WO2018042464A1 - Rapid enzymatic hydrolysis of substrates for production of fermentable sugars - Google Patents

Abstract

The present disclosure provides a process for rapid hydrolysis of a substrate the process comprises the steps of obtaining a substrate slurry; contacting the 10 substrate slurry with at least one enzyme to obtain a soluble fraction and an insoluble fraction; separating the soluble fraction from the insoluble fraction; recovering the at least one enzyme from the soluble fraction to obtain recovered enzymes; and treating the insoluble fraction with the recovered enzymes to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours.

Description

RAPID ENZYMATIC HYDROLYSIS OF SUBSTRATES FOR

PRODCUTION OF FERMENTABLE SUGARS

FIELD OF THE INVENTION

The invention relates to a process for hydrolysis of a substrate to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours.

BACKGROUND OF THE INVENTION

[001] Cellulose containing feed stocks such as agricultural wastes, grasses, and forestry wastes has received much attention for production of ethanol and due to its availability in large amounts, the desirability to avoid burning or land filling cellulosic waste materials. Ethanol offers several advantages over gasoline and preferred as a fuel when compared to gasoline. In addition, by product of the lignocellulosic biomass conversion process, i.e., lignin, can be converted to biogas thereby reducing the use of fossil fuels. Studies have shown that, taking the entire cycle into account, the use of ethanol produced from cellulose reduces the greenhouse gas emission. The cellulosic feedstocks that may be used for ethanol production include agricultural wastes, such as corn stover, wheat straw, barley straw, oat straw, oat hulls, canola straw, and soybean stover; grasses such as switch grass, miscanthus, cord grass, and reed canary grass; forestry wastes such as aspen wood and sawdust; and sugar processing residues such as bagasse and beet pulp.

[002] Conversion of these cellulosic feedstocks into ethanol involves an intermediate saccharification process which employs enzymes to hydrolyze the beta 1,4- glucosidic linkages and release the component sugars from the polymer. The action of these enzymes is impeded due to the tight physical packing of the cellulose polymer and the presence of lignin which reduces the accessibility for the enzymes. Therefore, these enzymes are generally required in large quantities to achieve complete hydrolysis of the substrate. Extensive studies have been conducted in this direction to develop robust saccharification methods which can efficiently release sugars with minimal usage of enzymes since these enzymes are one of the highest cost contributing factors.

[003] Efforts to reduce cost of saccharification process have been made by modifying the enzyme system for high specific activity and stability, optimizing ratio of enzymes in the preparation or by employing processes for recovery and reuse of enzymes.

[004] US20160010130 discloses a process for production of sugar solution, wherein slurry of the cellulose containing biomass is prepared with inactivated cellulase and hydrolysed by adding fungal derived cellulases and the sugars generated are separated after hydrolysis from the enzymes by employing ultrafiltration wherein the recovery of the enzymes is enhanced due to addition of inactivated cellulase.

[005] US 8728770 discloses a process of saccharification of pre-treated lignocellulosic material, wherein an electrolyte is added to the enzyme containing water and is subjected to enzymatic treatment followed by separation and recovery of the reaction product and enzyme containing solution from the treated suspension and recycling the recovered enzyme for the enzymatic saccharification process. However, this process suppresses the adsorption of enzymes to the lignocellulose raw material or the reaction residue which reduces the saccharification efficiency of the enzymes.

[006] US20110039319 discloses a process for hydrolysis of cellulose to fermentable sugars, wherein the released sugars are fermented using appropriate microorganism preferable yeast and the beer is distilled out to separate and remove the fermentation product from the top of the distillation column and the cellulase are recovered from the bottom of the distillation column and recycled back to the hydrolysis step. However, long retention of the enzyme with the sugars results in loss of enzyme activity and therefore requires supplementation of fresh enzyme to compensate for the loss of enzyme activity.

[007] US20120036768 discloses a method for producing alcohol from lignocellulosic biomass in two steps, wherein the biomass is treated with a first enzyme mixture to form a hydrolysis mixture followed by thickening the mixture by separating the filtrate and hydrolysing the thickened mixture by a second enzyme mixture to provide fermentable sugar mixture which can be converted to ethanol.

[008] Most of the reports and technologies known in the art make use of catalytic enzymes in free soluble form that cannot be recovered for reuse. Further, often the substrates namely cellulosic and/or hemicellulosic polymers and products of hydrolysis thereof, have tendencies to 'inhibit, the enzymes' actions.

Such a use of these enzymes makes them less attractive for use on a commercial scale or makes the use of the enzymes more expensive than often desired.

Therefore, for reasons of cost, amount of enzymes used per unit weight of cellulose and/or hemicellulose hydrolyzed is often kept to a minimum, which in turn reduces the rate of hydrolysis reactions and increases the reaction times.

Shortcomings of the enzyme process can be improved making it the obvious choice for new process development.

[009] For the reasons stated above, there is a significant need in the art for a process that provides rapid and complete enzymatic hydrolysis of substrate to soluble and fermentable sugars with increased yield and complete enzyme recovery and recycling.

SUMMARY OF INVENTION

[0010] In an aspect of the present disclosure, there is provided a process for rapid hydrolysis of a substrate comprising the steps of: (i) obtaining a substrate slurry; (ii) contacting the substrate slurry with at least one enzyme to obtain a soluble fraction and an insoluble fraction; (iii) separating the soluble fraction from the insoluble fraction; (iv) recovering the at least one enzyme from the soluble fraction to obtain recovered enzymes; (v) treating the insoluble fraction with the recovered enzymes to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours.

[0011] In an aspect of the present disclosure, there is provided a process for rapid hydrolysis of a substrate comprising the steps of: (i) obtaining a substrate slurry; (ii) contacting the substrate slurry with at least one enzyme to obtain a soluble fraction and an insoluble fraction; (iii) separating the soluble fraction from the insoluble fraction; (iv) recovering the at least one enzyme from the soluble fraction to obtain recovered enzymes; (v) treating the insoluble fraction, and optionally fresh substrate slurry with the recovered enzymes to obtain a second soluble and a second insoluble fraction; (vi) repeating the steps (iii) to (v) on the second insoluble fraction so as to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours.

BRIEF DESCRIPTION OF THE DRAWING

[0012] The detailed description is described with reference to the accompanying figures. In the figures, the left-most digit(s) of a reference number identifies the figure in which the reference number first appears. The same numbers are used throughout the drawings to reference like features and components.

[0013] Figure 1 illustrates schematic representation of enzymatic hydrolysis of the substrate, in accordance with an embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

[0014] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.

Definitions:

[0015] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below. [0016] The articles "a", "an" and "the" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0017] The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as "consists of only".

[0018] Throughout this specification, unless the context requires otherwise the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0019] The term "including" is used to mean "including but not limited to". "Including" and "including but not limited to" are used interchangeably.

[0020] The term "substrate" used herein refers to initial compound or substance on which enzyme acts and transforms into one or more fermentable sugars. The substrate used in the present invention includes but is not limited to cellulose, holocellulose, hemicellulose and its derivative thereof. The substrate also includes polysaccharides, oligosaccharides, disaccharides etc. which can be converted into fermentable sugars. The substrate has polysaccharide content in range of 82- 100% w/w.

[0021] The term "substrate slurry" used herein refers to suspension of substrate in pumpable form. The terms "substrate slurry" and "slurry" are used interchangeably in the specification. The slurry is an acidified slurry obtained by suspending treated biomass in acidified water of pH 5 to make slurry of concentration ranging from 2 -35% w/v. The water in the slurry depends on the slurry concentration, i.e., for 5% w/v slurry, the water content is 95% and for 25% slurry the water concentration is 75%.

[0022] The term "soluble sugar" used herein refers to sugars containing more than one repeating glucosyl units or repeating xylose units including but not limited to cellobiose, xylobiose, cellotriose, cellotetraose etc.

[0023] The term "fermentable sugar" used herein refers to C5 and C6 sugars including but not limited to xylose, arabinose, mannose, glucose, galactose etc. [0024] The term "soluble fraction or hydrolysate" used herein refers to the liquid fraction obtained after hydrolysis of substrate, wherein hydrolysate comprises soluble sugars, fermentable sugars and enzymes. The terms "soluble fraction" or "hydrolysate" can be used interchangeably in the specification.

[0025] The term "insoluble fraction or unhydrolysed substrate" used herein refers to the solid residue obtained after hydrolysis of substrate, wherein insoluble fraction comprises unhydrolysed residue and sugars with >6 repeating glucosyl or xylose units. The terms "insoluble fraction" or "unhydrolysed substrate" can be used interchangeably in the specification.

[0026] The term "partial hydrolysis" used herein refers to at least 30% hydrolysis of substrate is achieved.

[0027] The term "complete hydrolysis" used herein refers to at least 90% hydrolysis of substrate is achieved.

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0029] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure as described herein.

[0030] In view of the problems associated with the enzymatic hydrolysis as mentioned in the background section, the present disclosure provides as process which overcomes the drawbacks such as longer hydrolysis time, high enzyme consumption, lower sugar conversion efficiency, complicated process and equipments, high processing cost within a short span of time.

[0031] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of substrate, wherein substrate is selected from the group consisting of cellulose, cellulose derivatives, holocellulose, holocellulose derivatives, hemicellulose, hemicellulose derivatives, and combinations thereof. Substrate includes polysaccharides, oligosaccharides, disaccharides, etc., which can be converted into fermentable sugars. The substrate includes or is derivable from agricultural waste, grasses, forest waste, municipal solid waste, industrial waste, algae, food waste, food processing waste, sewage waste, and combinations thereof.

[0032] In an embodiment of the present disclosure, there is provided a process that provides rapid and complete enzymatic hydrolysis for production of one or more fermentable sugars from substrates wherein the process involving a step of enzyme recovery, wherein the recovered enzyme is recycled to further process unhydrolysed substrate and/or fresh substrate, thereby resulting in increased overall hydrolysis of the substrate. Hydrolysis is carried in one or more steps to achieve partial or complete hydrolysis. Preferably two-step process of enzymatic hydrolysis is carried to achieve rapid and complete hydrolysis of substrate to obtain at least 95% fermentable sugar/s of hydrolysed substrate (soluble fraction). The process also provides rapid and complete hydrolysis of substrate, wherein complete hydrolysis of at least 90% substrate is achieved in less than 5 hours. The soluble fraction (hydrolysate) obtained after each hydrolysis step comprises at least 95% fermentable sugar. Fermentable sugars can be separated by any known conventional methods and used further for production of one or more value added products.

[0033] In an embodiment of the present disclosure, there is provided a process a process for rapid hydrolysis of substrate comprising of the steps of: (i) preparing substrate slurry with substrate concentration in the range of 2% to 35%; (ii) treating the substrate slurry of step (i) with enzyme having concentration in the range of 30 to 100 mg protein/gm of substrate for time period in the range of 0.5 hour to 3 hours to obtain soluble fraction (I) and insoluble fraction (I); wherein at least 30% hydrolysis is achieved; (iii) separating soluble fraction (I) from insoluble fraction (I) by known conventional methods; wherein soluble fraction (I) comprises at least 95% fermentable sugars of the hydrolysed substrate; (iv)recovering enzymes from soluble fraction (I); (v) treating insoluble fraction (I) with recovered enzyme from step (iv) for the time period in the range of 0.5 hour to 3 hours to obtain soluble fraction (II) and insoluble fraction (II); wherein at least 90% hydrolysis is achieved and the soluble fraction (II) comprises at least 95% fermentable sugars of hydrolysed substrate; and (vi) optionally repeating the steps (iv) to (v) to achieve complete hydrolysis of the substrate in less than 5 hours.

[0034] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate comprising: (i) obtaining a substrate slurry; (ii) contacting the substrate slurry with at least one enzyme to obtain a soluble fraction and an insoluble fraction; (iii) separating the soluble fraction from the insoluble fraction; (iv) recovering the at least one enzyme from the soluble fraction to obtain recovered enzymes; and (v) treating the insoluble fraction with the recovered enzymes to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours.

[0035] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate comprising: (i) obtaining a substrate slurry; (ii) contacting the substrate slurry with at least one enzyme to obtain a soluble fraction and an insoluble fraction; (iii) separating the soluble fraction from the insoluble fraction; (iv) recovering the at least one enzyme from the soluble fraction to obtain recovered enzymes; and (v) treating the insoluble fraction with the recovered enzymes to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 3 hours.

[0036] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein treating the insoluble fraction in step (v) results in a second soluble, and a second insoluble fraction.

[0037] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the at least 90% hydrolysis of the substrate is achieved by repeating the steps (iii) to (v) at least once.

[0038] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the at least 90% hydrolysis of the substrate is achieved by repeating the steps (ii) to (v) at least once on the second insoluble fraction, wherein the at least one enzyme in step (ii) is the recovered enzymes from step (iv).

[0039] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the substrate has a polysaccharide content in the range of 80 - 95%.

[0040] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the substrate slurry comprises substrate, wherein the substrate has a concentration in the range of 2 - 35% with respect to the substrate slurry.

[0041] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein contacting the substrate slurry with the at least one enzyme is carried out at a temperature in the range of 40°C to 60°C at pH in the range of 4.5 to 5.5.

[0042] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein contacting the substrate slurry with the at least one enzyme is carried out at a temperature in the range of 45°C to 55°C at pH in the range of 4.5 to 5.0.

[0043] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein contacting the substrate slurry with the at least one enzyme is carried out at a temperature of 50°C at pH of 4.8.

[0044] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein contacting the substrate slurry with at the least one enzyme is carried out for a period in the range of 0.5 hours to 3 hours.

[0045] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein contacting the substrate slurry with at the least one enzyme is carried out for a period in the range of 0.5 hours to 2 hours. [0046] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein contacting the substrate slurry with at the least one enzyme is carried out for a period of 1 hour.

[0047] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the at least one enzyme is used at a concentration in the range of 10 to 100 mg protein/gm of substrate.

[0048] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the at least one enzyme is used at a concentration in the range of 25 to 95 mg protein/gm of substrate.

[0049] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the at least one enzyme is used at a concentration of 95 mg protein/gm of substrate.

[0050] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the at least one enzyme is used at a concentration of 93 mg protein/gm of substrate.

[0051] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the at least one enzyme is used at a concentration of 62 mg protein/gm of substrate.

[0052] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the at least one enzyme is used at a concentration of 60 mg protein/gm of substrate.

[0053] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the at least one enzyme is selected from the group consisting of cellulases, pectinases, amylases, maltases, xylanases, lactases, and combinations thereof.

[0054] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein separating the soluble fraction from the insoluble fraction is carried by conventional method selected from the group consisting of filtration, centrifugation, and decantation. [0055] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein treating the insoluble fraction with the recovered enzymes to achieve at least 90% hydrolysis of the substrate is carried out at a temperature in the range of 40°C to 60°C with a pH in the range of 4.5 to 5.5.

[0056] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein treating the insoluble fraction with the recovered enzymes to achieve at least 90% hydrolysis of the substrate is carried out at a temperature in the range of 45°C to 55°C with a pH in the range of 4.5 to 5.0.

[0057] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein treating the insoluble fraction with the recovered enzymes to achieve at least 90% hydrolysis of the substrate is carried out at a temperature of 50°C with a pH of 4.8.

[0058] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein treating the insoluble fraction with the recovered enzymes to achieve at least 90% hydrolysis of the substrate is carried out for a period in the range of 0.5 hours to 3 hours.

[0059] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein treating the insoluble fraction with the recovered enzymes to achieve at least 90% hydrolysis of the substrate is carried out for a period in the range of 0.5 hours to 2 hours.

[0060] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein treating the insoluble fraction with the recovered enzymes to achieve at least 90% hydrolysis of the substrate is carried out for a period of 1 hour.

[0061] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein treating the insoluble fraction in step (v) results in a second soluble, and a second insoluble fraction is carried out at a temperature in the range of 40°C to 60°C with a pH in the range of 4.5 to 5.5. [0062] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein treating the insoluble fraction in step (v) results in a second soluble, and a second insoluble fraction is carried out at a temperature in the range of 45°C to 55°C with a pH in the range of 4.5 to 5.0.

[0063] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein treating the insoluble fraction in step (v) results in a second soluble, and a second insoluble fraction is carried out at a temperature of 50°C with a pH of 4.8.

[0064] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein treating the insoluble fraction in step (v) results in a second soluble, and a second insoluble fraction is carried out for a period in the range of 0.5 hours to 3 hours.

[0065] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein treating the insoluble fraction in step (v) results in a second soluble, and a second insoluble fraction is carried out for a period in the range of 0.5 hours to 2 hours.

[0066] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein treating the insoluble fraction in step (v) results in a second soluble, and a second insoluble fraction is carried out for a period of 1 hour.

[0067] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the at least 90% hydrolysis of the substrate is achieved by repeating the steps (iii) to (v) at least once is carried out at a temperature in the range of 40°C to 60°C with a pH in the range of 4.5 to 5.5.

[0068] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the at least 90% hydrolysis of the substrate is achieved by repeating the steps (iii) to (v) at least once is carried out at a temperature in the range of 45°C to 55°C with a pH in the range of 4.5 to 5.0. [0069] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the at least 90% hydrolysis of the substrate is achieved by repeating the steps (iii) to (v) at least once is carried out at a temperature of 50°C with a pH of 4.8.

[0070] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the at least 90% hydrolysis of the substrate is achieved by repeating the steps (iii) to (v) at least once is carried out for a period in the range of 0.5 hours to 3 hours.

[0071] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the at least 90% hydrolysis of the substrate is achieved by repeating the steps (iii) to (v) at least once is carried out for a period in the range of 0.5 hours to 2 hours.

[0072] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the at least 90% hydrolysis of the substrate is achieved by repeating the steps (iii) to (v) at least once is carried out for a period of 1 hour.

[0073] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein recovering the at least one enzyme from the soluble fraction is carried out by a conventional method selected from the group consisting of adsorption, membrane filtration, chromatography, precipitation, aqueous two phase extraction, and combinations thereof.

[0074] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein recovering the at least one enzyme is carried out using membrane filtration selected from the group consisting of microfiltration, ultrafiltration, nanofiltration, and combinations thereof.

[0075] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the recovered enzyme in step (v), has a concentration in the range of 20 - 300 mg protein/gm of substrate. [0076] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the recovered enzyme in step (v), has a concentration in the range of 100 - 300 mg protein/gm of substrate.

[0077] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the recovered enzyme used for hydrolysis of substrate has concentration of at least two times higher than the concentration of at least one enzyme.

[0078] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the process is selected from the group consisting of batch process, fed-batch process, continuous process, and combinations thereof.

[0079] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein soluble fraction and second soluble fraction comprise fermentable sugars, enzyme and soluble sugars. In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate comprising the steps of: (i) obtaining a substrate slurry; (ii) contacting the substrate slurry with at least one enzyme to obtain a soluble fraction and an insoluble fraction; (iii) separating the soluble fraction from the insoluble fraction; (iv) recovering the at least one enzyme from the soluble fraction to obtain recovered enzymes; (v) treating the insoluble fraction, and optionally the substrate slurry with the recovered enzymes to obtain a second soluble and a second insoluble fraction; and (vi) repeating the steps (iii) to (v) on the second insoluble fraction so as to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours.

[0080] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate comprising the steps of: (i) obtaining a substrate slurry; (ii) contacting the substrate slurry with at least one enzyme to obtain a soluble fraction and an insoluble fraction; (iii) separating the soluble fraction from the insoluble fraction; (iv) recovering the at least one enzyme from the soluble fraction to obtain recovered enzymes; (v) treating the insoluble fraction, and optionally the substrate slurry with the recovered enzymes to obtain a second soluble and a second insoluble fraction; and (vi) repeating the steps (iii) to (v) on the second insoluble fraction so as to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 3 hours.

[0081] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate having a polysaccharide content in the range of 80 - 95%, the process comprising steps of: (i) obtaining a substrate slurry; (ii) contacting the substrate slurry with at least one enzyme to obtain a soluble fraction and an insoluble fraction; (iii) separating the soluble fraction from the insoluble fraction; (iv) recovering the at least one enzyme from the soluble fraction to obtain recovered enzymes; (v) treating the insoluble fraction, and optionally the substrate slurry with the recovered enzymes to obtain a second soluble and a second insoluble fraction; and (vi) repeating the steps (iii) to (v) on the second insoluble fraction so as to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours.

[0082] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate comprising the steps of: (i) obtaining a substrate slurry comprising the substrate having a concentration in the range of 2 - 35% with respect to the substrate slurry; (ii) contacting the substrate slurry with at least one enzyme to obtain a soluble fraction and an insoluble fraction; (iii) separating the soluble fraction from the insoluble fraction; (iv) recovering the at least one enzyme from the soluble fraction to obtain recovered enzymes; (v) treating the insoluble fraction, and optionally the substrate slurry with the recovered enzymes to obtain a second soluble and a second insoluble fraction; and (vi) repeating the steps (iii) to (v) on the second insoluble fraction so as to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours.

[0083] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate comprising the steps of: (i) obtaining a substrate slurry; (ii) contacting the substrate slurry with at least one enzyme is carried out at a temperature in the range of 40°C to 60°C at pH in the range of 4.5 to 5.5 to obtain a soluble fraction and an insoluble fraction; (iii) separating the soluble fraction from the insoluble fraction; (iv) recovering the at least one enzyme from the soluble fraction to obtain recovered enzymes; (v) treating the insoluble fraction, and optionally the substrate slurry with the recovered enzymes to obtain a second soluble and a second insoluble fraction; and (vi) repeating the steps (iii) to (v) on the second insoluble fraction so as to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours.

[0084] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate comprising the steps of: (i) obtaining a substrate slurry; (ii) contacting the substrate slurry with at least one enzyme is carried out for a period in the range of 0.5 hours to 3 hours to obtain a soluble fraction and an insoluble fraction; (iii) separating the soluble fraction from the insoluble fraction; (iv) recovering the at least one enzyme from the soluble fraction to obtain recovered enzymes; (v) treating the insoluble fraction, and optionally the substrate slurry with the recovered enzymes to obtain a second soluble and a second insoluble fraction; and (vi) repeating the steps (iii) to (v) on the second insoluble fraction so as to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours.

[0085] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate comprising the steps of: (i) obtaining a substrate slurry; (ii) contacting the substrate slurry with at least one enzyme at a concentration in the range of 10 to 100 mg protein/gm of substrate to obtain a soluble fraction and an insoluble fraction; (iii) separating the soluble fraction from the insoluble fraction; (iv) recovering the at least one enzyme from the soluble fraction to obtain recovered enzymes; (v) treating the insoluble fraction, and optionally the substrate slurry with the recovered enzymes to obtain a second soluble and a second insoluble fraction; and (vi) repeating the steps (iii) to (v) on the second insoluble fraction so as to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours.

[0086] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate comprising the steps of: (i) obtaining a substrate slurry; (ii) contacting the substrate slurry with at least one enzyme selected from the group consisting of cellulases, pectinases, amylases, maltases, xylanases, lactases, and combinations thereof to obtain a soluble fraction and an insoluble fraction; (iii) separating the soluble fraction from the insoluble fraction; (iv) recovering the at least one enzyme from the soluble fraction to obtain recovered enzymes; (v) treating the insoluble fraction, and optionally the substrate slurry with the recovered enzymes to obtain a second soluble and a second insoluble fraction; and (vi) repeating the steps (iii) to (v) on the second insoluble fraction so as to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours.

[0087] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate comprising the steps of: (i) obtaining a substrate slurry; (ii) contacting the substrate slurry with at least one enzyme to obtain a soluble fraction and an insoluble fraction; (iii) separating the soluble fraction from the insoluble fraction is carried by conventional method selected from the group consisting of filtration, centrifugation, and decantation; (iv) recovering the at least one enzyme from the soluble fraction to obtain recovered enzymes; (v) treating the insoluble fraction, and optionally the substrate slurry with the recovered enzymes to obtain a second soluble and a second insoluble fraction; and (vi) repeating the steps (iii) to (v) on the second insoluble fraction so as to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours.

[0088] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate comprising the steps of: (i) obtaining a substrate slurry; (ii) contacting the substrate slurry with at least one enzyme to obtain a soluble fraction and an insoluble fraction; (iii) separating the soluble fraction from the insoluble fraction; (iv) recovering the at least one enzyme from the soluble fraction to obtain recovered enzymes; (v) treating the insoluble fraction, and optionally the substrate slurry with the recovered enzymes is carried out at a temperature in the range of 40°C to 60°C with a pH in the range of 4.5 to 5.5 to obtain a second soluble and a second insoluble fraction ; and (vi) repeating the steps (iii) to (v) on the second insoluble fraction so as to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours. [0089] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate comprising the steps of: (i) obtaining a substrate slurry; (ii) contacting the substrate slurry with at least one enzyme to obtain a soluble fraction and an insoluble fraction; (iii) separating the soluble fraction from the insoluble fraction; (iv) recovering the at least one enzyme from the soluble fraction to obtain recovered enzymes; (v) treating the insoluble fraction, and optionally the substrate slurry with the recovered enzymes is carried out for a period in the range of 0.5 hours to 3 hours to obtain a second soluble and a second insoluble fraction; and (vi) repeating the steps (iii) to (v) on the second insoluble fraction so as to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours.

[0090] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate comprising the steps of: (i) obtaining a substrate slurry; (ii) contacting the substrate slurry with at least one enzyme to obtain a soluble fraction and an insoluble fraction; (iii) separating the soluble fraction from the insoluble fraction; (iv) recovering the at least one enzyme from the soluble fraction is carried out by a conventional method selected from the group consisting of adsorption, membrane filtration, chromatography, precipitation, aqueous two phase extraction, and combinations thereof to obtain recovered enzymes; (v) treating the insoluble fraction, and optionally the substrate slurry with the recovered enzymes to obtain a second soluble and a second insoluble fraction; and (vi) repeating the steps (iii) to (v) on the second insoluble fraction so as to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours.

[0091] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate comprising the steps of: (i) obtaining a substrate slurry; (ii) contacting the substrate slurry with at least one enzyme to obtain a soluble fraction and an insoluble fraction; (iii) separating the soluble fraction from the insoluble fraction; (iv) recovering the at least one enzyme from the soluble fraction is carried out using membrane filtration selected from the group consisting of microfiltration, ultrafiltration, nanofiltration, and combinations thereof to obtain recovered enzymes; (v) treating the insoluble fraction, and optionally the substrate slurry with the recovered enzymes to obtain a second soluble and a second insoluble fraction; and (vi) repeating the steps (iii) to (v) on the second insoluble fraction so as to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours.

[0092] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate comprising the steps of: (i) obtaining a substrate slurry; (ii) contacting the substrate slurry with at least one enzyme to obtain a soluble fraction and an insoluble fraction; (iii) separating the soluble fraction from the insoluble fraction; (iv) recovering the at least one enzyme from the soluble fraction to obtain recovered enzymes; (v) treating the insoluble fraction, and optionally the substrate slurry with the recovered enzymes having a concentration in the range of 20 - 300 mg protein/gm of substrate to obtain a second soluble and a second insoluble fraction; and (vi) repeating the steps (iii) to (v) on the second insoluble fraction so as to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours.

[0093] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate comprising the steps of: (i) obtaining a substrate slurry; (ii) contacting the substrate slurry with at least one enzyme to obtain a soluble fraction and an insoluble fraction; (iii) separating the soluble fraction from the insoluble fraction; (iv) recovering the at least one enzyme from the soluble fraction to obtain recovered enzymes; (v) treating the insoluble fraction, and optionally the substrate slurry with the recovered enzymes to obtain a second soluble and a second insoluble fraction; and (vi) repeating the steps (iii) to (v) on the second insoluble fraction so as to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours, wherein the process is a batch process, fed-batch process, continuous process, or combinations thereof.In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein complete hydrolysis of substrate can be achieved in multiple steps in a period in the range of 2 - 5 hours. [0094] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate having a polysaccharide content in the range of 80 - 95%, the process comprising the steps of: (i) obtaining a substrate slurry having a substrate concentration in the range of 2 - 35% with respect to the substrate slurry; (ii) contacting the substrate slurry with at least one enzyme at a concentration in the range of 10 to 100 mg protein/gm of substrate, at a temperature in the range of 40°C to 60°C, at pH in the range of 4.5 to 5.5, for a period in the range of 0.5 hours to 3 hours to obtain a soluble fraction and an insoluble fraction ; (iii) separating the soluble fraction from the insoluble fraction is carried by conventional method selected from the group consisting of filtration, centrifugation, and decantation; (iv) recovering the at least one enzyme from the soluble fraction to obtain recovered enzymes; (v) treating the insoluble fraction, and optionally the substrate slurry with the recovered enzymes having a concentration in the range of 20 - 300 mg protein/gm of substrate to obtain a second soluble and a second insoluble fraction is carried out at a temperature in the range of 40°C to 60°C with a pH in the range of 4.5 to 5.5 for a period in the range of 0.5 hours to 3 hours; and (vi) repeating the steps (iii) to (v) on the second insoluble fraction so as to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours.

[0095] In an embodiment of the present disclosure, there is provided a process for rapid hydrolysis of a substrate as described herein, wherein at least one enzyme provides selected from the cellulases, pectinases, amylases, maltases, xylanases, lactases, and combinations thereof, wherein enzyme has a concentration in the range of 10 - 100 mg/g of substrate. Further, at least one enzyme can be used in immobilized form, wherein immobilization of the enzyme is carried out by known conventional methods.

[0096] In an embodiment of the invention there is provided recovery of the enzyme, wherein enzyme is recovered by known conventional methods. Further recovered enzyme is reused and recycled repetitively to achieve complete hydrolyse the substrate. Further, recovered enzyme can be reused and recycled to hydrolyse fresh substrate or insoluble fraction (unhydrolysed substrate) or combination thereof.

[0097] In an embodiment of the present disclosure there is provided a process for rapid hydrolysis of a substrate as described herein, wherein the hydrolysis of the substrate slurry is done by at least one enzyme. The hydrolysis (partial or complete) is achieved in one or more steps to produce one or more fermentable sugars. In another embodiment, at least 30% of substrate is hydrolysed to obtain soluble fraction (hydrolysate) comprising at least 95% fermentable sugar/s. In another embodiment, at least 60% of substrate is hydrolysed in one or more steps to achieve hydrolysis. In yet another embodiment at least 70% of substrate is hydrolysed to obtain soluble fraction (hydrolysate) comprising at least 95% fermentable sugar. In still another embodiment at least 90% hydrolysis of the substrate is achieved in one or more steps.

[0098] Although the subject matter has been described in considerable detail with reference to certain preferred implementation thereof, other implementations are possible.

EXAMPLES

[0099] 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:

General procedure for calculation of percentage hydrolysis: [00100] The percentage of hydrolysis was calculated on the basis of sugars released during enzymatic conversion. Samples were withdrawn from every reaction tanks and the solid concentration was determined. Due to hydrolysis the solid concentration in the tanks were reduced. The sugars released was measured using multiple analytical techniques well known in the art and the total conversion was calculated by the formula given below:

■ S olub le 's obtaine d i n sup ernatant '. ■· . 3 . -,„„

Hydrolysis < % ) = ———— —— * 100

Total olo ce llulose add ed

[00101] The end point of hydrolysis was considered when the substrate concentration was reduced by 90%. In case of continuous system the solid concentration build-up was monitored, if no build-up was observed, then more than 90% hydrolysis was achieved.

Example 2

Mode of operation: Batch process for hydrolysis of substrate

Pre-treated rice straw having polysaccharide content in the range of 80 - 95 % was mixed with water to make slurry (2-15% w/v), the pH and the temperature of the slurry was maintained at 4.8 and 50°C respectively and added to the reaction tank. The enzyme solution containing 60 to 95 mg protein/g of substrate was added to the reaction tank to achieve more than 50% hydrolysis in one hour to obtain soluble fraction (as fermentable sugars, soluble sugars) and insoluble fraction as unhydrolysed substrate. Soluble fraction was separated from insoluble fraction by decantation. The soluble fraction obtained after decantation was passed through a microfiltration unit (0.2 um pore size) for separation of residual solids and the clear permeate was subjected to ultrafiltration (5 kDa) to separate the enzyme from soluble fraction. The insoluble fraction obtained after decantation was collected in a second reaction tank which also received the enzyme recovered after ultrafiltration in a concentrated manner. The recovered enzyme was recycled to hydrolyse the insoluble fraction obtained after decantation with at least two times higher enzyme loading as compared to the first reaction step therefore achieving complete hydrolysis. The second soluble fraction obtained after hydrolysis in the second reactor was recycled into the main reaction tank and the second soluble fraction was then subjected to ultrafiltration to separate the fermentable sugars from the enzyme which can be recycled for hydrolysis of fresh substrate. The schematic representation of the process is depicted in Figure 1 wherein the generalised process for rapid hydrolysis of the substrate slurry is demonstrated. The substrate slurry (2-35% w/v) is treated with enzyme to obtain a soluble fraction and an insoluble fraction; the enzymes are recovered from the soluble fraction and recycled to treat the insoluble fraction.

[00102] The results obtained from the batch process are summarised in

Table 1.

Table 1: Batch mode of operation

CC= Carbohydrate content; PH= Partial hydrolysis; TH= Total hydrolysis; EC= Enzyme concentration

Example 2:

Mode of operation: Fed batch process for hydrolysis of substrate

[00103] Pre-treated wheat straw having polysaccharide content in the range of 80 - 95 % was made into 5% w/v slurry of pH 4.8 and temperature 50°C. This slurry was added into the reaction tank containing 93 mg protein/g of substrate, to achieve rapid solubilization of the slurry. Further, the Pre-treated substrate slurry was continuously added in to the reaction tank for one hour to achieve up to 35% substrate concentration. After partial hydrolysis, the slurry was subjected to decantation to separate the soluble fraction from the insoluble fraction. The soluble fraction obtained after decantation was passed through microfiltration and ultrafiltration (5 kDa) to separate the enzyme from soluble sugars. The insoluble fraction obtained after decantation was hydrolysed at a higher enzyme concentration as compared to the first reaction step therefore achieving more than 70% hydrolysis. The second soluble fraction obtained after hydrolysis in the receiver tank was recycled into the main reaction tank and then subjected to ultrafiltration to separate the fermentable sugars from the enzyme which can be recycled for hydrolysis of any residual substrate or fresh substrate. Table 2 below mentions the percentage hydrolysis of the substrate obtained at different steps when the mode of operation was Fed batch.

Table 2: Fed-Batch process

CC= Carbohydrate content; PH= Partial hydrolysis; TH= Total hydrolysis; EC= Enzyme concentration

Example 3:

Mode of operation: Continuous process for hydrolysis of substrate

[00104] Pre-treated rice straw having polysaccharide content in the range of 80 - 95 % was used to prepare slurry of 25% w/v concentration with pH 4.8 and temperature 50°C. This slurry was added to the reaction tank, simultaneously enzyme solution containing 95 mg protein/g of substrate was added, to achieve 47.2% hydrolysis in one hour. After hydrolysis soluble fraction and insoluble fraction was separated by decantation. The soluble fraction was processed through the membrane filtration system to separate the fermentable sugars and concentrate the enzymes. The recovered concentrated enzyme was recycled to hydrolyse the insoluble fraction to obtain second soluble fraction and second insoluble fraction, wherein 81.52% hydrolysis was achieved and enzyme loading was at least two times higher than initial enzyme loading in the first hydrolysis step. The second soluble fraction obtained after second hydrolysis was further separated from second insoluble fraction. The second soluble fraction was subjected to filtration to separate the sugars and recover the enzyme. The recovered enzyme was used to hydrolyse second insoluble fraction to obtain third soluble fraction and third insoluble fraction, wherein 94.46% hydrolysis was achieved. The third soluble fraction (containing at least 95% fermentable sugars of hydrolysed substrate) was separated from third insoluble fraction by decantation. The % enzymatic hydrolysis of the substrate is presented in Table 3 below.

[00105] Table 3

CC= Carbohydrate content; PH= Partial hydrolysis; TH= Total hydrolysis; EC= Enzyme concentration

Example 4:

Mode of operation: Multi step hydrolysis for hydrolysis of substrate

[00106] Pre-treated sugarcane bagasse having polysaccharide content in the range of 80 - 95 % was used to prepare a slurry of 10% substrate concentration and pH 4.8 and temperature 50°C was prepared in a reaction tank. The reaction tank was filled with enzyme solution containing 95 mg protein/g of cellulose to achieve rapid solubilization of biomass. Fresh biomass was added to this reaction slurry intermittently for one hour to increase the substrate concentration to 35%. The slurry containing fermentable sugars, soluble sugars, unhydrolysed residue and the enzyme was subjected to decantation and membrane filtration to separate soluble sugars, enzymes and the insoluble residue. The recovered enzyme was reused to hydrolyse the residual biomass after removal of sugars with at least two times higher enzyme loading as compared to the first step to achieve 71.10% hydrolysis. The slurry was further subjected to decantation and filtration to separate the soluble sugars, enzyme and the residue. The insoluble fraction was again treated with the recovered after intermittent sugar removal to achieve a total hydrolysis yield of 97.17%. The sugars obtained after each step was combined and was composed of 95.42% fermentable sugars.

[00107] The sugars produced were continuously removed to avoid product inhibition, which otherwise results in lower conversion of substrate to sugars.

[00108] Table 4

Enzyme concentration

Example 5:

Mode of operation: Batch without decantation (control)

[00109] Pre-treated rice straw was mixed with water to make slurry (25% w/v), the pH and the temperature of the slurry was maintained at 4.8 and 50°C respectively and added to the reaction tank. The enzyme solution containing 95 mg protein/g of substrate was added to the reaction tank. Samples were withdrawn every two hour to monitor the reaction and the system was allowed to run for 8 hours. The reaction was then terminated and the released sugars were analysed. [00110] It is clear from the above process and also from Table 5 that if process steps of the instant disclosure were not followed there was decrease in the percentage hydrolysis.

[00111] Table 5

[00112] The present disclosure thus provides a process for rapid enzymatic hydrolysis of substrate in a step wise manner to achieve complete hydrolysis of substrate (at least 90%) in less than 5 hours. In the first step, substrate slurry is partially hydrolysed (at least 30%) with enzymes for time period in the range of 0.5 hours to 3 hours to obtain a soluble fraction and insoluble fraction, wherein the soluble fraction comprises at least 95% fermentable sugar of hydrolysed substrate. The enzymes are recovered from soluble fraction and used for hydrolysis of the insoluble fraction in additional step/s for time period in the range of 0.5 hours to 3 hours to obtain another soluble fraction and another insoluble fraction, and repeating these steps until complete hydrolysis of substrate (at least 90%) is achieved. The soluble fraction obtained after the complete hydrolysis comprises at least 95% fermentable sugar and insoluble fraction comprises less than 5% unhydrolysed substrate. Further, the process of the present disclosure also involves recovery and recycling of enzymes to hydrolyse insoluble fraction to achieve complete hydrolysis. The process also involves intermittent removal of the soluble fraction so as to avoid product feedback inhibition, and attain at least 90% hydrolysis of the substrate.

[00113] The advantage of the present process showed high efficiency in terms of conversion, shorter reaction time and better utilization of enzyme and hence improved economics than known in the art. [00114] Although the subject matter has been described in considerable detail with reference to certain examples and implementations thereof, other implementations are possible.

Claims

A process for rapid hydrolysis of a substrate comprising the steps of:

(i) obtaining a substrate slurry;

(ii) contacting the substrate slurry with at least one enzyme to obtain a soluble fraction and an insoluble fraction;

(iii) separating the soluble fraction from the insoluble fraction;

(iv) recovering the at least one enzyme from the soluble fraction to obtain recovered enzymes; and

(v) treating the insoluble fraction with the recovered enzymes to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours.

The process as claimed in claim 1, wherein treating the insoluble fraction with the recovered enzymes in step (v) results in a second soluble, and a second insoluble fraction.

The process as claimed in claim 1, wherein the at least 90% hydrolysis of the substrate is achieved by repeating the steps (iii) to (v) at least once.

The process as claimed in claim 1, wherein the at least 90% hydrolysis of the substrate is achieved by repeating the steps (ii) to (v) at least once, wherein the at least one enzyme in step (ii) is the recovered enzymes from step (iv).

The process as claimed in claim 1, wherein the substrate has a polysaccharide content in the range of 80 - 95%.

The process as claimed in claim 1, wherein the substrate slurry comprises substrate, wherein the substrate has a concentration in the range of 2 - 35% (w/v) with respect to the substrate slurry.

The process as claimed in claim 1, wherein contacting the substrate slurry with the at least one enzyme is carried out at a temperature in the range of 40°C to 60°C at pH in the range of 4.5 to 5.5.

8. The process as claimed in claim 1, wherein contacting the substrate slurry with at the least one enzyme is carried out for a period in the range of 0.5 hours to 3 hours.

9. The process as claimed in any of the claim 7 or 8, wherein the at least one enzyme is used at a concentration in the range of 10 to 100 mg protein/gm of substrate.

10. The process as claimed in claim 1, wherein the at least one enzyme is selected from the group consisting of cellulases, pectinases, amylases, maltases, xylanases, lactases, and combinations thereof.

11. The process as claimed in claim 1, wherein separating the soluble fraction from the insoluble fraction is carried by conventional method selected from the group consisting of filtration, centrifugation, and decantation.

12. The process as claimed in claim 1, wherein treating the insoluble fraction with the recovered enzymes to achieve at least 90% hydrolysis of the substrate is carried out at a temperature in the range of 40°C to

60°C with a pH in the range of 4.5 to 5.5.

13. The process as claimed in claim 1, wherein treating the insoluble fraction with the recovered enzymes to achieve at least 90% hydrolysis of the substrate is carried out for a period in the range of 0.5 hours to 3 hours.

14. The process as claimed in claim 2, wherein treating the insoluble fraction in step (v) results in a second soluble, and a second insoluble fraction is carried out at a temperature in the range of 40°C to 60°C with a pH in the range of 4.5 to 5.5.

15. The process as claimed in claim 2, wherein treating the insoluble fraction in step (v) results in a second soluble, and a second insoluble fraction is carried out for a period in the range of 0.5 hours to 3 hours. 16. The process as claimed in claim 3, wherein the at least 90% hydrolysis of the substrate is achieved by repeating the steps (iii) to (v) at least once is carried out at a temperature in the range of 40°C to 60°C with a pH in the range of 4.5 to 5.5.

17. The process as claimed in claim 3, wherein the at least 90% hydrolysis of the substrate is achieved by repeating the steps (iii) to (v) at least once is carried out for a period in the range of 0.5 hours to 3 hours.

18. The process as claimed in claim 1, wherein recovering the at least one enzyme from the soluble fraction is carried out by a conventional method selected from the group consisting of adsorption, membrane filtration, chromatography, precipitation, aqueous two phase extraction, and combinations thereof.

19. The process as claimed in claim 18, wherein recovering the at least one enzyme is carried out using membrane filtration selected from the group consisting of microfiltration, ultrafiltration, nanofiltration, and combinations thereof.

20. The process as claimed in claim 1, wherein the recovered enzyme in step (v), has a concentration in the range of 20 - 300 mg protein/gm of substrate.

21. The process as claimed in any of the claims 1-20, wherein the process is selected from the group consisting of batch process, fed-batch process, continuous process, and combinations thereof.

22. A process for rapid hydrolysis of a substrate comprising the steps of:

(i) obtaining a substrate slurry;

(ii) contacting the substrate slurry with at least one enzyme to obtain a soluble fraction and an insoluble fraction;

(iii) separating the soluble fraction from the insoluble fraction;

(iv) recovering the at least one enzyme from the soluble fraction to obtain recovered enzymes;

(v) treating the insoluble fraction, and optionally the substrate slurry with the recovered enzymes to obtain a second soluble and a second insoluble fraction; and (vi) repeating the steps (iii) to (v) on the second insoluble fraction so as to achieve at least 90% hydrolysis of the substrate in a time period in the range of 2 - 5 hours.

23. The process as claimed in claim 1, 2, 3 or 22, wherein soluble fraction and second soluble fraction comprise fermentable sugars, enzyme and soluble sugars.

24. The process as claimed in claim 23, wherein the fermentable sugars is more than 95%.