as “syrup” or "raw juice," which forms the basis for further processing. It can be further purified to produce crystallized sugar or processed to obtain other sugarcane-based products, such as molasses or ethanol.

[0068] To obtain crystalline sugar, the raw juice is subjected to boiling and cooling processes called as crystallization stage. This process facilitates the formation of sugar crystals from the juice. The resulting mixture of sugar crystals and residual syrup is then separated through centrifugation. The separated sugar crystals are further dried and refined to produce various types of sugar, such as granulated sugar or brown sugar.

[0069] Throughout the sugarcane milling process, by-products such as bagasse, molasses, and filter cake are generated and can be utilized for various purposes, including as a source of energy in power generation or as animal feed or fertilizers.

[0070] The byproduct of sugarcane milling or diffusion, called bagasse, is the fibrous residue left after the juice extraction process. Bagasse is often utilized as a renewable energy source, providing fuel for the mill's operations, or serving as a feedstock for biomass power generation.

[0071] Sucrose extraction efficiency depends on many factors, including, inter alia, the amount of fiber in the sugarcane. Typically, more than 90% of the sucrose content of the sugarcane is extracted out of the biomass material. More typically more than 95% of the sucrose content is extracted.

[0072] In an embodiment of the invention, the extraction efficiency of the process is above 0.2% compared to conventional methods.

[0073] Sugarcane juice comprises mainly sucrose, however as a result of the conditions of the process some of the sucrose is inverted into fructose and glucose.

[0074] The remaining biomass, after processing, is referred to as sugarcane bagasse or bagasse. Typically, bagasse comprises (on a washed and dried basis); 45%-55% wt cellulose, 20%-25% wt hemicellulose and 18%-24% wt lignin. Additional components such as ash and waxes are less than 5% wt. Bagasse is mainly used for providing energy (more than sufficient for the sugar mill), to generate electricity by burning, as cattle feed and for intermediate and low-quality paper. Bagasse could theoretically be used for the production of fermentable carbohydrates, e.g., for the production of ethanol.

[0075] In an embodiment of the invention, the %polarization (POL) of juice is increased in the range of 0.0001-5%.

[0076] In an embodiment of the invention, the %polarization (POL) of bagasse is reduced in the range of 0.0001-5%.

[0077] In an embodiment of the invention, the %polarization (POL) of cane is reduced in the range of 0.0001-5%.

[0078] In an embodiment of the invention, the %moisture of bagasse is reduced in the range of 0.01-15%.

[0079] In an embodiment of the invention, the yield of juice is increased more than 0.01%.

[0080] Enzyme Compositions

[0081] Preferably, the compositions are enriched in the polypeptides useful according to the invention. The term "enriched" indicates that the enzymatic activity of the composition has been increased, e.g., with an enrichment factor of at least 1.1 , such as at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 2.0, at least 3.0, at least 4.0, at least 5.0, at least 10.

[0082] In an embodiment of the invention, the enzyme composition comprises of one or more hydrolytic enzymes The enzyme compositions comprise one or more hydrolytic enzymes that is useful in extraction of cane juice from sugar cane and / or bagasse.

[0083] In an embodiment of the invention, the enzyme composition comprises one or more (e.g., several) proteins selected from the group consisting of a cellulase, a GH61 polypeptide having cellulolytic enhancing activity, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.

[0084] In an embodiment of the invention, the cellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[0085] In an embodiment of the invention, the hemicellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase.

[0086] In an embodiment of the invention, the enzyme composition comprises one or more (e.g., several) cellulolytic enzymes.

[0087] In an embodiment of the invention, the enzyme composition comprises or further comprises one or more (e.g., several) hemicellulolytic enzymes.

[0088] In an embodiment of the invention, the enzyme composition comprises of a GH62 polypeptide having arabinofuranosidase activity and a GH10 xylanase.

[0089] In an embodiment the invention the enzyme composition comprises of a GH62 polypeptide having arabinofuranosidase activity and a GH11 xylanase.

[0090] In an embodiment the invention the enzyme composition comprises of a GH43 polypeptide having arabinofuranosidase activity and a GH 10 xylanase.

[0091] In an embodiment the invention the enzyme composition comprises of a GH43 polypeptide having arabinofuranosidase activity and a GH11 xylanase.

[0092] In an embodiment the invention arabinofuranosidase and xylanase activity are contemplated, it is at present contemplated that the xylanase is used in one or more of the following amounts (dosage ranges): 0.01-200; 0.05-100; 0.1-50; 0.2-20; 0.1-1 ; 0.2-2; 0.5-5; or 1- 10 wherein all these ranges are mg xylanase protein per kg substrate (ppm). It is at present contemplated that the arabinofuranosidase is administered in one or more of the following amounts (dosage ranges): 0.01-200; 0.05-100; 0.1-50; 0.2-20; 0.1-1 ; 0.2-2; 0.5-5; or 1-10 wherein all these ranges are mg arabinofuranosidase protein per kg substrate (ppm). It is further contemplated that the ratio of the GH10 or 11 xylanase to GH62 arabinofuranosidase is in the range of 100: 1 to 1 : 100 xylanase: arabinofuranosidase such as the ranges 50: 1 to 1 :50, 50: 1 to 1 :10, 25:1 to 1 :5, 10:1 to 1 :2 or such as 10:1 to 1 :50, 5:1 to 1 :25, 2:1 to 1 :10 xylanase: arabinofuranosidase.

[0093] Polypeptides Having Arabinofuranosidase Activity

[0094] Preferred embodiments of the aspect of the invention relating to the GH62 polypeptide having arabinofuranosidase activity are disclosed herein below. Additional details of preferred GH62 polypeptides having arabinofuranosidase activity are found in PCT / CN2015 / 071015 filed 19 January 2015.

[0095] In an embodiment, the GH62 polypeptide having arabinofuranosidase activity has a sequence identity to the polypeptide of SEQ ID NO: 2 of at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. Xylanase (GH10 and GH11 Polypeptides)

[0096] Exemplary embodiments relating to the GH10 or GH11 polypeptide having xylanase activity are disclosed herein below, alternatively referred to as Family 10 xylanase and Family 11 xylanase, respectively.

[0097] In an embodiment, the GH10 polypeptide having xylanase activity such as the xylanase from Aspergillus aculeatus (Xyl II) as disclosed in WO 1994 / 021785 as SEQ ID NO: 5. In an embodiment, the GH10 polypeptide having xylanase activity such as the xylanase from Clostridium acetobutylicum as disclosed in J. Bacteriol. 2001 , 183(16):4823 as Swissprot:Q97TP5.

[0098] In an embodiment, the GH10 polypeptide having xylanase activity such as the xylanase from Aspergillus aculeatus as disclosed as SEQ ID NO: 8 in WO 2005 / 059084.

[0099] In an embodiment, the GH11 polypeptide having xylanase activity such as the xylanase from Thermomyces lanuginosus as disclosed as SEQ ID NO: 2 in WO1996 / 23062.

[0100] In an embodiment, the GH11 polypeptide having xylanase activity such as the xylanase from Dictyoglomus thermophilum as disclosed as SEQ ID NO: 305 in WO2011 / 057140.

[0101] In an embodiment, the GH11 polypeptide having xylanase activity such as the xylanase from Paenibacillus Pabuli as disclosed as SEQ ID NO: 2 in W02005 / 079585.

[0102] In an embodiment, the GH11 polypeptide having xylanase activity such as the xylanase from Fusarium oxysporum called FoxXyn6 as disclosed as SEQ ID NO: 2 in WO2014 / 019220.

[0103] In an embodiment, the GH11 polypeptide having xylanase activity such as the xylanase from Fusarium oxysporum called AclXyn5 as disclosed as SEQ ID NO: 7 in WO2014 / 020143.

[0104] In an embodiment, the GH11 polypeptide having xylanase activity is the xylanase from Cornyascus such as Corynascus thermophilus, from Scytalidium such as Scytalidium thermophilum, from Penicillium such as Penicillium oxalicum as disclosed in WO 2013 / 075642.

[0105] In an embodiment, the GH10 polypeptide having xylanase activity includes the xylanase from Talaromyces leycettanus as disclosed in WO 2013 / 019827.

[0106] In an embodiment, the GH10 polypeptide having xylanase activity includes the xylanase from Trichophaea saccata as disclosed in WO 2011 / 057083.

[0107] In one embodiment, the cellulolytic composition comprises a xylanase.

[0108] In a preferred embodiment of the invention, the xylanase is a Family 10 xylanase.

[0109] Examples of xylanases useful in the processes of the present invention include, but are not limited to, xylanases from Aspergillus aculeatus (GeneSeqP:AAR63790; WO 94 / 21785), Aspergillus fumigatus (WO 2006 / 078256), Penicillium pinophilum (WO 2011 / 041405), Penicillium sp. (WO 2010 / 126772), Thielavia terrestris NRRL 8126 (WO 2009 / 079210), and Trichophaea saccata GH10 (WO 2011 / 057083).

[0110] One or more (e.g., several) components of the enzyme composition may be wild-type proteins, recombinant proteins, or a combination of wild-type proteins and recombinant proteins. For example, one or more (e.g., several) components may be native proteins of a cell, which is used as a host cell to express recombinantly one or more (e.g., several) other components of the enzyme composition. One or more (e.g., several) components of the enzyme composition may be produced as monocomponents, which are then combined to form the enzyme composition. The enzyme composition may be a combination of multicomponent and monocomponent protein preparations. The enzymes used in the process of the present invention may be in any form suitable for use, such as, for example, a fermentation broth formulation or a cell composition, a cell lysate with or without cellular debris, a semi-purified or purified enzyme preparation, or a host cell as a source of the enzymes. The enzyme composition may be a dry powder or granulate, a nondusting granulate, a liquid, a stabilized liquid, or a stabilized protected enzyme. Liquid enzyme preparations may, for instance, be stabilized by adding stabilizers such as a sugar, a sugar alcohol or another polyol, and / or lactic acid or another organic acid according to established processes.

[0111] The polypeptides having cellulolytic enzyme activity or hemicellulolytic enzyme activity as well as other proteins / polypeptides useful in the degradation of the cellulosic material, e.g., GH61 polypeptides having cellulolytic enhancing activity (collectively hereinafter “polypeptides having enzyme activity”) can be derived or obtained from any suitable origin, including, bacterial, fungal, yeast, plant, or mammalian origin. The term “obtained” also means herein that the enzyme may have been produced recombinantly in a host organism employing methods described herein, wherein the recombinantly produced enzyme is either native or foreign to the host organism or has a modified amino acid sequence, e.g., having one or more (e.g., several) amino acids that are deleted, inserted and / or substituted, i.e., a recombinantly produced enzyme that is a mutant and / or a fragment of a native amino acid sequence or an enzyme produced by nucleic acid shuffling processes known in the art. Encompassed within the meaning of a native enzyme are natural variants and within the meaning of a foreign enzyme are variants obtained recombinantly, such as by site-directed mutagenesis or shuffling.

[0112] Chemically modified or protein engineered mutants of polypeptides having enzyme activity may also be used.

[0113] One or more (e.g., several) components of the enzyme composition may be a recombinant component, i.e., produced by cloning of a DNA sequence encoding the single component and subsequent cell transformed with the DNA sequence and expressed in a host (see, for example, WO 91 / 17243 and WO 91 / 17244). The host is preferably a heterologous host (enzyme is foreign to host), but the host may under certain conditions also be a homologous host (enzyme is native to host). Monocomponent cellulolytic proteins may also be prepared by purifying such a protein from a fermentation broth.

[0114] Enzymes may be added in an effective amount, which can be adjusted according to the practitioner and particular process needs. In general, enzyme may be present in an amount of 0.0001-1 mg enzyme protein per kg of sugar cane, such as 0.001-0.1 mg enzyme protein per kg of sugar cane. In particular embodiments, the enzyme may be present in an amount of, e.g., 1 pg, 2.5 pg, 5 pg, 10 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, 50 pg, 75 pg, 100 pg, 125 pg, 150 pg, 175 pg, 200 pg, 225 pg, 250 pg, 275 pg, 300 pg, 325 pg, 350 pg, 375 pg, 400 pg, 450 pg, 500 pg, 550 pg, 600 pg, 650 pg, 700 pg, 750 pg, 800 pg, 850 pg, 900 pg, 950 pg, 1000 pg enzyme protein per kg of sugar cane.

[0115] Formulating agent

[0116] The enzyme composition of the invention may be formulated as a liquid or a solid. For a liquid formulation, the formulating agent may comprise a polyol (such as e.g. glycerol, ethylene glycol or propylene glycol), a salt (such as e.g. sodium chloride, sodium benzoate, potassium sorbate) or a sugar or sugar derivative (such as e.g. dextrin, glucose, sucrose, and sorbitol). Thus in one embodiment, the composition is a liquid composition comprising the polypeptide of the invention and one or more formulating agents selected from the list consisting of glycerol, ethylene glycol, 1 ,2-propylene glycol, 1 ,3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, dextrin, glucose, sucrose, and sorbitol.

[0117] For a solid formulation, the formulation may be for example as a granule, spray dried powder or agglomerate. The formulating agent may comprise a salt (organic or inorganic zinc, sodium, potassium or calcium salts such as e.g. such as calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch or a sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol).

[0118] In an embodiment, the solid composition is in granulated form. The granule may have a matrix structure where the components are mixed homogeneously. However, the granule typically comprises a core particle and one or more coatings, which typically are salt and / or wax coatings. The core particle can either be a homogeneous blend of xylanase of the invention optionally combined with one or more additional enzymes and optionally together with one or more salts or an inert particle with the xylanase of the invention optionally combined with one or more additional enzymes applied onto it.

[0119] In an embodiment, the material of the core particles are selected from the group consisting of inorganic salts (such as calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch or a sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol), sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol), small organic molecules, starch, flour, cellulose and minerals. The salt coating is typically at least 1 pm thick and can either be one particular salt or a mixture of salts, such as Na2SO4, K2SO4, MgSO4and / or sodium citrate. Other examples are those described in e.g. WO 2008 / 017659, WO 2006 / 034710, WO 1997 / 05245, WO 1998 / 54980, WO 1998 / 55599, WO 2000 / 70034 or polymer coating such as described in WO 2001 / 00042.

[0120] In another embodiment, the composition is a solid composition comprising the xylanase of the invention and one or more formulating agents selected from the list consisting of sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch and cellulose. In a preferred embodiment, the formulating agent is selected from one or more of the following compounds: sodium sulfate, dextrin, cellulose, sodium thiosulfate and calcium carbonate. In a preferred embodiment, the solid composition is in granulated form. In an embodiment, the solid composition is in granulated form and comprises a core particle, an enzyme layer comprising the xylanase of the invention and a salt coating.

[0121] In a further embodiment, the formulating agent is selected from one or more of the following compounds: glycerol, ethylene glycol, 1 , 2-propylene glycol or 1 , 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch and cellulose. In a preferred embodiment, the formulating agent is selected from one or more of the following compounds: 1 , 2-propylene glycol, 1 , 3-propylene glycol, sodium sulfate, dextrin, cellulose, sodium thiosulfate and calcium carbonate.

[0122] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

[0123] The invention is further disclosed in the following numbered paragraphs.

[0124] Paragraph 1 . A process for improving yield of sugar cane juice comprising steps of: a. contacting a substrate derived from sugarcane with an enzyme composition comprising one or more hydrolytic enzymes in presence of imbibition water; b. separating the mixture of sugar cane juice and bagasse; c. clarifying the separated sugar cane juice; and d. recovering the sugar from sugar cane juice.

[0125] Paragraph 2. The process according to paragraph 1 , wherein the process efficiency is above 0.2%.

[0126] Paragraph 3. The process according to paragraph 1 , wherein the substrate is sugar cane or shredded sugarcane or bagasse or milling intermediate or diffusion intermediate.

[0127] Paragraph 4. The process according to paragraph 1 , wherein the recovered sugar has 99% of sucrose.

[0128] Paragraph 5. The process according to paragraph 1 , wherein contacting the substrate with an enzyme composition is done before or during milling stage. Paragraph 6. The process according to paragraph 1 , wherein contacting the substrate with an enzyme composition is done before or during diffusion stage.

[0129] Paragraph 7. The process according to paragraph 1 , wherein contacting the substrate with an enzyme composition is done before clarification step.

[0130] Paragraph 8. The process according to paragraph 1 , wherein contacting the substrate with an enzyme composition is done before, during and / or after shredding step.

[0131] Paragraph 9. The process according to any of the preceding paragraphs, wherein the enzyme composition comprises cellulases, xylanases and / or arabinofuranosidases.

[0132] Paragraph 10. The process according to any of the preceding paragraphs, wherein the enzyme composition further comprises protease and / or alpha-amylase.

[0133] Paragraph 11 . The process according to any of the preceding paragraphs, wherein the xylanase is a GH10 polypeptide with xylanase activity and / or a GH11 polypeptide with xylanase activity.

[0134] Paragraph 12. The process according to any of the preceding paragraphs, wherein the arabinofuranosidase is a GH61 polypeptide with arabinofuranosidase activity and / or a GH62 polypeptide with arabinofuranosidase activity.

[0135] Paragraph 13. The process according to any of the preceding paragraphs, wherein the sugar cane is contacted with said one or more hydrolytic enzymes at a temperature which is within the range of 20-80°C.

[0136] Paragraph 14. The process according to paragraph 1 , wherein the recovered sugar is refined by conventional methods.

[0137] Paragraph 15. The process according to paragraph 1 , wherein the % polarization (POL) of juice is increased in the range of 0.0001-5%.

[0138] Paragraph 16. The process according to paragraph 1 , wherein the % polarization (POL) of bagasse is reduced in the range of 0.0001-5%.

[0139] Paragraph 17. The process according to paragraph 1 , wherein the % polarization (POL) of cane is reduced in the range of 0.0001-5%.

[0140] Paragraph 18. The process according to paragraph 1 , wherein the % moisture of bagasse is reduced in the range of 0.01-15%.

[0141] Paragraph 19. The process according to paragraph 1 , wherein the yield of juice is increased more than 0.01%.

[0142] Paragraph 20. The process according to paragraph 1 , wherein the imbibition water added is reduced in the range of 0.5-15% w / w to per / kg of sugarcane.

[0143] Paragraph 21. The process according to paragraph 1 , wherein the process increases sugarcane crushing rate.

[0144] Paragraph 22. The process of any one of preceding paragraphs, wherein the xylanase is selected from the group consisting of: (a) a xylanase comprising or consisting of the polypeptide of SEQ ID NO: 1 ; (b) a xylanase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 1 ; c) a fragment of the polypeptide of (a) or (b); wherein the polypeptide has xylanase activity.

[0145] Paragraph 23. The process of any one of preceding paragraphs, wherein the arabinofuranosidase is selected from the group consisting of: (a) an arabinofuranosidase comprising or consisting of the polypeptide of SEQ ID NO: 2; (b) an arabinofuranosidase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 2; c) a fragment of the polypeptide of (a) or (b); wherein the polypeptide has arabinofuranosidase activity.

[0146] Paragraph 24. The process of any one of preceding paragraphs, wherein the enzyme composition is a cellulase composition produced from Trichoderma reesei.

[0147] The invention is further illustrated by the following examples.

[0148] EXAMPLES

[0149] Enzymes:

[0150] Enzyme composition: A Trichoderma reesei cellulase preparation comprising a xylanase of SEQ ID NO: 1 and an arabinofuranosidase of SEQ ID NO: 2.

[0151] To extract the juice from sugarcane, the sugarcanes procured from the USA were cleaned from the outside by removing the leaves and roots. The ends of the canes were removed so that the contaminated cane portion were removed. The canes were then shredded together using a sugarcane shredder ensuring that the juice was not extracted during the shredding. The leftover pieces of the cane from the shredded cane were removed and the prepared cane was aliquoted 250 g each in the steel containers. The primary juice was extracted in the beaker using a labscale roller mill and the solids were collected back in the container. To the solids, 27% to 30wt % warm water maintained at 45°C was added along with or without the enzyme blend. The lid of the container was closed tightly and the mixture in the container mixed for 2 minutes. The juice from the mixture was then extracted again using a roller mill and collected in the beaker along with the primary juice. The solids after the second milling were weighed and stored as Bagasse, whereas the combined juice together after the first and second milling stage was weighed and stored as mixed juice for analysis purposes. Analysis of streams: % Wet Bagasse: The % Wet Bagasse is the ratio of Bagasse obtained after each treatment to the prepared cane applied for the respective treatment. It can be calculated as:

[0152] Analysis of % Poloarization (POL) in Bagasse: 50g of bagasse weighed accurately in the mixer-juicer jar. To this, 400g of water was added accurately. The mixture was then mixed at 7000-10000 rpm for 5 min. after 5 min, the 150g of liquor was collected into a 250ml conical flask, and 0.5 g of Lead subacetate was dispersed into the liquor and filtered through filter paper. The filtrate was collected, transferred to a 400 mm polarimeter tube and analysed for angle of rotation using a polarimeter. The % POL in Bagasse after moisture correction was calculated using the below formula:

[0153] % POL in Bagasse = °A * (% Moisture in Bagasse * 0.0016 * 100 + 1.032)

[0154] Where °A is the angle of rotation

[0155] The filtrate is also analysed for Brix using a Brix meter.

[0156] The % POL in the Bagasse on a cane basis can be calculated as:

[0157] Analysis of Moisture in Bagasse & Juice: 3 g of bagasse and 5 g of Juice samples were weighed accurately in the pre-weighed pans. The pans are transferred in the hot-air oven set at 105°C for 4 hours. After 4 hours, the pans were cooled to room temperature using a desiccator. The weight of the pan + dry sample has been noted.

[0158] The moisture in bagasse or juice was calculated using the below formula:

[0159] Where,

[0160] W is the weight of the sample taken for analysis

[0161] W1 is the weight of the Pan + Sample after drying

[0162] P is the weight of the pan taken for analysis

[0163] Analysis of % POL in the Juice: A 100ml of Juice was aliquoted into a 250ml conical flask, and 2 g of Lead subacetate was dispersed into the juice and filtered through filter paper. The filtrate was collected, transferred to a 200mm polarimeter tube, and analysed for the angle of rotation using a polarimeter. The % POL in juice is calculated using the below formula: 26.03 n Ju ce — — — — —

[0164] J99.718 * 1.1 * 100

[0165] Where °A is the angle of rotation Extraction efficiency: The extraction efficiency is the ratio of POL extracted in the juice to the total POL detected. The total POL detected is the sum of % POL in Bagasse (Cane Basis) and % POL in Juice (Cane Basis)

[0166] The extraction efficiency was calculated as:

[0167] Extraction of sugar in the sugarcane juice thereby reducing the sugar losses in the bagasse was conducted. The three variables assessed during the experiment are a) Addition of enzyme / s during the milling process, b) Variation in the dose of enzyme, c) Variation in the imbibition water.

[0168] Table 1 : %wet bagasse

[0169] Table 1 summarizes the % bagasse generated. The percentage values are calculated based on the quantity of cane taken for the individual treatment and summarised as average for three treatments each (refer the section “Analysis of streams”). When enzyme treatments are compared with the control at the respective level of imbibition, it was observed that the wet bagasse quantity was reduced by 0.8 % to 2.0% on a cane basis. It was also observed that the bagasse quantity for control treatments increased with a reduction in imbibition water (From 25.3% to 26.0%). This could be due to more left-over POL in the bagasse which is not true when treated with the enzyme. When treated with the enzyme either at a 100ppm dose or 200ppm dose, the bagasse quantity was the same (between 24.0% to 24.4%). It can be concluded that adding enzymes increases juice extraction even though, the imbibition was reduced.

[0170] Table 2: Extraction of % juice

[0171] Table 2 summarizes the % juice extracted. The percentage values were calculated based on the quantity of cane taken for the individual treatment and summarised as average for three treatments each (refer the section “Analysis of streams”). When enzyme treatments were compared with the control at the respective level of imbibition, it was observed that the juice quantity increased by 0.8 % to 2.1% on a cane basis. It can be concluded that adding enzymes increases juice extraction even though, the imbibition is reduced.

[0172] Table 3: % moisture in bagasse Table 3 above summarizes the moisture left in the bagasse (refer the section “Analysis of streams”). From the table, it can be observed that irrespective of imbibition water, the moisture in control treatments remains within 49.9% to 50.6%. It can be assumed that for control, the change in imbibition does not make an impact on the moisture content in the bagasse. When, the enzyme treatments at 28wt% and 27wt% imbibition water compared with control at 30% imbibition, the delta in the moisture in the bagasse increases from 1.5% to 2.4%. At 28% imbibition water, the 200ppm dose of enzyme does not provide added advantage in further reduction in the moisture in bagasse compared to the 100ppm dose. Whereas further reduction in moisture in the bagasse is possible by reducing the imbibition water to 27% or more.

[0173] Table 4: % POL in Bagasse(Cane Basis)

[0174] Table 4 summarizes the % POL in the bagasse on a cane basis. The percentage values were calculated based on the quantity of cane taken for the individual treatment and summarised as average for three treatments each, (refer the section “Analysis of streams”). When enzyme treatments are compared with the control at the respective level of imbibition, the residual % POL in bagasse has been reduced by 0.2 to 0.27% compared to the respective control. At imbibition, 30% and 28%, when treated with the enzyme at 100 and 200 ppm dose, the residual % POL in the bagasse was reduced to 0.47% on a cane basis, but when the imbibition was reduced further to 27%, the enzyme treatment at 100 ppm dose reduced the % POL in bagasse only up to 0.53% on cane basis whereas, the enzyme treatment at 200 ppm dose still reduced the % POL in bagasse to 0.47% on cane basis. From this, it can be concluded that higher enzyme dose is required to get the desired reduction in % POL in bagasse when imbibition is further reduced. On the other hand, it was also observed that the % POL in the bagasse for control treatment increased with a reduction in imbibition water. It can be assumed that if Imbibition water is not sufficient or reduced for control treatment, the residual % POL in the bagasse may increase and hence lower % POL extraction in the juice was achieved.

[0175] Table 5: % POL in Juice (Cane Basis)

[0176] Table 5 summarizes the % POL in juice on a cane basis. The percentage value were calculated based on the quantity of cane taken for the individual treatment and summarised as average for three treatments each, (refer the section “Analysis of streams”). When enzyme treatments were compared with the control at the respective level of imbibition, it was observed that the % POL in the juice was increased by 0.21 % to 0.26% on a cane basis. When, the enzyme treatments at 28% and 27% imbibition water compared with control at 30% imbibition, the delta in % POL in juice remains between +0.18 to +0.21% except for enzyme treatment at 100 ppm dose at 27% imbibition water where the delta is +0.14%.

[0177] Table 6: % Extraction Efficiency

[0178]

[0179] Table 6 summarises the efficiency of extraction. The extraction efficiency can be calculated using % POL in the juice and % POL in the bagasse (Please refer the section “Analysis of streams” for more details). For control treatments, it can be observed that the extraction efficiency improves with an increase in imbibition water. At all three imbibition levels, the extraction efficiency can be improved by at least 1 .26% using enzymes.

[0180] While the preferred embodiments of the invention have been illustrated and described, it will be clear that the invention is not so limited. Numerous modifications, changes, variations, substitutions and equivalents will occur to those skilled in the art without departing from the spirit and scope of the present invention as described in the claims.

Claims

CLAIMS:1 . A process for improving yield of sugar cane juice comprising steps of: a. contacting a substrate derived from sugarcane with an enzyme composition comprising one or more hydrolytic enzymes in presence of imbibition water; b. separating the mixture of sugar cane juice and bagasse; c. clarifying the separated sugar cane juice; and d. recovering the sugar from sugar cane juice.

2. The process according to claim 1 , wherein the substrate is sugar cane or shredded sugarcane or bagasse or milling intermediate or diffusion intermediate.

3. The process according to any of the preceding claims, wherein the hydrolytic enzymes comprises cellulases, xylanases and / or arabinofuranosidases.

4. The process according to any of the preceding claims, wherein the enzyme composition further comprises protease and / or alpha-amylase.

5. The process according to any of the preceding claims, wherein the xylanase is a GH10 polypeptide with xylanase activity and / or a GH11 polypeptide with xylanase activity.

6. The process according to any of the preceding claims, wherein the arabinofuranosidase is a GH61 polypeptide with arabinofuranosidase activity and / or a GH62 polypeptide with arabinofuranosidase activity.

7. The process according to any of the preceding claims, wherein the sugar cane is contacted with said one or more hydrolytic enzymes at a temperature which is within the range of 20-80°C.

8. The process according to claim 1 , wherein the % polarization (POL) of juice is increased in the range of 0.0001-5%.

9. The process according to claim 1 , wherein the % polarization (POL) of bagasse is reduced in the range of 0.0001-5%.

10. The process according to claim 1 , wherein the % polarization (POL) of cane is reduced in the range of 0.0001-5%.11 . The process according to claim 1 , wherein the % moisture of bagasse is reduced in the range of 0.01-15%.

12. The process according to claim 1 , wherein the yield of juice is increased more than 0.01%.

13. The process of any one of preceding claims, wherein the xylanase is selected from the group consisting of: (a) a xylanase comprising or consisting of the polypeptide of SEQ ID NO: 1 ; (b) a xylanase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 1 ; c) a fragment of the polypeptide of (a) or (b); wherein the polypeptide has xylanase activity.

14. The process of any one of preceding claims, wherein the arabinofuranosidase is selected from the group consisting of: (a) an arabinofuranosidase comprising or consisting of the polypeptide of SEQ ID NO: 2; (b) an arabinofuranosidase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 2; c) a fragment of the polypeptide of (a) or (b); wherein the polypeptide has arabinofuranosidase activity.

15. The process of any one of preceding claims, wherein the cellulase composition is produced from Trichoderma reesei.

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