PROCESS FOR PREPARING TAGATOZA FROM BIOMASS

Abstract

The invention relates to a process for the preparation of tagatose from sugars derived from various substrates such as biomass or any carbohydrate source. The invention comprises methods for the preparation of tagatose using a series of enzymes and enzyme fusion to convert biomass such as, but not limited to, starch, maltose, maltodextrin, cellulose, sucrose from juice, as well as monosaccharides such as, but not limited to, glucose and / or fructose, and similar. The invention also focuses on the use of enzymes in single form and / or in fusion with the enzyme(s) involved in sequential steps, to achieve the economical and efficient conversion of sugars from biomass to tagatose.

Description

[0001] “PROCESS FOR PREPARTION OF TAGATOSE FROM BIOMASS”

[0002] TECHNICAL FIELD OF THE INVENTION:

[0003] The present invention relates to a process for preparation of tagatose from sugars derived from biomass or any carbohydrate source. The invention includes the methods of Tagatose production using series of enzymes for conversion of biomass such as and not limited to Starch, maltose, maltodextrin, cellulose, sucrose from juice and monosaccharide sugar such as and not limited to Glucose and / or fructose, etc. The invention also focuses on application of enzymes in alone and / or in fusion with enzyme(s) involved in sequential step, for the economical, efficient conversion of biomass sugars to Tagatose.

[0004] BACKGROUND AND PRIOR ART OF THE INVENTION:

[0005] Tagatose is a low calorie, natural sweetener with 92% sweetness of sucrose, giving only 38% calories (1.5 kcal / g). It is a naturally occurring monomeric saccharide, ketose sugar. Tagatose naturally occurs in very low amounts in fruits, cacao, and dairy products. Tagatose is used as a bulk sweetener, and like other sugars (sucrose, glucose, fructose) it is also used as a humectant, texturizer and stabilizer. It is also useful in formulating dietetic foods with a low glycemic index, used in ready-to eat breakfast cereals, diet soft drinks, low / non-fat ice cream and frozen yogurt, hard and soft confectioneries, frostings, chewing gum, formula diets for meal replacement, and dietary supplements. Tagatose is stable in the foods to which it is added during the processing and storage of food

[0006] GRAS Notice (GRN) No. 977 document describes the cascade enzymatic methodology for Tagatose production, wherein each enzyme conversion was performed as individual reaction using immobilized enzymes, referring to the patent WO2017 / 059278A1. The enzymes are derived from non-pathogenic source microbes, and hence making the production process safe. WO2017 / 059278A1 discloses method for tagatose production from biomass derived sugars, by series of conversion steps catalysed by cascade of enzymes. Sugar phosphate route of sugar conversion was achieved using isomerase, epimerase and phosphatases enzyme.

[0007] Some of the major drawbacks of the existing conventional method for converting tagatose from biomass using fusion enzyme comprising

[0008] 1) Loss of sugar product intermediates in each purification step of respective enzyme conversion. More loss is anticipated in case of more number of process steps

[0009] 2) The loss of sugar product leading to reduction in yield of Tagatose production.

[0010] Hence, the present inventors have come up with a cost-effective and high conversion rate process for preparation of tagatose from sugars derived from biomass using fusion enzyme, which that addresses all the concerns and limitations of the prior art. This remains the objective of the invention.

[0011] SUMMARY OF THE INVENTION:

[0012] In accordance with the above objective the present invention provides a short, cost-effective and high conversion rate process for Tagatose production using series of enzymes for conversion biomass such as and not limited to Starch, maltose, maltodextrin, cellulose, sucrose from juice and monosaccharide sugar such as and not limited to Glucose and / or fructose, etc.

[0013] In an aspect, the present invention provides a short, cost effective and high conversion rate process for the preparation of tagatose from sugar derived from biomass using fusion enzyme comprsing;

[0014] 1. Converting the biomass to starch;

[0015] 2. Converting starch of step (1) to Glucose 1 -phosphate ; 3. Converting Glucose 1 -Phosphate of step (2) to glucose-6-phosphate

[0016] 4. Converting glucose-6-phosphate of step (3) to fructose-6-phosphate;

[0017] 5. Converting fructose-6-phosphate of step (4) to tagatose-6-phosphate; and

[0018] 6. Converting tagatose-6-phosphate of step (5) to Tagatose.

[0019] In another aspect, the present invention provides the application of enzymes alone and / or in fusion with enzyme(s) involved in sequential step, for the economical, efficient conversion of biomass sugars to Tagatose.

[0020] In another aspect, the present invention provides the application of enzymes alone and / or in fusion with enzyme(s) involved in sequential step, for the economical, efficient conversion of biomass sugars to Tagatose.

[0021] In an aspect, the present invention provides a process of sequential enzymatic conversions for efficient production of Tagatose from Biomass substrates such as and not limited to sucrose, starch, cellulose using series of enzymes in single or in combinations as fusion proteins.

[0022] In another aspect, the enzymes involved in the sequential conversion steps are either single or in fusion with subsequent enzyme(s) catalysing two or multiple enzyme conversions using single fusion enzymes, thereby reducing the number of steps.

[0023] In another aspect, the present invention provides a short, cost effective and high conversion rate process of sequential enzymatic conversions for efficient production of Tagatose from Biomass substrates such as and not limited to sucrose, starch, cellulose using series of enzymes alone or in combinations as fusion proteins / enzymes.

[0024] In another aspect, the present invention provides a short, cost effective and high conversion rate process for the preparation of tagatose from sugar derived from biomass using fusion enzyme comprising combination of the steps: a. Converting the carbohydrate source to a mixture of glucose and fructose in the presence of extracellular enzymes selected from invertase; b. Converting the glucose in the mixture of step (a) to fructose through enzymatic conversion in the presence of Glucose isomerase (GI) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 1; c. Converting the fructose obtained in step (b) to fructose 6 phosphate in the presence of phosphor fructokinase (PPFK) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 2; d. Converting the fructose 6 phosphate produced in the step (c) to Tagatose 6- phosphate in the presence of Fructose: Tagatose eldolase (GATY) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 6; and e. Converting the Tagatose 6-phosphate obtained in step (d) to Tagatose in the presence of enzyme Tagatose 6 phosphate phosphatase (T6PP) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 7

[0025] In another aspect, the process for the preparation of Tagatose from the said carbohydrate substrate using fusion enzyme comprising combination of the steps: a. Converting the carbohydrate source to glucose using extracellular enzymes selected from invertase, b. Converting the glucose produced in the step (a) in the presence of polyphosphate to glucose 6-phosphate (G6P) by the enzyme glucokinase comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 2 c. Converting the glucose 6-phosphate (G6P) produced in step (b) to fructose 6 phosphate (F6P) in the presence of phosphogluco isomerase (PGI) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 5 d. Converting the fructose 6 phosphate produced in the step (c) to Tagatose 6- phosphate in the presence of Fructose: Tagatose eldolase (GATY) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 6; and e. Converting the Tagatose 6-phosphate obtained in step (d) to Tagatose in the presence of enzyme Tagatose 6 phosphate phosphatase (T6PP) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 7

[0026] In another aspect, the process for the preparation of Tagatose from carbohydrate source such as and not limited to Starch, Maltose, Amltodextrin, glucan, using fusion enzyme comprising combination of the steps: a. Converting the carbohydrate source to glucose- 1 phosphate in the presence of glycogen / glucan phosphorylase (GP) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 3, with or without addition Pullulanase (SEQ ID 8) for enhancing starch conversion; b. converting the glucose- 1 phosphate obtained in step (a) to glucose 6- phosphate in the presence of the enzyme phosphoglucomutase (PGM) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 4; c. converting the glucose 6-phosphate (G6P) produced in step (b) to fructose 6 phosphate (F6P) in the presence of phosphoglucoisomerase (PGI) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 5; d. Converting the fructose 6 phosphate produced in the step (c) to Tagatose 6- phosphate in the presence of Fructose: Tagatose eldolase (GATY) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 6; and e. Converting the Tagatose 6-phosphate obtained in step (d) to Tagatose in the presence of enzyme Tagatose 6 phosphate phosphatase (T6PP) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ

[0027] ID: 7

[0028] In another aspect, the process for the preparation of Tagatose from carbohydrate source such as and not limited to cellulose, cellobiose, using fusion enzyme comprising combination of the steps: a. Converting the carbohydrate source to glucose using extracellular enzymes selected from Cellulase, Exo and Endo glucanase, Glucosidase, and the like; b. Converting the glucose produced in the step (a) in the presence of polyphosphate to glucose 6-phosphate (G6P) by the enzyme glucokinase comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 2; c. converting the glucose 6-phosphate (G6P) produced in step (b) to fructose 6 phosphate (F6P) in the presence of phosphoglucoisomerase (PGI) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 5; d. Converting the fructose 6 phosphate produced in the step (c) to Tagatose 6- phosphate in the presence of Fructose: Tagatose eldolase (GATY) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 6; and e. Converting the Tagatose 6-phosphate obtained in step (d) to Tagatose in the presence of enzyme Tagatose 6 phosphate phosphatase (T6PP) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 7.

[0029] In yet another aspect, the enzymes used in sequential steps of bioconversion are generated as fusion protein using linkers such as and not limited to flexible linker, rigid linker, and the like.

[0030] In another aspect, the fusion enzymes are introduced in the reaction mixture in either free or immobilized form or combination thereof. In yet another aspect, the enzymes reaction is carried out in the presence of inorganic polyphosphate or ATP.

[0031] In yet another aspect, the said enzymes are used in free or immobilized form or combination thereof.

[0032] In yet another aspect, the enzymes are added as individual enzyme and / or fusion enzymes, either in a single pot reaction or in sequential enzyme / fusion nzyme addition to achieve tagatose production.

[0033] In yet another aspect, the present invention provides the process for the preparation of the fusion enzymes includes combination of the steps: a. Preparing recombinant cells for expressing the recombinant fusion proteins encoding the enzymes Fructose: Tagatose aldolase and Tagatose 6 phosphate phosphatase; b. Culturing the recombinant cells at 37°C followed by inducing fusion protein expression under different concentration of IPTG and at a temperature of 20- 30°C; and c. Extracting and isolating the fusion proteins from culture by causing cell lysis and after separation of soluble and insoluble fractions.

[0034] In another aspect, the present invention provides process of glucose generation from cellulose or sucrose or conversion of fructose to glucose, and further conversion to Tagatose by series of enzyme conversions.

[0035] In another aspect, the present invention provides a process that involves multi- step bioconversion of biomass sugars to tagatose using the fusion enzymes for step 2 and 3, and another fusion enzyme for step 4 and 5.

[0036] In yet another aspect, the present invention discloses desiging of fusion enzyme by fusing more than one enzyme using flexible or rigid linkers. In an another aspect, the present invention provides the thermostsble enzymes which are derived from non-pathogenic microbes, either in native form or in truncated / modified form.

[0037] DETAILED DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 : Depcts the flowchart with the enzymes involved in conversion of biomass sugars to Turanose production, wherein the biomass / biomass sugars includes but not limited to starch, maltose, maltodextrin, cellulose, sucrose, glucose, fructose, etc.

[0039] Figure 2: Depicts the expression profile of Tagatose production enzymes Fructose : Tagatose aldolase (GATY) and Tagatose 6-phosphate phosphatase (T6PP), as separated on SDS-PAGE gel and stained by Coomassie stain.

[0040] Figure 3: Depicts the appearance of Tatose 6-phosphate (T6P) by the conversion of Fructose-6-phosphate (F6P) catalysed by GATY enzyme, as observed on HPLC chromatogram (separation of sugar phosphates)

[0041] Figure 4: Depicts the confirmation of Tagatose formed from enzyme conversion reaction analysed against standard Tagatose sugar, as observed form HPLC chromatogram (separation of sugars)

[0042] Figure 5: Depicts novel process of Tagatose production from Biomass

[0043] DETAILED DESCRIPTION OF THE INVENTION

[0044] The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

[0045] The present invention provides a short and cost effective process for preparation of the alternate sugars such as tagatose from the sugars derived from biomass using fusion enzymes in high yield which the prior art processes have failed to achieve.

[0046] The present invention details the economical, efficient conversion process of Tagatose production using series of enzymes for conversion of biomass such as and not limited to Starch, maltose, maltodextrin, cellulose, sucrose from juice and monosaccharide sugar such as and not limited to Glucose and / or fructose, etc.

[0047] The invention also focuses on application of enzymes alone and / or in fusion with enzyme(s) involved in sequential step, for the economical and efficient conversion of biomass sugars to Tagatose.

[0048] Accordingly, the present invention relates to the short and cost effective process for the preparation of tagatose from biomass using fusion enzyme comprsing,

[0049] 1. Converting the biomass to starch;

[0050] 2. Converting starch of step (1) to Glucose 1 -phosphate;

[0051] 3. Converting Glucose 1 -phosphate of step (2) to glucose-6-phosphate;

[0052] 4. Converting glucose-6-phosphate of step (3) to fructose-6-phosphate;

[0053] 5. Converting fructose-6-phosphate of step (4) to tagatose-6-phosphate; and

[0054] 6. Converting tagatose-6-phosphate of step (5) to tagatose.

[0055] In an embodiment, the present invention provides a process of sequential enzymatic conversions for efficient production of Tagatose from Biomass substrates such as and not limited to sucrose, starch, cellulose using series of enzymes in single or in combinations as fusion proteins.

[0056] In another embodiement, the enzymes involved in the sequential conversion steps are either single or in fusion with subsequent enzyme(s) catalysing two or multiple enzyme conversions using single fusion enzymes, thereby reducing the number of steps. In yet another embodiment, the present invention provides a short, cost effective and high conversion rate process for the preparation of tagatose from sugar derived from biomass using fusion enzyme comprising combination of the steps: a. Converting the carbohydrate source to a mixture of glucose and fructose in the presence of extracellular enzymes selected from invertase; b. Converting the glucose in the mixture of step (a) to fructose through enzymatic conversion in the presence of Glucose isomerase (GI) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 1; c. Converting the fructose obtained in step (b) to fructose 6 phosphate in the presence of phosphor fructokinase (PPFK) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 2; d. Converting the fructose 6 phosphate produced in the step (c) to Tagatose 6- phosphate in the presence of Fructose: Tagatose eldolase (GATY) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 6; and e. Converting the Tagatose 6-phosphate obtained in step (d) to Tagatose in the presence of enzyme Tagatose 6 phosphate phosphatase (T6PP) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 7.

[0057] In another embodiment, the process for the preparation of tagatose from said carbohydrate substrate using fusion enzyme comprises combination of the steps: a. Converting the carbohydrate source to glucose using extracellular enzymes selected from invertase; b. Converting the glucose produced in the step (a) in the presence of polyphosphate to glucose 6-phosphate (G6P) by the enzyme glucokinase comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 2; c. Converting the glucose 6-phosphate (G6P) produced in step (b) to fructose 6 phosphate (F6P) in the presence of phosphogluco isomerase (PGI) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 5; d. Converting the fructose 6 phosphate produced in the step (c) to Tagatose 6- phosphate in the presence of Fructose: Tagatose eldolase (GATY) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 6; e. Converting the Tagatose 6-phosphate obtained in step (d) to Tagatose in the presence of enzyme Tagatose 6 phosphate phosphatase (T6PP) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 7;

[0058] In another embodiment, the process for the preparation of tagatose from carbohydrate source such as and not limited to Starch, Maltose, Amltodextrin, glucan, etc., using fusion enzyme comprising combination of the steps: a. Converting the carbohydrate source to glucose- 1 phosphate in the presence of glycogen / glucan phosphorylase (GP) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 3, with or without addition Pullulanase (SEQ ID 8) for enhancing starch conversion b. Converting the glucose- 1 phosphate obtained in step (a) to glucose 6- phosphate in the presence of the enzyme phosphoglucomutase (PGM) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 4 c. Converting the glucose 6-phosphate (G6P) produced in step (b) to fructose 6 phosphate (F6P) in the presence of phosphoglucoisomerase (PGI) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 5 d. Converting the fructose 6 phosphate produced in the step (c) to Tagatose 6- phosphate in the presence of Fructose: Tagatose eldolase (GATY) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 6 e. Converting the Tagatose 6-phosphate obtained in step (d) to Tagatose in the presence of enzyme Tagatose 6 phosphate phosphatase (T6PP) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 7

[0059] In another embodiment, the process for the preparation of tagatose from sugar derived from carbohydrate source such as and not limited to cellulose, cellobiose, etc., using fusion enzyme comprises combination of the steps: a. Converting the carbohydrate source to glucose using extracellular enzymes selected from Cellulase, Exo and Endo glucanase, Glucosidase, etc. b. Converting the glucose produced in the step (a) in the presence of polyphosphate to glucose 6-phosphate (G6P) by the enzyme glucokinase comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 2 c. Converting the glucose 6-phosphate (G6P) produced in step (b) to fructose 6 phosphate (F6P) in the presence of phosphoglucoisomerase (PGI) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 5 d. Converting the fructose 6 phosphate produced in the step (c) to Tagatose 6- phosphate in the presence of Fructose: Tagatose eldolase (GATY) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 6 e. Converting the Tagatose 6-phosphate obtained in step (d) to Tagatose in the presence of enzyme Tagatose 6 phosphate phosphatase (T6PP) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 7 In yet another embodiment, the enzymes used in sequential steps of bioconversion are generated as fusion protein using linkers such as and not limited to flexible linker, rigid linker, etc.

[0060] In another embodiment, the fusion enzymes are introduced in the reaction mixture in either free or immobilized form or combination thereof.

[0061] In another embodiment, the enzymes reaction is carried out in the presence of inorganic polyphosphate or ATP.

[0062] In yet another embodiment, the said enzymes are used in free or immobilized form or combination thereof.

[0063] In another embodiment, the enzymes are added as individual enzyme and / or fusion enzymes, either in a single pot reaction or in sequential enzyme / fusion nzyme addition to achieve tagatose production.

[0064] In yet another embodiment, the present invention provides the process for the preparation of the fusion enzymes includes combination of the steps: a. Preparing recombinant cells for expressing the recombinant fusion proteins encoding the enzymes Fructose: Tagatose aldolase and Tagatose 6 phosphate phosphatase; b. Culturing the recombinant cells at 37°C followed by inducing fusion protein expression under different concentration of IPTG and at a temperature of 20- 30°C; and c. Extracting and isolating the fusion proteins from culture by causing cell lysis and after separation of soluble and insoluble fractions.

[0065] In another embodiment, the process of the present invention involves reduced multi-step bioconversion of tagatose from biomass by application of fusion enzyme and combinations therof.

[0066] In an embodiment, the present invention discloses the designing fusion enzymes by fusing more than one enzyme using flexible or rigid linkers. In another embodiment, the enzymes are modified for parameters including but not limited to enhanced activity, thermotolerance, shift in equilibrium of substrate and product, etc., wherein the modification may include and not limited to point mutation, truncation, domain swapping, etc.

[0067] In an embodiment, the present invention discloses the thermostable enzymes which are derived from non-pathogenic microbe.

[0068] In another embodiment, the present invention discloses the reducing equilibrium constraints of each enzyme by fusing the enzymes together with linker, thereby product formed in first step will be continuously taken up as substrate for second step and so on. This drives the conversion flow in forward direction leading increased product formation.

[0069] The Novel process of Tagatose production from Biomass is an Enzymatic conversion of multi-step process, involves in-house enzymes. Enzymes are unique in the sense of efficient conversion of glucose and related sugars to form Tagatose, which generally is produced from Galactose.

[0070] Enzymes are used in immobilized form as a processing aid in sugar conversion, thereby reducing protein contamination in final sugar product. Fusion enzymes have advantages in immobilized form, where in process steps are reduced and loss of intermediates is minimized.

[0071] Another embodiment of the invention describes the advantages of the fusion enzymes generation and usage in series of Bioconversion reactions for production of Tagatose. The enzymes in series of steps either as individual enzymes or as fusion enzymes results in a simple process for the production of D-Tagatose with following advantages:

[0072] 1. The invention helps in overcoming the equilibrium limitations of conventional isomerization and epimerization processes and results in higher conversion efficiencies. 2. The number of enzymes required for the production of Tagatose from glucose / fructose is reduced.

[0073] 3. The process does not require the utilization of expensive ATP and complex systems for its regeneration.

[0074] 4. Multiple complex and expensive separation systems for sugars and other intermediates is avoided.

[0075] 5. Higher conversion rate of raw material to Tagatose is possible as the reaction equilibrium is overcome.

[0076] 6. Provides benefit on operational cost reduction, due to the reduced number of process steps.

[0077] Examples

[0078] Example 1: E.coli expression of Tagatose production enzymes including Fructose: Tagatose aldolase (GATY) and Tagatose 6-phosphate phosphatase (T6PP) as individual enzyme:

[0079] Enzymes meant for Tagatose production including Fructose:Tagatose aldolase (GATY) and Tagatose 6-phosphate phosphatase (T6PP), were expressed as recombinant protein from E.coli expression host cells. Protein expression was under control of IPTG induction (T7lac promoter). Protein expression was induced by expression under different concentration of IPTG and at 25°C (culture growth at 37°C).

[0080] Protein expression from culture analysed by cell lysis and separation of soluble and insoluble fractions. Sample analysis was performed by electrophoresis on 10% polyacrylamide gel under standard conditions, as shown in figure 2.

[0081] Example 2: Enzyme activity for conversion of Tagatose from biomass substrate, starch as an example of biomass, using series of enzyme addition:

[0082] Enzymes meant for starch conversion to Tatose including Glucan phosphorylase (GP), Phospho gluco mutase (PGM), Phospho gluco isomerase (PGI) and GATY, T6PP were used in conversion reaction in single pot reaction both by enzyme addition at a time and by sequential addition of enzymes

[0083] Result: single pot reaction of both processes gave similar results, as per the sugar and sugar phosphate analysis and HPLC analysis (as depicted in figure 3 and 4).

[0084] Example 3:

[0085] Generation of GATY and T6PP with linker fragment, as an example of Fusion enzyme generation:

[0086] Inorder to reduce the number of process steps, enzymes involved in sugar conversion to Tagatose, were fused in combination via linker sequence to generate Fusion proteins. As an example of fusion enzyme, GATY and T6PP were fused in following combinations using the linker sequences,

[0087] • Rigid Linker: A(EAAAK)4ALEA(EAAAK)4A

[0088] • Flexible Peptide Linker: (GGGGS)s

[0089] The gene fusion combinations are, i. GATY C-terminal - Linker - T6PP N-terminal sequence ii. T6PP C-terminal - Linker - GATY N-terminal sequence iii. GATY C-terminal - Linker - T6PP C-terminal sequence iv. GATY N-terminal - Linker - T6PP N-terminal sequence

[0090] Fusion constructs generated at gene level were expressed under IPTG inducible lac promoter to get the fusion enzyme GATY-T6PP.

[0091] Result: Based on activity analysis, fusion enzyme of the order “GATY C-terminal - Linker - T6PP N-terminal sequence” with flexible linker showed active conversion of F6P to T6P to Tagatose.

[0092] SEQUENCE LISTING:

[0093] Sequence Listing Information:

[0094] DTD Version: Vl_3

[0095] File Name: Sequence lisitng_202341018195.xml

[0096] Software Name: WIPO Sequence

[0097] Software Version: 2.3.0

[0098] Production Date: 2024-09-17

[0099] General Information:

[0100] Current application / IP Office: IN

[0101] Current application / Application number: 202341018195

[0102] Current application / Filing date: 2023-09-17

[0103] Current application / Applicant file reference: GNANLex_18195

[0104] Earliest priority application / IP Office: IN

[0105] Earliest priority application / Application number: 202341018195

[0106] Earliest priority application / Filing date: 2023-09-17

[0107] Applicant name: Fertis India Pvt. Ltd.

[0108] Applicant name / Language: en

[0109] Invention title: PROCESS FOR PREPARTION OF TAGATOSE FROM

[0110] BIOMASS ( en )

[0111] Sequence Total Quantity: 8

[0112] Sequences:

[0113] Sequence Number (ID): 1

[0114] Length: 388

[0115] Molecule Type: AA

[0116] Features Location / Qualifiers: - source, E.388

[0117] > mol type, protein

[0118] > organism, Isomerase

[0119] Residues:

[0120] MSYQPTPEDR FTFGLWTVGW QGRDPFGDAT RRALDPVETV

[0121] QRLAELGAHG VTFHDDDLIP 60

[0122] FGSSDTERES HIKRFRQALD ATGMTVPMAT TNLFTHPVFK

[0123] DGAFTANDRD VRRYALRKTI 120

[0124] RNIDLAAELG AKTYVAWGGR EGAESGAAKD VRSALDRMKE

[0125] AFDLLGEYVT SQGYDLRFAI 180

[0126] EPKPNEPRGD ILLPTVGHAL AFIERLERPE LYGVNPEVGH

[0127] EQMAGLNFPH GIAQALWAGK 240

[0128] LFHIDLNGQS GIKYDQDLRF GAGDLRSAFW LVDLLESAGY

[0129] EGPRHFDFKP PRTEDLDGVW 300

[0130] ASAAGCMRNY LILKERAAAF RADPEVQAAL RASRLDQLAQ

[0131] PTAADGLEDL LADRAAFEDF 360

[0132] DVEAAAARGM AFERLDQLAM DHLLGARG 388

[0133] Sequence Number (ID): 2

[0134] Length: 273

[0135] Molecule Type: AA

[0136] Features Location / Qualifiers:

[0137] - source, 1..273

[0138] > mol type, protein

[0139] > organism, Polyphosphate fructokinase Residues:

[0140] MTTHTSTAQR SSKYPTTIGI DVGGTGMKGG LVRLGSGPKA

[0141] GTLRGDRFRI PTPQPAMPEP 60

[0142] VAATLAAITA ELDGREKAPK PSAPLGVCFP SIVKDGVCLS

[0143] ANNIDHSWIG TDLRGRFEEH 120 LRRPVTVVND ADAAGLAEAR YGAGRGVRGL VHVITLGTGI

[0144] GGAMIHDGVL VPNFEVGSLE 180

[0145] LDGVMAESRA SAKARERDQL SWAEYAERLQ RFFSHVERIF

[0146] SPDLFIVGGG ISKRPDDYLP 240

[0147] LLRLRTPIVP AQLQNNAGIV GAALAAHGSL ADG 273

[0148] Sequence Number (ID): 3

[0149] Length: 819

[0150] Molecule Type: AA

[0151] Features Location / Qualifiers:

[0152] - source, 1..819

[0153] > mol type, protein

[0154] > organism, Glucan phosphorylase Residues:

[0155] MNVLGRITAM PDLPEPLEGL KEIAYNLWWS WNPEAAELFQ

[0156] ELDPALWKRF RGNPVKLLLE 60

[0157] LDPARLEALS ASGYAARVQA TREALRAYLE ARRTKRGPLV

[0158] AYFSAEYGFH SSLPIYAGGL 120

[0159] GVLAGDHVKA ASDLGLNLVG VGLFYHEGYF HQRLSPEGEQ

[0160] VEVYEPLRPE ELPLVPVQDA 180

[0161] EGRPVRVAVE FPGRLVHVGG YRVQVGAVPV YLLTTDLPEN

[0162] APEDRQITAR LYAAGLEARI 240

[0163] QQELVLGLGG VRFLRALGLA PAFFHMNEGH SAFLGLERLR

[0164] ELVAEGYPFR EALELVRASA 300

[0165] LFTTHTPVPA GHDVFPLDLV DRYLGGFWEK LGVDRDTFLG

[0166] LGLEEKPWGP VFSMSNLALR 360

[0167] TAAQANGVSR LHGEVSRNMF RHLWPGLLGE EVPIGHVTNG

[0168] VHTWTFLHPR LRRHYAEVFG 420

[0169] PEWVERPEDP ETWRVEGLGE AFWRIRQDLK LFLVREVRQR

[0170] LYEQRRRNGE GPARLREAEK 480 ALDPEALTIG FARRFATYKR AVLLFKDPER LLRILKGPYP

[0171] VQFVFAGKAH PKDEAGKAYL 540

[0172] KELVSKIREY GLEDRMVVLE DYDMYLARVL THGSDVWLNT

[0173] PRRPMEASGT SGMKAALNGA 600

[0174] LNLSVLDGWW AEAYNGKNGF AIGDERVYES EEAQDVADAQ

[0175] ALYDLLESEV IPLFYAKGLE 660

[0176] GYSSGWMSMV HESLRTVGPY FSAGRMVRDY LALYERGALW

[0177] EKEARARLEA LKAFAEALPA 720

[0178] FHALGVRPEV PGDLTLNGGR LEVGAVLEGE VPEGLRPHLR

[0179] VQLVVRRLGG GLEVVDLEEV 780

[0180] APGRYRTAFR PTRPGSYTYG LRLALLHPVT GRVEWVRWA

[0181] 819

[0182] Sequence Number (ID): 4

[0183] Length: 456

[0184] Molecule Type: AA

[0185] Features Location / Qualifiers:

[0186] - source, 1..456

[0187] > mol type, protein

[0188] > organism, Phospho gluco mutase

[0189] Residues:

[0190] MGKLFGTFGV RGIANEEITP EFALKIGMAF GTLLKREGRE

[0191] RPLVWGRDT RVSGEMLKDA 60

[0192] LISGLLSTGC DVIDVGIAPT PAIQWATNHF NADGGAVITA

[0193] SHNPPEYNGI KLLEPNGMGL 120

[0194] KKEREAIVEE LFFSEDFHRA KWNEIGELRK EDIIKPYIEA

[0195] IKNRVDVEAI KKRRPFVVVD 180

[0196] TSNGAGSLTL PYLLRELGCK VVSVNAHPDG HFPARNPEPN

[0197] EENLKGFMEI VKALGADFGV 240

[0198] AQDGDADRAV FIDENGRFIQ GDKTFALVAD AVLRENGGGL

[0199] LVTTIATSNL LDDIAKRNGA 300 KVMRTKVGDL IVARALLENN GTIGGEENGG VIFPDFVLGR

[0200] DGAMTTAKIV EIFAKSGKKF 360

[0201] SELIDELPKY YQFKTKRHVE GDRKAIVAKV AELAEKKGYK

[0202] IDTTDGTKII FDDGWVLVRA 420

[0203] SGTEPIIRIF SEAKSEEKAR EYLELGIKLL EEALKG 456

[0204] Sequence Number (ID): 5

[0205] Length: 189

[0206] Molecule Type: AA

[0207] Features Location / Qualifiers:

[0208] - source, 1..189

[0209] > mol type, protein

[0210] > organism, Phospho gluco isomerase

[0211] Residues:

[0212] MYKEPFGVKV DFETGIIEGA KKSVRRLSDM EGYFVDERAW

[0213] KELVEKEDPV VYEVYAVEQE 60

[0214] EKEGDLNFAT TVLYPGKVGK EFFFTKGHFH AKLDRAEVYV

[0215] ALKGKGGMLL QTPEGDAKWI 120

[0216] SMEPGTVVYV PPYWAHRTVN IGDEPFIFLA IYPADAGHDY

[0217] GTIAEKGFSK IVIEENGEVK 180

[0218] VVDNPRWKK 189

[0219] Sequence Number (ID): 6

[0220] Length: 429

[0221] Molecule Type: AA

[0222] Features Location / Qualifiers:

[0223] - source, 1..429

[0224] > mol type, protein

[0225] > organism, Fructose:Tagatose aldolase (GATY)

[0226] Residues: MSPQNPLIGL FKNREKEFKG IISVCSSNEI VLEAVLKRMK

[0227] DTNLPIIIEA TANQVNQFGG 60

[0228] YSGLTPSQFK ERVIKIAQKV DFPLERIILG GDHLGPFVWR

[0229] DQEPEIAMEY AI<QMII<EYII< 120

[0230] AGFTKIHIDT SMPLKGENSI DDEIIAKRTA VLCRIAEECF EKISINNPYI

[0231] TRPVYVIGAD 180

[0232] VPPPGGESSI CQTITTKDEL ERSLEYFKEA FKKEGIEHVF

[0233] DYVVAVVANF GVEFGSDEIV 240

[0234] DFDMEKVKPL KELLAKYNIV FEGHSTDYQT KENLKRMVEC

[0235] GIAILKVGPA LTFTLREALV 300

[0236] ALSHIEEEIY SNEKEKLSRF REVLLNTMLT CKDHWSKYFD

[0237] ENDKLIKSKL LYSYLDRWRY 360

[0238] YFENESVKSA VYSLIGNLEN VKIPPWLVSQ YFPSQYQKMR

[0239] KKDLKNGAAD LILDKIGEVI 420

[0240] DHYVYAVKE 429

[0241] Sequence Number (ID): 7

[0242] Length: 223

[0243] Molecule Type: AA

[0244] Features Location / Qualifiers:

[0245] - source, 1..223

[0246] > mol type, protein

[0247] > organism, Tagatose 6-phosphate phosphatase (T6PP)

[0248] Residues:

[0249] MFKPKAIAVD IDGTLTDRKR ALNCRAVEAL RKVKIPVILA

[0250] TGNISCFARA AAKLIGVSDV 60

[0251] VICENGGVVR FEYDGEDIVL GDKEKCVEAV RVLEKHYEVE

[0252] LLDFEYRKSE VCMRRSFDIN 120

[0253] EARKLIEGMG VKLVDSGFAY HIMDADVSKG KALKFVAERL

[0254] GISSAEFAVI GDSENDIDMF 180 RVAGFGIAVA NADERLKEYA DLVTPSPDGE GWEALQFLG LLR

[0255] 223

[0256] Sequence Number (ID): 8

[0257] Length: 718

[0258] Molecule Type: AA

[0259] Features Location / Qualifiers:

[0260] - source, 1..718

[0261] > mol type, protein

[0262] > organism, Pullulanase

[0263] Residues:

[0264] MLHISRTFAA YLDEMDQIVV LAPKSLGFDG MAPLTLVAPS

[0265] GEEIPLSVQH VEDCGETVKY 60

[0266] VCRFASAFEF GATYWVRSCR GEETDVQIGA VVRTPAFDDR

[0267] FFYDGPLGVE YSKEQAVFRV 120

[0268] WAPTATAVNV KLVHPHLGEI RCVPLERGEC GVWSAAVPGD

[0269] WERACYTYIA CINRVWREAV 180

[0270] DPYATAVSIN GEFGVVIDWE KTKLTPPSSP LPPLCSPTDA

[0271] ILYELSIRDF TSHPDSGAVH 240

[0272] KGKYLGLAET NTSGPNGTAT GLSYVKELGV THVQLMPFMD

[0273] FAGVDERDPQ AAYNW GYNPL 300

[0274] HLYAPEGSYA TDPADPYARI VELKQAIHTL HENGLRVVMD

[0275] AVYNHVYDRE QSPLEKLVPG 360

[0276] YYFRYDAYGQ PANGTGVGND IASERRMARR WIVDSVVFWA

[0277] KEYGLDGFRF DLMGVHDIET 420

[0278] MKAVRDALDT IDPSILVYGE GWDLPTPLPP EQKATMANAN

[0279] QLPRFAYFND RFRDAVKGST 480

[0280] FHLPDRGFAL GNPGGREQVK LAIAGSLRAL GGLFCHPRQS

[0281] INYVECHDNH TFWDKMEAAN 540

[0282] HDEPEWLRRK RQKLATAIVL LAQGIPFLHS GQEFYRTKGG

[0283] DGNSYRSPDA VNQLDWGRKS 600 RYEDDVRYVQ GLIALRRAHG AFRLATEAEV LRHLTFLEPL

[0284] PPSVIAYRLH DVAVYGPWDE 660

[0285] IIVIHHNEEK KEAIHLPDER EWDIVCDGQR SGAAPFRRVR

[0286] GRLELDGIGT WVLVKTDA 718

Claims

WE CLAIM:

1. A short, cost effective and high conversion rate process of sequential enzymatic conversions for efficient production of Tagatose from Biomass substrates such as and not limited to sucrose, starch, cellulose using series of enzymes alone or in combinations as fusion proteins / enzymes.

2. The process as claimed in claim 1, wherein the enzymes involved in the sequential conversion steps are either alone or in fusion with subsequent enzyme(s) catalysing two or multiple enzyme conversions using fusion enzymes, thereby reducing the number of steps.

3. The process sas claimed in claim 1, wherein the production of Tagatose from sugar derived from from carbohydrate source such as and not limited to sucrose or juice, using fusion enzyme comprising combination of the steps: a. Converting the carbohydrate source to a mixture of glucose and fructose in the presence of extracellular enzymes selected from invertase; b. Converting the glucose in the mixture of step (a) to fructose through enzymatic conversion in the presence of Glucose isomerase (GI) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 1; c. Converting the fructose obtained in step (b) to fructose 6 phosphate in the presence of phosphor fructokinase (PPFK) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 2; d. Converting the fructose 6 phosphate produced in the step (c) to Tagatose 6-phosphate in the presence of Fructose: Tagatose eldolase (GATY) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 6; and e. Converting the Tagatose 6-phosphate obtained in step (d) to Tagatose in the presence of enzyme Tagatose 6 phosphate phosphatase (T6PP) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 7.

4. The process as claimed in claim 1, wherein the production of Tagatose from the said carbohydrate substrate using fusion enzyme comprising combination of the steps: a. Converting the carbohydrate source to glucose using extracellular enzymes selected from invertase; b. Converting the glucose produced in the step (a) in the presence of polyphosphate to glucose 6-phosphate (G6P) by the enzyme glucokinase comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 2; c. Converting the glucose 6-phosphate (G6P) produced in step (b) to fructose 6 phosphate (F6P) in the presence of phosphogluco isomerase (PGI) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 5; d. Converting the fructose 6 phosphate produced in the step (c) to Tagatose 6-phosphate in the presence of Fructose: Tagatose eldolase (GATY) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 6; and e. Converting the Tagatose 6-phosphate obtained in step (d) to Tagatose in the presence of enzyme Tagatose 6 phosphate phosphatase (T6PP) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 7.

5. The process as claimed in claim 1, wherein the production of Tagatose from carbohydrate source such as and not limited to Starch, Maltose, Amltodextrin, glucan, using fusion enzyme comprising combination of the steps: a. Converting the carbohydrate source to glucose- 1 phosphate in the presence of glycogen / glucan phosphorylase (GP) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 3, with or without addition Pullulanase (SEQ ID 8) for enhancing starch conversion; b. converting the glucose- 1 phosphate obtained in step (a) to glucose 6- phosphate in the presence of the enzyme phosphoglucomutase (PGM)comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 4; c. converting the glucose 6-phosphate (G6P) produced in step (b) to fructose 6 phosphate (F6P) in the presence of phosphoglucoisomerase (PGI) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 5; d. Converting the fructose 6 phosphate produced in the step (c) to Tagatose 6-phosphate in the presence of Fructose: Tagatose eldolase (GATY) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 6; and e. Converting the Tagatose 6-phosphate obtained in step (d) to Tagatose in the presence of enzyme Tagatose 6 phosphate phosphatase (T6PP) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 7.

6. The process as claimed in claim 1, wherein the production of Tagatose from carbohydrate source such as and not limited to cellulose, cellobiose, using fusion enzyme comprising combination of the steps: a. Converting the carbohydrate source to glucose using extracellular enzymes selected from Cellulase, Exo and Endo glucanase, Glucosidase, and the like; b. Converting the glucose produced in the step (a) in the presence of polyphosphate to glucose 6-phosphate (G6P) by the enzyme glucokinase comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 2; c. converting the glucose 6-phosphate (G6P) produced in step (b) to fructose 6 phosphate (F6P) in the presence of phosphoglucoisomerase (PGI) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 5; d. Converting the fructose 6 phosphate produced in the step (c) to Tagatose 6-phosphate in the presence of Fructose: Tagatose eldolase (GATY)comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 6; and e. Converting the Tagatose 6-phosphate obtained in step (d) to Tagatose in the presence of enzyme Tagatose 6 phosphate phosphatase (T6PP) comprising the amino acid sequence which has atleast 85% sequence similarity to SEQ ID: 7.

7. The process as claimed in claim 1, wherein the enzymes used in sequential steps of bioconversion are generated as fusion protein using linkers such as and not limited to flexible linker, rigid linker, and the like.

8. The process as claimed in claim 1, wherein the fusion enzymes are introduced in the reaction mixture in either free or immobilized form or combination thereof.

9. The process as claimed in claim 1, wherein the enzymes reaction is carried out in the presence of inorganic polyphosphate or ATP.

10. The process as claimed in claim 1, wherein the said enzymes are used in free or immobilized form or combination thereof.

11. The process as claimed in claim 1 to 6, wherein the enzymes are added as individual enzyme and / or fusion enzymes, either in a single pot reaction or in sequential enzyme / fusion enzyme addition to achieve tagatose production.

12. The process as claimed in claim 1, wherein the process for the preparation of the fusion enzymes comprises: a. Preparing recombinant cells for expressing the recombinant fusion proteins encoding the enzymes Fructose: Tagatose aldolase and Tagatose 6 phosphate phosphatase; b. Culturing the recombinant cells at 37°C followed by inducing fusion protein expression under different concentration of IPTG and at a temperature of 20- 30°C; and c. Extracting and isolating the fusion proteins from culture by causing cell lysis and after separation of soluble and insoluble fractions.