JPWO2021011881A5

JPWO2021011881A5 -

Info

Publication number: JPWO2021011881A5
Application number: JP2022502510A
Authority: JP
Publication date: 2023-07-11

Claims

An immobilized enzyme composition for the preparation of hexoses, wherein at least two, at least three, at least four, at least five of the following enzymes immobilized on at least one carrier or mixture of carriers: , at least 6 carriers, at least 7, or at least 8:
a) alpha glucan phosphorylase (αGP), phosphoglucomutase (PGM), and optionally 1,4-glucan transferase (4-GT), and b) an enzyme from among a combination of enzymes selected from :
(i) phosphoglucoisomerase (PGI), fructose-6-phosphate epimerase (F6PE), and tagatose-6-phosphate phosphatase (T6PP) for preparing tagatose;
(ii) phosphoglucoisomerase (PGI), picose-6-phosphate epimerase (P6PE), and picose-6-phosphate phosphatase (P6PP) for the preparation of allulose;
(iii) phosphoglucoisomerase (PGI), P6PE, allose-6-phosphate isomerase (A6PI), and allose-6-phosphate phosphatase (A6PP) for the preparation of allose;
(iv) phosphoglucoisomerase (PGI), mannose-6-phosphate isomerase (M6PI) or phosphoglucose/phosphomannose isomerase (PGPMI), and mannose 6-phosphate phosphatase (M6PP) for the preparation of mannose;
(v) phosphoglucoisomerase (PGI), F6PE, galactose 6-phosphate isomerase (Gal6PI), and galactose 6-phosphate phosphatase (Gal6PP) for the preparation of galactose;
(vi) PGI and fructose 6-phosphate phosphatase (F6PP) for the preparation of fructose;
(vii) PGI, P6PE, altrose 6-phosphate isomerase (Alt6PI), and altrose 6-phosphate phosphatase (Alt6PP) for the preparation of altrose;
(viii) PGI, F6PE, talose 6-phosphate isomerase (Tal6PI), and talose 6-phosphate phosphatase (Tal6PP) for the preparation of talose;
(ix) PGI, F6PE, sorbose 6-phosphate epimerase (S6PE), and sorbose 6-phosphate phosphatase (S6PP) for the preparation of sorbose;
(x) PGI, F6PE, S6PE, gulose 6-phosphate isomerase (Gul6PI), and gulose 6-phosphate phosphatase (Gul6PP) for preparing gulose;
(xi) PGI, F6PE, S6PE, idose 6-phosphate isomerase (I6PI), and idose 6-phosphate phosphatase (I6PP) to prepare idose, and (xii) inositol 3 to prepare inositol - phosphate synthase (IPS) and inositol monophosphatase (IMP).

2. The immobilized enzyme composition of claim 1, wherein the weight (w/w) % of each enzyme relative to the total weight of said enzymes ranges from 0.1% to 40%.

10-30% (αGP), 0-10% (4GT), 10-30% (PGM) and, if present, 0.1-10% (PGI). enzyme composition.

4. The immobilized enzyme composition of claim 3 , further comprising PGI, F6PE, and T6PP to prepare tagatose.

The weight (w/w)% of each enzyme relative to the total weight of the enzyme is 10-30% (αGP), 0-10% (4GT), 10-30% (PGM), 0.1-10% (PGI ), 15-35% (F6PE), and T6PP (25-45%), wherein the total weight of enzymes in said composition totals 100% w/w relative to the total weight of said enzymes. Item 5. The immobilized enzyme composition according to item 4.

the αGP comprises the amino acid sequence of SEQ ID NO: 1, or a fragment thereof;
the 4-GT comprises the amino acid sequence of SEQ ID NO: 6, or a fragment thereof;
the PGM comprises the amino acid sequence of SEQ ID NO: 2, or a fragment thereof;
the PGI comprises the amino acid sequence of SEQ ID NO: 3, or a fragment thereof;
said F6PE comprises the amino acid sequence of SEQ ID NO: 4, or a fragment thereof, and
wherein said T6PP comprises the amino acid sequence of SEQ ID NO: 5, or a fragment thereof;
6. The immobilized enzyme composition of claim 5.

4. The immobilized enzyme composition of claim 3 , further comprising PGI, P6PE, and P6PP to prepare allulose.

The weight (w/w)% of each enzyme relative to the total weight of the enzyme is 10-30% (αGP), 0-10% (4GT), 10-30% (PGM), 0.1-10% (PGI ), 0.1-10% (P6PE), and (45-65%) P6PP, wherein the total weight of enzymes in the composition totals 100% w/w based on the total weight of the enzymes. 8. The immobilized enzyme composition of claim 7.

the αGP comprises the amino acid sequence of SEQ ID NO: 1, or a fragment thereof;
the 4-GT comprises the amino acid sequence of SEQ ID NO: 6;
the PGM comprises the amino acid sequence of SEQ ID NO: 2;
the PGI comprises the amino acid sequence of SEQ ID NO: 3;
said P6PE comprises the amino acid sequence of SEQ ID NO: 7, and
wherein the P6PP comprises the amino acid sequence of SEQ ID NO:8;
9. The immobilized enzyme composition of claim 8.

The immobilized enzyme composition according to any one of claims 1 to 9, wherein the total weight ( W/W ) % of said enzyme relative to the weight of said carrier is between 2.5% and 12.5%.

The immobilized enzyme composition of any one of claims 1-10, wherein the carrier is a weakly basic anion exchange resin.

12. The immobilized enzyme composition of claim 11, wherein said carrier comprises phenol formaldehyde polycondensate.

13. The immobilized enzyme composition of claim 11 or 12, wherein said carrier comprises a tertiary amine functional group and said composition is optionally DUOLITE(TM) A568.

13. The immobilized enzyme composition of claim 11 or 12, wherein the carrier comprises secondary amine functional groups and the composition is optionally DUOLITE(TM) PWA7.

The immobilized enzyme composition of any one of claims 1-10, wherein the carrier comprises a His-tag affinity resin.

Contacting a starch derivative with an immobilized enzyme composition according to any one of claims 1 to 15 under suitable reaction conditions to convert said starch derivative to a hexose. enzymatic process to

wherein said process steps are carried out at temperatures ranging from about 40° C. to about 85° C., pH ranging from about 5.0 to about 8.0, and/or from about 0.5 hours to about 48 hours. 17. The process of paragraph 16 .

18. The process of claim 16 or 17 , wherein the process steps are performed in a single bioreactor, multiple bioreactors arranged in series, or multiple bioreactors arranged in parallel.

the steps of the process are performed at a phosphate concentration of about 0.1 mM to about 150 mM, ATP-free, NAD(P)(H)-free, wherein the phosphate is recycled within the enzymatic cascade reaction; and /or at least one step of said process involves a highly energetically favorable chemical reaction.