CN112442521A

CN112442521A - Method for fixing carbon compound by catalyzing xylose with enzyme method

Info

Publication number: CN112442521A
Application number: CN201910823381.6A
Authority: CN
Inventors: 游淳; 李运杰; 李国玮
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2019-09-02
Filing date: 2019-09-02
Publication date: 2021-03-05
Anticipated expiration: 2039-09-02
Also published as: CN112442521B

Abstract

The invention discloses a method for fixing a carbon compound by using xylose, belonging to the field of enzyme catalysis. The invention takes xylose or biomass hydrolysate rich in xylose and a carbon compound (formaldehyde and methanol) as substrates, and the substrates are efficiently converted into fructose 6-phosphate by in vitro multi-enzyme molecular machine catalysis in a multi-enzyme reaction system. The method adopts an in-vitro multi-enzyme catalytic system to fix the carbon compound, obviously improves the conversion efficiency of the raw materials compared with a microbial method, and has the advantages of high reaction speed and simple and convenient operation. By adopting the method for fixing the carbon compound by using the xylose, the invention further discloses a preparation method of a series of high value-added products, which comprise fructose 6-phosphate, fructose 1,6-diphosphate, inositol, psicose, mannose and tagatose.

Description

Method for fixing carbon compound by catalyzing xylose with enzyme method

Technical Field

The invention belongs to the field of enzyme catalysis, and mainly relates to a method for preparing a high value-added product by catalyzing xylose and a carbon compound through enzyme catalysis.

Background

In recent years, efficient use of monocarbon compounds has received much attention. The formaldehyde, the methanol, the formic acid and the methane are all optional raw materials with abundant reserves and low price. Microbially immobilized one-carbon compounds are produced by catalyzing formaldehyde and Ribulose5-phosphate to hexulose6-phosphate mainly through the Ribulose monophosphate pathway (RuMP), specifically, hexulose6-phosphate synthase (HPS), and hexulose6-phosphate is isomerized into fructose 6-phosphate by the action of hexulose6-phosphate isomerase (PHI), and fructose 6-phosphate enters the central metabolic pathway and participates in various metabolic activities. Most studies are currently carried out to produce chemicals using naturally occurring methylotrophic bacteria with methanol or other reduced monocarbon compounds as the sole carbon and energy source, but with very low yields and lacking in gene manipulation tools. Engineering bacteria such as escherichia coli, corynebacterium glutamicum, saccharomyces cerevisiae and the like which can use methanol as a unique carbon source are researched and constructed, but efficient strains which can completely use methanol as a unique carbon source for growth and production are not obtained.

Biomass is a renewable energy source, and is widely distributed and abundant in reserves. At present, a great amount of straws are burnt in situ or used as fuel, which not only causes waste of resources, but also causes environmental pollution, so how to efficiently utilize the biomass becomes a research hotspot. Xylose is one of the major components of biomass, and accounts for about 20-30% of its dry weight. The straws are treated by acid hydrolysis or gas explosion, and a large amount of xylose can be released by controlling the treatment conditions, but the xylose can be mostly utilized by microorganisms after being detoxified by a biological method or a physical method.

The in vitro multi-enzyme reaction catalysis platform has the advantages of high product yield, high reaction speed, simple reaction process, easy operation, certain toxicity tolerance and the like, so that development and construction of an in vitro multi-enzyme catalysis reaction system are urgently needed, and a non-grain carbon source xylose is adopted to fix a carbon compound to prepare high-value-added products such as fructose 6-phosphate, fructose 1,6-diphosphate, inositol, psicose, mannose, tagatose and the like.

Disclosure of Invention

In order to solve the above problems, the present invention provides a method for immobilizing a carbon compound using xylose, comprising the steps of:

(1) converting xylose into xylulose by adopting xylose isomerase as an enzyme; and

(2) xylulose is converted into xylulose-5-phosphate under the catalysis of xylulokinase and polyphosphate kinase; and

(3) ribulose-5-phosphate is converted into ribulose-5-phosphate by adopting the catalysis of ribulose-phosphate 3-epimerase; and

(4) ribulose5-phosphate and formaldehyde are converted into 6-phosphohexulose by adopting 6-phosphohexulose synthetase for catalysis; and

(5) the 6-hexulose phosphate is converted into fructose 6-phosphate by using 6-hexulose phosphate isomerase as an enzyme.

Further, the method comprises the step of converting methanol to formaldehyde, wherein the converting step is catalyzed by methanol dehydrogenase, NADH oxidase or by alcohol oxidase, catalase.

By adopting the method for fixing the carbon compound by xylose, the invention further provides a series of high value-added products, including a preparation method of fructose 6-phosphate, fructose 1,6-diphosphate, inositol, psicose, mannose and tagatose, wherein the preparation method comprises the following steps:

(1) provides a method for preparing fructose 6-phosphate by catalyzing xylose and formaldehyde through in vitro multi-enzyme reaction:

xylose and formaldehyde are used as substrates, Xylose isomerase (Xylose isomerase, EC 5.3.1.5), Xylulokinase (Xylulokinase, EC2.7.1.17), Ribulose phosphate 3-epimerase (Ribulose-phosphate 3-epimerase, EC 5.1.3.1), 6-hexulose phosphate synthase (3-hexulose6-phosphate synthase, EC4.1.2.43), 6-hexulose phosphate isomerase (6-phospho3-hexuloisomerase, EC 5.3.1.27), Polyphosphate kinase (EC 2.7.4.1) are added to establish a multi-enzyme reaction system, and enzyme catalytic reaction is carried out. Polyphosphate kinase is added into a reaction system, and the aim is to use cheap polyphosphate to replace expensive Adenosine Triphosphate (ATP) so as to greatly reduce the production cost.

In the experiment for converting xylose and formaldehyde into fructose 6-phosphate by in vitro multi-enzyme catalysis, in a reaction system, the concentrations of xylose and formaldehyde are respectively 1-1000mM, the dosage of xylose isomerase is 0.1-10000U/mL, the dosage of xylulokinase is 0.1-10000U/mL, the dosage of ribulose phosphate 3-epimerase is 0.1-10000U/mL, the dosage of 6-phosphohexulose synthase is 0.1-10000U/mL, the dosage of 6-phosphohexulose isomerase is 0.1-10000U/mL, and the dosage of polyphosphate kinase is 0.1-10000U/mL.

Preferably, the concentration of xylose is 50mM, the concentration of formaldehyde is 60mM, the dosage of xylose isomerase is 10U/mL, the dosage of xylulokinase is 10U/mL, the dosage of ribulose phosphate 3-epimerase is 10U/mL, the dosage of 6-hexulose phosphate synthase is 10U/mL, the dosage of 6-hexulose phosphate isomerase is 10U/mL, and the dosage of polyphosphate kinase is 10U/mL.

The enzyme catalysis reaction system is carried out at the temperature of 20-90 ℃ and the reaction time is 1-100 hours.

The multi-enzyme reaction system also comprises buffer solution, magnesium salt, manganese salt, polyphosphoric acid and Adenosine Diphosphate (ADP).

Preferably, the buffer solution is Tris-HCl buffer solution, the pH value of the buffer solution is 4.0-9.0, and the concentration of the buffer solution is 20-500 mM; magnesium salt is magnesium sulfate with concentration of 0.01-500 mM; manganese salt is manganese sulfate, and the concentration is 0.01-500 mM; the polyphosphate is sodium polyphosphate with the concentration of 5-500 mM; adenosine Diphosphate (ADP) at a concentration of 0.01-500 mM;

it will be understood by those skilled in the art that various buffers can be used in the present invention, such as HEPES buffer, Tris-HCl buffer, MOPS buffer, citrate buffer, phosphate buffer, etc.; various polyphosphoric acids and polyphosphates may be used in the present invention, such as sodium hexametaphosphate, sodium polyphosphate, and the like; various magnesium and manganese salts can be used in the present invention, such as magnesium chloride, magnesium sulfate, manganese chloride, manganese sulfate, and the like. The source of xylose can be pure xylose or biomass hydrolysate containing a large amount of xylose.

(2) Provides a method for preparing fructose 1,6-diphosphate by catalyzing xylose and formaldehyde through in vitro multi-enzyme reaction:

xylose isomerase (Xylose isomerase, EC 5.3.1.5), Xylulokinase (Xylulokinase, EC2.7.1.17), Ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1), 6-phosphohexulose synthase (3-hexose 6-phosphosynthase, EC4.1.2.43), 6-phosphohexuloisomerase (6-phosphohexuloisomerase, EC 5.3.1.27), Polyphosphate kinase (EC 2.7.4.1) and 6-phosphofructokinase (6-phosphofructokinase, EC 2.7.1.11) were added as substrates to establish a multienzyme reaction system, and enzyme-catalyzed reaction was carried out.

In the experiment for converting xylose and formaldehyde into fructose 1,6-diphosphate by in vitro multi-enzyme catalysis, in a reaction system, the concentrations of xylose and formaldehyde are respectively 1-1000mM, the dosage of xylose isomerase is 0.1-10000U/mL, the dosage of xylulokinase is 0.1-10000U/mL, the dosage of ribulose phosphate 3-epimerase is 0.1-10000U/mL, the dosage of 6-phosphohexulose synthase is 0.1-10000U/mL, the dosage of 6-phosphohexuloisomerase is 0.1-10000U/mL, the dosage of 6-phosphofructokinase is 0.1-10000U/mL, and the dosage of polyphosphate kinase is 0.1-10000U/mL.

Preferably, the concentration of xylose is 50mM, the concentration of formaldehyde is 60mM, the dosage of xylose isomerase is 10U/mL, the dosage of xylulokinase is 10U/mL, the dosage of ribulose phosphate 3-epimerase is 10U/mL, the dosage of 6-hexulose phosphate synthase is 10U/mL, the dosage of 6-hexulose phosphate isomerase is 10U/mL, the dosage of 6-phosphofructokinase is 10U/mL, and the dosage of polyphosphate kinase is 10U/mL.

(3) Provides a method for preparing inositol by catalyzing xylose and formaldehyde through in vitro multi-enzyme reaction:

xylose isomerase (Xylose isomerase, EC 5.3.1.5), Xylulokinase (Xylulokinase, EC2.7.1.17), Ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1), 6-phosphohexulose synthase (3-hexose 6-phosphosynthase, EC4.1.2.43), 6-phosphohexulose isomerase (6-phosphohexuloisomerase, EC 5.3.1.27), glucose phosphate isomerase (phosphoisomerase, EC 5.3.1.9), Inositol 1-phosphate synthase (Inositol-1-phosphosynthase, EC5.5.1.4) and Inositol monophosphatase (Inositol monophosphokinase, EC3.1.3.25), Polyphosphate kinase (Polyphosphate EC2.7.4.1) were added as substrates to carry out a reaction.

In the experiment for converting xylose and formaldehyde into inositol through in-vitro multi-enzyme catalysis, in a reaction system, the concentrations of xylose and formaldehyde are respectively 1-1000mM, the dosage of xylose isomerase is 0.1-10000U/mL, the dosage of xylulokinase is 0.1-10000U/mL, the dosage of ribulose phosphate 3-epimerase is 0.1-10000U/mL, the dosage of 6-phosphohexulose synthase is 0.1-10000U/mL, the dosage of 6-phosphohexulose isomerase is 0.1-10000U/mL, the dosage of phosphoglucose isomerase is 0.1-10000U/mL, the dosage of inositol 1-phosphate synthase is 0.1-10000U/mL, the dosage of inositol monophosphatase is 0.1-10000U/mL, and the dosage of polyphosphate kinase is 0.1-10000U/mL.

Preferably, the concentration of xylose is 50mM, the concentration of formaldehyde is 60mM, the dosage of xylose isomerase is 10U/mL, the dosage of xylulokinase is 10U/mL, the dosage of ribulose phosphate 3-epimerase is 10U/mL, the dosage of 6-phosphohexulose synthase is 10U/mL, the dosage of 6-phosphohexulose isomerase is 10U/mL, the dosage of phosphoglucose isomerase is 10U/mL, the dosage of inositol 1-phosphate synthase is 10U/mL, the dosage of phytase is 10U/mL, and the dosage of polyphosphate kinase is 10U/mL.

(4) Provides a method for preparing allulose by catalyzing xylose and formaldehyde through in vitro multi-enzyme reaction:

xylose isomerase (Xylose isomerase, EC 5.3.1.5), Xylulokinase (Xylulokinase, EC2.7.1.17), Ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1), 6-phosphohexulose synthase (3-hexulose 6-phosphosynthase, EC4.1.2.43), 6-phosphohexulose isomerase (6-phosphohexuloisomerase, EC 5.3.1.27), Polyphosphate kinase (EC 2.7.4.1), psicose 6-phosphate 3-epimerase (D-alloulose 6-phosphosynthase, EC 5.1.3.) were added as substrates to perform a phosphatase-catalyzed reaction.

In the above-mentioned multienzyme reaction system, the phosphatase may be phytase, alkaline phosphatase, acid phosphatase, psicose 6-phosphate specific phosphatase, preferably psicose 6-phosphate specific phosphatase.

In the experiment for converting xylose and formaldehyde into psicose by in vitro multi-enzyme catalysis, in a reaction system, the concentrations of xylose and formaldehyde are respectively 1-1000mM, the dosage of xylose isomerase is 0.1-10000U/mL, the dosage of xylulokinase is 0.1-10000U/mL, the dosage of ribulose phosphate 3-epimerase is 0.1-10000U/mL, the dosage of 6-phosphohexulose synthase is 0.1-10000U/mL, the dosage of 6-phosphohexulose isomerase is 0.1-10000U/mL, the dosage of polyphosphate kinase is 0.1-10000U/mL, the dosage of psicose 6-phosphohexulose 3-epimerase is 0.1-10000U/mL, and the dosage of phosphatase is 0.1-10000U/mL.

Preferably, the concentration of xylose is 50mM, the concentration of formaldehyde is 60mM, the dosage of xylose isomerase is 10U/mL, the dosage of xylulokinase is 10U/mL, the dosage of ribulose phosphate 3-epimerase is 10U/mL, the dosage of 6-hexulose phosphate synthase is 10U/mL, the dosage of 6-hexulose phosphate isomerase is 10U/mL, the dosage of polyphosphate kinase is 10U/mL, the dosage of psicose 6-phosphate 3-epimerase is 10U/mL, and the dosage of phosphatase is 10U/mL.

(5) Provides a method for preparing mannose by catalyzing xylose and formaldehyde through in vitro multi-enzyme reaction:

xylose and formaldehyde are used as substrates, Xylose isomerase (Xylose isomerase, EC 5.3.1.5), Xylulokinase (Xylulokinase, EC2.7.1.17), Ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1), 6-hexulose phosphate synthase (3-hexulose6-phosphate synthase, EC4.1.2.43), 6-hexulose phosphate isomerase (6-phospho3-hexuloisomerase, EC 5.3.1.27), Polyphosphate kinase (EC 2.7.4.1), Mannose 6-phosphate isomerase (manose 6-phosphoisomerase, EC 5.3.1.8) are added, and phosphatase establishes a multienzyme reaction system to perform enzyme catalytic reaction.

In the above-mentioned multi-enzyme reaction system, the phosphatase may be phytase, alkaline phosphatase, acid phosphatase, mannose 6-phosphate specific phosphatase, preferably mannose 6-phosphate specific phosphatase.

In the experiment for converting xylose and formaldehyde into mannose by in vitro multi-enzyme catalysis, in a reaction system, the concentrations of xylose and formaldehyde are respectively 1-1000mM, the dosage of xylose isomerase is 0.1-10000U/mL, the dosage of xylulokinase is 0.1-10000U/mL, the dosage of ribulose phosphate 3-epimerase is 0.1-10000U/mL, the dosage of 6-phosphohexulose synthase is 0.1-10000U/mL, the dosage of 6-phosphohexulose isomerase is 0.1-10000U/mL, the dosage of polyphosphate kinase is 0.1-10000U/mL, the dosage of mannose 6-phosphoisomerase is 0.1-10000U/mL, and the dosage of phosphatase is 0.1-10000U/mL.

Preferably, the concentration of xylose is 50mM, the concentration of formaldehyde is 60mM, the dosage of xylose isomerase is 10U/mL, the dosage of xylulokinase is 10U/mL, the dosage of ribulose phosphate 3-epimerase is 10U/mL, the dosage of 6-hexulose phosphate synthase is 10U/mL, the dosage of 6-hexulose phosphate isomerase is 10U/mL, the dosage of polyphosphate kinase is 10U/mL, the dosage of mannose 6-phosphate isomerase is 10U/mL, and the dosage of phosphatase is 10U/mL.

(6) Provides a method for preparing tagatose by catalyzing xylose and formaldehyde through in vitro multi-enzyme reaction:

xylose isomerase (Xylose isomerase, EC 5.3.1.5), Xylulokinase (Xylulokinase, EC2.7.1.17), Ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1), 6-hexulose phosphate synthase (3-hexulose6-phosphate synthase, EC4.1.2.43), 6-hexulose phosphate isomerase (6-phospho3-hexuloisomerase, EC 5.3.1.27), Polyphosphate kinase (EC 2.7.4.1), tagatose6-phosphate 4-epimerase (tagatose 6-phospho 4-epimerase, EC 5.1.3.40) were added to a substrate of Xylose and formaldehyde to establish a phosphatase multi-enzyme reaction system for enzyme catalytic reaction.

In the above-mentioned multi-enzyme reaction system, the phosphatase may be phytase, alkaline phosphatase, acid phosphatase, tagatose6-phosphate specific phosphatase, preferably tagatose6-phosphate specific phosphatase.

In the experiment for converting xylose and formaldehyde into tagatose by in vitro multienzyme catalysis, in a reaction system, the concentrations of xylose and formaldehyde are respectively 1-1000mM, the dosage of xylose isomerase is 0.1-10000U/mL, the dosage of xylulokinase is 0.1-10000U/mL, the dosage of ribulose phosphate 3-epimerase is 0.1-10000U/mL, the dosage of 6-phosphohexulose phosphate synthase is 0.1-10000U/mL, the dosage of 6-phosphohexulose isomerase is 0.1-10000U/mL, the dosage of polyphosphate kinase is 0.1-10000U/mL, the dosage of tagatose6-phosphate 4-epimerase is 0.1-10000U/mL, and the dosage of phosphatase is 0.1-10000U/mL.

Preferably, the concentration of xylose is 50mM, the concentration of formaldehyde is 60mM, the dosage of xylose isomerase is 10U/mL, the dosage of xylulokinase is 10U/mL, the dosage of ribulose phosphate 3-epimerase is 10U/mL, the dosage of 6-hexulose phosphate synthase is 10U/mL, the dosage of 6-hexulose phosphate isomerase is 10U/mL, the dosage of polyphosphate kinase is 10U/mL, the dosage of tagatose6-phosphate epimerase is 10U/mL, and the dosage of phosphatase is 10U/mL.

(7) Another method for preparing fructose 6-phosphate by catalyzing xylose and methanol through in vitro multi-enzyme reaction is provided:

using Xylose and Methanol as substrates, Methanol dehydrogenase (EC 1.1.1.1), NADH oxidase (NADH oxidase, EC1.6.3.4), Xylose isomerase (Xylose isomerase, EC 3.1.5), Xylulokinase (Xylokinase, EC2.7.1.17), Ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1), 6-hexulose phosphate synthase (3-hexulose6-phosphate synthase, EC4.1.2.43), 6-hexulose phosphate isomerase (6-phospho 3-hexokinase, EC 5.3.1.27), Polyphosphate kinase (Polyphosphate kinase, EC2.7.4.1) were added to establish a multi-enzyme reaction system, and enzyme catalytic reaction was performed.

In the experiment for converting xylose and methanol into fructose 6-phosphate by in vitro multi-enzyme catalysis, in a reaction system, the concentrations of xylose and methanol are respectively 1-1000mM, the dosage of methanol dehydrogenase is 0.1-10000U/mL, the dosage of NADH oxidase is 0.1-10000U/mL, the dosage of xylose isomerase is 0.1-10000U/mL, the dosage of xylulokinase is 0.1-10000U/mL, the dosage of ribulose phosphate 3-epimerase is 0.1-10000U/mL, the dosage of 6-hexulose phosphate synthase is 0.1-10000U/mL, the dosage of 6-hexulose phosphate isomerase is 0.1-10000U/mL, and the dosage of polyphosphate kinase is 0.1-10000U/mL.

Preferably, the concentration of xylose is 50mM, the concentration of methanol is 60mM, the dosage of methanol dehydrogenase is 10U/mL, the dosage of NADH oxidase is 10U/mL, the dosage of xylose isomerase is 10U/mL, the dosage of xylulokinase is 10U/mL, the dosage of ribulose phosphate 3-epimerase is 10U/mL, the dosage of 6-phosphohexulose synthase is 10U/mL, the dosage of 6-phosphohexulose isomerase is 10U/mL, and the dosage of polyphosphate kinase is 10U/mL.

The multi-enzyme reaction system also comprises buffer solution, magnesium salt, manganese salt, polyphosphoric acid, Adenosine Diphosphate (ADP), Nicotinamide Adenine Dinucleotide (NAD)⁺) A reducing agent. Preferably, the buffer solution is Tris-HCl buffer solution, the pH value of the buffer solution is 4.0-9.0, and the concentration of the buffer solution is 20-500 mM; magnesium salt is magnesium sulfate with concentration of 0.01-500 mM; manganese salt is manganese sulfate with concentration of0.01-500 mM; the polyphosphate is sodium polyphosphate with the concentration of 5-500 mM; adenosine Diphosphate (ADP) at a concentration of 0.01-500 mM; nicotinamide Adenine Dinucleotide (NAD)⁺) In a concentration of 0.01-500 mM; the reducing agent is Dithiothreitol (DTT) with concentration of 0.001-100 mM.

(8) Provides another method for preparing fructose 1,6-diphosphate by catalyzing xylose and methanol through in vitro multi-enzyme reaction:

a multienzyme reaction system was established by adding Methanol dehydrogenase (EC 1.1.1.1), NADH oxidase (NADH oxidase, EC1.6.3.4), Xylose isomerase (Xylose isomerase, EC 5.3.1.5), Xylulokinase (Xylokinase, EC2.7.1.17), Ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1), 6-hexulose phosphate synthase (3-hexose 6-phosphate synthase, EC4.1.2.43), 6-hexulose phosphate isomerase (6-phospho 3-hexokinase, EC 5.3.1.27), Polyphosphate kinase (Polyphosphate kinase, EC2.7.4.1) and 6-phosphofructokinase (6-phosphofructokinase, EC 2.7.1.11) to Xylose and Methanol as substrates.

In the experiment of converting xylose and methanol into fructose 1,6-diphosphate by in vitro multi-enzyme catalysis, in a reaction system, the concentration of xylose and methanol is 1-1000mM respectively, the dosage of the added alcohol dehydrogenase is 0.1-10000U/mL, the dosage of NADH oxidase is 0.1-10000U/mL, the dosage of xylose isomerase is 0.1-10000U/mL, the dosage of xylulokinase is 0.1-10000U/mL, the dosage of ribulose phosphate 3-epimerase is 0.1-10000U/mL, the dosage of 6-phosphohexulose synthase is 0.1-10000U/mL, the dosage of 6-phosphohexuloisomerase is 0.1-10000U/mL, the dosage of 6-phosphofructokinase is 0.1-10000U/mL, and the dosage of polyphosphate kinase is 0.1-10000U/mL.

Preferably, the concentration of xylose is 50mM, the concentration of methanol is 60mM, the dosage of alcohol dehydrogenase is 10U/mL, the dosage of NADH oxidase is 10U/mL, the dosage of xylose isomerase is 10U/mL, the dosage of xylulokinase is 10U/mL, the dosage of ribulose phosphate 3-epimerase is 10U/mL, the dosage of 6-hexulose phosphate synthase is 10U/mL, the dosage of 6-hexulose phosphate isomerase is 10U/mL, the dosage of 6-phosphofructokinase is 10U/mL, and the dosage of polyphosphate kinase is 10U/mL.

The multi-enzyme reaction system also comprises buffer solution, magnesium salt, manganese salt, polyphosphoric acid, Adenosine Diphosphate (ADP), Nicotinamide Adenine Dinucleotide (NAD)⁺) A reducing agent. Preferably, the buffer solution is Tris-HCl buffer solution, the pH value of the buffer solution is 4.0-9.0, and the concentration of the buffer solution is 20-500 mM; magnesium salt is magnesium sulfate with concentration of 0.01-500 mM; manganese salt is manganese sulfate, and the concentration is 0.01-500 mM; the polyphosphate is sodium polyphosphate with the concentration of 5-500 mM; adenosine Diphosphate (ADP) at a concentration of 0.01-500 mM; nicotinamide Adenine Dinucleotide (NAD)⁺) In a concentration of 0.01-500 mM; the reducing agent is Dithiothreitol (DTT) with concentration of 0.001-100 mM.

It will be understood by those skilled in the art that various buffers can be used in the present invention, such as HEPES buffer, Tris-HCl buffer, MOPS buffer, citrate buffer, phosphate buffer, etc.; various polyphosphoric acids and polyphosphates may be used in the present invention, such as sodium hexametaphosphate, sodium polyphosphate, and the like; various magnesium and manganese salts can be used in the present invention, such as magnesium chloride, magnesium sulfate, manganese chloride, manganese sulfate, and the like. The source of xylose can be pure xylose or biomass hydrolysate containing a large amount of xylose. (9) Another method for preparing inositol by catalyzing xylose and methanol through in vitro multi-enzyme reaction is provided:

using Xylose and Methanol as substrates, Methanol dehydrogenase (EC 1.1.1.1), NADH oxidase (NADH oxidase, EC1.6.3.4), Xylose isomerase (XYlose isomerase, EC 5.3.1.5), Xylulokinase (XYLUKEN, EC2.7.1.17), Ribulose phosphate 3-epimerase (Ribulose-phosphate 3-epimerase, EC 5.1.3.1), 6-hexulose phosphate synthase (3-hexulose6-phosphate synthase, EC 2), 6-hexulose phosphate isomerase (6-phospho 3-hexokinase, EC 5.3.1.27), glucose phosphate isomerase (phosphoglucokinase, EC 5.3.1.9), Inositol 1-phosphosynthase (Inphos 1-phosphokinase, EC 54), polyphosphokinase (EC 82), and polyphosphokinase (EC EC2.7.4.1) were added to carry out a reaction.

In the experiment for converting xylose and methanol into inositol through in-vitro multi-enzyme catalysis, in a reaction system, the concentrations of xylose and methanol are respectively 1-1000mM, the dosage of alcohol dehydrogenase is 0.1-10000U/mL, the dosage of NADH oxidase is 0.1-10000U/mL, the dosage of xylose isomerase is 0.1-10000U/mL, the dosage of xylulokinase is 0.1-10000U/mL, the dosage of ribulose phosphate 3-epimerase is 0.1-10000U/mL, the dosage of 6-phosphohexulose synthase is 0.1-10000U/mL, the dosage of 6-phosphohexulose isomerase is 0.1-10000U/mL, the dosage of glucose isomerase is 0.1-10000U/mL, and the dosage of inositol 1-phosphate synthase is 0.1-10000U/mL, the dosage of the phytase is 0.1-10000U/mL, and the dosage of the polyphosphate kinase is 0.1-10000U/mL.

Preferably, the concentration of xylose is 50mM, the concentration of methanol is 60mM, the amount of alcohol dehydrogenase is 10U/mL, the amount of NADH oxidase is 10U/mL, the amount of xylose isomerase is 10U/mL, the amount of xylulokinase is 10U/mL, the amount of ribulose phosphate 3-epimerase is 10U/mL, the amount of 6-phosphohexulose synthase is 10U/mL, the amount of 6-phosphohexulose isomerase is 10U/mL, the amount of phosphoglucose isomerase is 10U/mL, the amount of inositol 1-phosphate synthase is 10U/mL, the amount of phytase is 10U/mL, and the amount of polyphosphate kinase is 10U/mL.

(10) Another method for preparing psicose by catalyzing xylose and methanol through in vitro multi-enzyme reaction is provided:

a reaction was carried out by adding Methanol dehydrogenase (Methanol dehydrogenase, EC1.1.1.1), NADH oxidase (NADH oxidase, EC1.6.3.4), Xylose isomerase (XYlose isomerase, EC 5.3.1.5), Xylulokinase (XYLUKINAse, EC2.7.1.17), Ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1), 6-hexulose phosphate synthase (3-hexulose6-phosphate synthase, EC4.1.2.43), 6-hexulose phosphate isomerase (6-phospho 3-hexokinase, EC 5.3.1.27), Polyphosphate kinase (polyphosphokinase, EC2.7.4.1), psicose 6-phospho 3-epimerase (D-phospho 6-epimerase, EC 1.5), and carrying out a phosphatase-catalyzed reaction using Xylose and Methanol as substrates.

In the experiment for converting xylose and methanol into psicose by in vitro multi-enzyme catalysis, in a reaction system, the concentrations of xylose and methanol are respectively 1-1000mM, the dosage of alcohol dehydrogenase is 0.1-10000U/mL, the dosage of NADH oxidase is 0.1-10000U/mL, the dosage of xylose isomerase is 0.1-10000U/mL, the dosage of xylulokinase is 0.1-10000U/mL, the dosage of ribulose phosphate 3-epimerase is 0.1-10000U/mL, the dosage of 6-phosphohexulose synthase is 0.1-10000U/mL, the dosage of 6-phosphohexulose isomerase is 0.1-10000U/mL, the dosage of polyphosphate kinase is 0.1-10000U/mL, the dosage of psicose 6-phosphate 3-epimerase is 0.1-10000U/mL, the amount of phosphatase is 0.1-10000U/mL.

Preferably, the concentration of xylose is 50mM, the concentration of methanol is 60mM, the amount of alcohol dehydrogenase is 10U/mL, the amount of NADH oxidase is 10U/mL, the amount of xylose isomerase is 10U/mL, the amount of xylulokinase is 10U/mL, the amount of ribulose phosphate 3-epimerase is 10U/mL, the amount of 6-hexulose phosphate synthase is 10U/mL, the amount of 6-hexulose phosphate isomerase is 10U/mL, the amount of polyphosphate kinase is 10U/mL, the amount of psicose 6-phosphate 3-epimerase is 10U/mL, and the amount of phosphatase is 10U/mL.

The multi-enzyme reaction system also comprises buffer solution, magnesium salt, manganese salt, polyphosphoric acid, Adenosine Diphosphate (ADP), Nicotinamide Adenine Dinucleotide (NAD)⁺) A reducing agent. Preferably, the buffer is Tris-HCl buffer, the pH value of the buffer is 4.0-9.0, and the concentration of the buffer is 20-500 mM; magnesium salt is magnesium sulfate with concentration of 0.01-500 mM; manganese salt is manganese sulfate, and the concentration is 0.01-500 mM; the polyphosphate is sodium polyphosphate with the concentration of 5-500 mM; adenosine Diphosphate (ADP) at a concentration of 0.01-500 mM; nicotinamide Adenine Dinucleotide (NAD)⁺) In a concentration of 0.01-500 mM; the reducing agent is beta-mercaptoethanol or Dithiothreitol (DTT) with concentration of 0.001-100 mM.

(11) Another method for preparing mannose by catalyzing xylose and methanol through in vitro multi-enzyme reaction is provided:

using Xylose and Methanol as substrates, Methanol dehydrogenase (C1.1.1.1), NADH oxidase (NADH oxidase, EC1.6.3.4), Xylose isomerase (Xylose isomerase, EC 5.3.1.5), Xylulokinase (Xylokinase, EC2.7.1.17), Ribulose phosphate 3-epimerase (Ribulose-phosphate 3-epimerase, EC 5.1.3.1), 6-hexulose phosphate synthase (3-hexulose6-phosphate synthase, EC4.1.2.43), 6-hexulose phosphate isomerase (6-phospho 3-hexokinase, EC 5.3.1.27), Polyphosphate kinase (polyphosphokinase, EC2.7.4.1), Mannose 6-phosphoisomerase (Mannose 6-phosphoisomerase, EC 5.3.1.8), and a multi-phosphatase catalytic reaction system were added to carry out the reaction.

In the experiment for converting xylose and methanol into mannose by in vitro multi-enzyme catalysis, in a reaction system, the concentrations of xylose and methanol are respectively 1-1000mM, the dosage of alcohol dehydrogenase is 0.1-10000U/mL, the dosage of NADH oxidase is 0.1-10000U/mL, the dosage of xylose isomerase is 0.1-10000U/mL, the dosage of xylulokinase is 0.1-10000U/mL, the dosage of ribulose phosphate 3-epimerase is 0.1-10000U/mL, the dosage of 6-phosphohexulose synthase is 0.1-10000U/mL, the dosage of 6-phosphohexuloisomerase is 0.1-10000U/mL, the dosage of polyphosphate kinase is 0.1-10000U/mL, the dosage of mannose 6-phosphoisomerase is 0.1-10000U/mL, the amount of phosphatase is 0.1-10000U/mL.

Preferably, the concentration of xylose is 50mM, the concentration of methanol is 60mM, the amount of alcohol dehydrogenase is 10U/mL, the amount of NADH oxidase is 10U/mL, the amount of xylose isomerase is 10U/mL, the amount of xylulokinase is 10U/mL, the amount of ribulose phosphate 3-epimerase is 10U/mL, the amount of 6-phosphohexulose synthase is 10U/mL, the amount of 6-phosphohexulose isomerase is 10U/mL, the amount of polyphosphate kinase is 10U/mL, the amount of mannose 6-phosphate isomerase is 10U/mL, and the amount of phosphatase is 10U/mL.

The multi-enzyme reaction system also comprises buffer solution, magnesium salt, manganese salt, polyphosphoric acid, Adenosine Diphosphate (ADP), Nicotinamide Adenine Dinucleotide (NAD)⁺) A reducing agent. Preferably, the buffer solution is Tris-HCl buffer solution, the pH value of the buffer solution is 4.0-9.0, and the concentration of the buffer solution is 20-500 mM; magnesium salt is magnesium sulfate with concentration of 0.01-500 mM; manganese salt is manganese sulfate, and the concentration is 0.01-500 mM; the polyphosphate is sodium polyphosphate with the concentration of 5-500 mM; adenosine Diphosphate (ADP) at a concentration of 0.01-500 mM; nicotinamide Adenine Dinucleotide (NAD)⁺) In a concentration of 0.01-500 mM; the reducing agent is beta-mercaptoethanol or Dithiothreitol (DTT) with concentration of 0.001-100 mM.

(12) Another method for preparing tagatose by catalyzing xylose and methanol through in vitro multi-enzyme reaction is provided:

using Xylose and Methanol as substrates, Methanol dehydrogenase (EC 1.1.1.1), NADH oxidase (NADH oxidase, EC1.6.3.4), Xylose isomerase (XYlose isomerase, EC 5.3.1.5), Xylulokinase (XYLUKEINase, EC2.7.1.17), Ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1), 6-hexulose phosphate synthase (3-hexulose6-phosphate synthase, EC4.1.2.43), 6-hexulose phosphate isomerase (6-phospho 3-hexokinase, EC 5.3.1.27), Polyphosphate kinase (Polyphosphate kinase, EC2.7.4.1), tagatose6-phosphate 4-epimerase (tagatose-6-phosphoisomerase, EC 5.1.3.40), and a multi-phosphatase system were added to carry out the reaction.

In the experiment for converting xylose and methanol into tagatose by in vitro multienzyme catalysis, in a reaction system, the concentrations of xylose and formaldehyde are respectively 1-1000mM, the dosage of alcohol dehydrogenase is 0.1-10000U/mL, the dosage of NADH oxidase is 0.1-10000U/mL, the dosage of xylose isomerase is 0.1-10000U/mL, the dosage of xylulokinase is 0.1-10000U/mL, the dosage of ribulose phosphate 3-epimerase is 0.1-10000U/mL, the dosage of 6-phosphohexulose synthase is 0.1-10000U/mL, the dosage of 6-phosphohexulose isomerase is 0.1-10000U/mL, the dosage of polyphosphate kinase is 0.1-10000U/mL, the dosage of tagatose6-phosphate 4-epimerase is 0.1-10000U/mL, the amount of phosphatase is 0.1-10000U/mL.

Preferably, the concentration of xylose is 50mM, the concentration of methanol is 60mM, the amount of alcohol dehydrogenase is 10U/mL, the amount of NADH oxidase is 10U/mL, the amount of xylose isomerase is 10U/mL, the amount of xylulokinase is 10U/mL, the amount of ribulose phosphate 3-epimerase is 10U/mL, the amount of 6-hexulose phosphate synthase is 10U/mL, the amount of 6-hexulose phosphate isomerase is 10U/mL, the amount of polyphosphate kinase is 10U/mL, the amount of tagatose6-phosphate 4-epimerase is 10U/mL, and the amount of phosphatase is 10U/mL.

(13) Provides a method for preparing fructose 6-phosphate by catalyzing xylose and methanol through in vitro multi-enzyme reaction:

xylose and methanol are used as substrates, and Alcohol oxidase (Alcohol oxidase, EC 1.1.3.13), Catalase (Catalase, EC 1.11.1.6), Xylose isomerase (Xylose isomerase, EC 3.1.5), Xylulokinase (xylokinase, EC2.7.1.17), Ribulose phosphate 3-epimerase (Ribulose 5-phospho 3-epimerase, EC 5.1.3.1), 6-phosphohexulose synthase (3-hexose 6-phospho synthase, EC4.1.2.43), 6-phosphohexulose isomerase (6-phospho 3-hexokinase, EC 5.3.1.27), Polyphosphate kinase (polyphosphokinase, EC2.7.4.1) are added to establish a multi-enzyme reaction system to perform enzyme catalytic reaction.

In the experiment for converting xylose and methanol into fructose 6-phosphate by in vitro multi-enzyme catalysis, in a reaction system, the concentrations of xylose and methanol are respectively 1-1000mM, the dosage of alcohol oxidase is 0.1-10000U/mL, the dosage of catalase is 0.1-10000U/mL, the dosage of xylose isomerase is 0.1-10000U/mL, the dosage of xylulokinase is 0.1-10000U/mL, the dosage of ribulose phosphate 3-epimerase is 0.1-10000U/mL, the dosage of 6-phosphohexulose synthase is 0.1-10000U/mL, the dosage of 6-phosphohexulose isomerase is 0.1-10000U/mL, and the dosage of polyphosphate kinase is 0.1-10000U/mL.

Preferably, the concentration of xylose is 50mM, the concentration of methanol is 60mM, the dosage of alcohol oxidase is 10U/mL, the dosage of catalase is 10U/mL, the dosage of xylose isomerase is 10U/mL, the dosage of xylulokinase is 10U/mL, the dosage of ribulose phosphate 3-epimerase is 10U/mL, the dosage of 6-phosphohexulose synthase is 10U/mL, the dosage of 6-phosphohexuloisomerase is 10U/mL, and the dosage of polyphosphate kinase is 10U/mL.

The multi-enzyme reaction system also comprises buffer solution, magnesium salt, manganese salt, polyphosphoric acid, Adenosine Diphosphate (ADP) and Flavin Adenine Dinucleotide (FAD). Preferably, the buffer solution is Tris-HCl buffer solution, the pH value of the buffer solution is 4.0-9.0, and the concentration of the buffer solution is 20-500 mM; magnesium salt is magnesium sulfate with concentration of 0.01-500 mM; manganese salt is manganese sulfate, and the concentration is 0.01-500 mM; the polyphosphate is sodium polyphosphate with the concentration of 5-500 mM; adenosine Diphosphate (ADP) at a concentration of 0.01-500 mM; the concentration of Flavin Adenine Dinucleotide (FAD) is 0.00001-10 mM.

(14) Provides a method for preparing fructose 1,6-diphosphate by catalyzing xylose and methanol through in vitro multi-enzyme reaction:

using Xylose and methanol as substrates, a reaction was carried out by adding Alcohol oxidase (Alcohol oxidase, EC 1.1.3.13), Catalase (Catalase, EC 1.11.1.6), Xylose isomerase (Xylose isomerase, EC 5.3.1.5), Xylulokinase (Xylulokinase, EC2.7.1.17), Ribulose phosphate 3-epimerase (Ribulose 5-phospho 3-epimerase, EC 5.1.3.1), 6-phosphohexulose synthase (3-phospho 6-phospho synthase, EC4.1.2.43), 6-phosphohexulose isomerase (6-phospho 3-hexolosomerase, EC 5.3.1.27), Polyphosphate kinase (Polyphosphate enzyme, EC2.7.4.1) and 6-phosphofructokinase (6-phosphofructokinase, EC 2.7.1.11) to establish a reaction system.

In the experiment of the invention for converting xylose and methanol into fructose 1,6-diphosphate by in vitro multi-enzyme catalysis, in a reaction system, the concentration of xylose and methanol is 1-1000mM respectively, the dosage of alcohol oxidase is 0.1-10000U/mL, the dosage of catalase is 0.1-10000U/mL, the dosage of xylose isomerase is 0.1-10000U/mL, the dosage of xylulokinase is 0.1-10000U/mL, the dosage of ribulose phosphate 3-epimerase is 0.1-10000U/mL, the dosage of 6-phosphohexulose synthase is 0.1-10000U/mL, the dosage of 6-phosphohexuloisomerase is 0.1-10000U/mL, the dosage of 6-phosphofructokinase is 0.1-10000U/mL, and the dosage of polyphosphate kinase is 0.1-10000U/mL.

Preferably, the concentration of xylose is 50mM, the concentration of formaldehyde is 60mM, the dosage of alcohol oxidase is 10U/mL, the dosage of catalase is 10U/mL, the dosage of xylose isomerase is 10U/mL, the dosage of xylulokinase is 10U/mL, the dosage of ribulose phosphate 3-epimerase is 10U/mL, the dosage of 6-phosphohexulose synthase is 10U/mL, the dosage of 6-phosphohexuloisomerase is 10U/mL, the dosage of 6-phosphofructokinase is 10U/mL, and the dosage of polyphosphate kinase is 10U/mL.

The multi-enzyme reaction system also comprises buffer solution, magnesium salt, manganese salt, polyphosphoric acid, Adenosine Diphosphate (ADP) and Flavin Adenine Dinucleotide (FAD). Preferably, the buffer is Tris-HCl buffer, the pH value of the buffer is 4.0-9.0, and the concentration of the buffer is 20-500 mM; magnesium salt is magnesium sulfate with concentration of 0.01-500 mM; manganese salt is manganese sulfate, and the concentration is 0.01-500 mM; the polyphosphate is sodium polyphosphate with the concentration of 5-500 mM; adenosine Diphosphate (ADP) at a concentration of 0.01-500 mM; the concentration of Flavin Adenine Dinucleotide (FAD) is 0.00001-10 mM.

(15) Provides a method for preparing inositol by catalyzing xylose and methanol through in vitro multi-enzyme reaction:

using Xylose and methanol as substrates, an Alcohol oxidase (Alcohol oxidase, EC 1.1.3.13), a Catalase (Catalase, EC 1.11.1.6), a Xylose isomerase (Xylose isomerase, EC 5.3.1.5), a Xylulokinase (Xylulokinase, EC2.7.1.17), a Ribulose phosphate 3-epimerase (Ribulose 5-phosphoesterase, EC 5.1.3.1), a 6-phosphohexulose synthase (3-phosphohexulose 6-phosphosynthase, EC4.1.2.43), a 6-phosphohexulose isomerase (6-phospho 3-phosphoinosomerase, EC 5.3.1.27), a Phosphoglucose isomerase (phosphosynthase, EC 5.3.1.9), a 1-phosphosynthase (Inphosphosynthase, EC 1-phosphokinase, EC 82), a polyphosphoinose phosphatase (phosphokinase, EC3.1.3.25) and a polyphosphokinase (phosphoinose synthase, EC2.7.4.1) were added to carry out a reaction.

In the experiment for converting xylose and methanol into inositol through in-vitro multi-enzyme catalysis, in a reaction system, the concentrations of xylose and methanol are respectively 1-1000mM, the dosage of alcohol oxidase is 0.1-10000U/mL, the dosage of catalase is 0.1-10000U/mL, the dosage of xylose isomerase is 0.1-10000U/mL, the dosage of xylulokinase is 0.1-10000U/mL, the dosage of ribulose phosphate 3-epimerase is 0.1-10000U/mL, the dosage of 6-phosphohexulose synthase is 0.1-10000U/mL, the dosage of 6-phosphohexulose isomerase is 0.1-10000U/mL, the dosage of glucose phosphate isomerase is 0.1-10000U/mL, and the dosage of inositol 1-phosphate synthase is 0.1-10000U/mL, the dosage of the phytase is 0.1-10000U/mL, and the dosage of the polyphosphate kinase is 0.1-10000U/mL.

Preferably, the concentration of xylose is 50mM, the concentration of methanol is 60mM, the dosage of alcohol oxidase is 10U/mL, the dosage of catalase is 10U/mL, the dosage of xylose isomerase is 10U/mL, the dosage of xylulokinase is 10U/mL, the dosage of ribulose phosphate 3-epimerase is 10U/mL, the dosage of 6-phosphohexulose synthase is 10U/mL, the dosage of 6-phosphohexulose isomerase is 10U/mL, the dosage of phosphoglucose isomerase is 10U/mL, the dosage of inositol 1-phosphate synthase is 10U/mL, the dosage of inositol monophosphatase is 10U/mL, and the dosage of polyphosphate kinase is 10U/mL.

It will be understood by those skilled in the art that various buffers can be used in the present invention, such as HEPES buffer, Tris-HCl buffer, MOPS buffer, citrate buffer, phosphate buffer, etc.; various polyphosphoric acids and polyphosphates may be used in the present invention, such as sodium hexametaphosphate, sodium polyphosphate, and the like; various magnesium and manganese salts can be used in the present invention, such as magnesium chloride, magnesium sulfate, manganese chloride, manganese sulfate, and the like. The source of xylose can be pure xylose or biomass hydrolysate containing a large amount of xylose. (16) Provides a method for preparing psicose by catalyzing xylose and methanol through in vitro multi-enzyme reaction:

using Xylose and methanol as substrates, Alcohol oxidase (Alcohol oxidase, EC 1.1.3.13), Catalase (Catalase, EC 1.11.1.6), Xylose isomerase (Xylose isomerase, EC 5.3.1.5), Xylulokinase (Xylulokinase, EC2.7.1.17), Ribulose phosphate 3-epimerase (Ribulose 5-phosphoestere 3-epimerase, EC 5.1.3.1), 6-phosphohexulose synthase (3-phosphoestere 6-phosphoestersynthase, EC4.1.2.43), 6-phosphohexuloisomerase (6-phosphoestere 3-hexolosomerase, EC 5.3.1.27), Polyphosphate kinase (Polyphosphate, EC2.7.4.1), allulose 6-phosphoestere 3-epimerase (D-phosphoestere 6-epimerase, EC 1.5.5) were added to carry out a reaction catalyzed by a polymerase enzyme system.

In the experiment for converting xylose and methanol into psicose by in vitro multi-enzyme catalysis, in a reaction system, the concentrations of xylose and methanol are respectively 1-1000mM, the dosage of alcohol oxidase is 0.1-10000U/mL, the dosage of catalase is 0.1-10000U/mL, the dosage of xylose isomerase is 0.1-10000U/mL, the dosage of xylulokinase is 0.1-10000U/mL, the dosage of ribulose phosphate 3-epimerase is 0.1-10000U/mL, the dosage of 6-phosphohexulose synthase is 0.1-10000U/mL, the dosage of 6-phosphohexuloisomerase is 0.1-10000U/mL, the dosage of polyphosphate kinase is 0.1-10000U/mL, the dosage of psicose 6-phosphate 3-epimerase is 0.1-10000U/mL, the amount of phosphatase is 0.1-10000U/mL.

Preferably, the concentration of xylose is 50mM, the concentration of methanol is 60mM, the dosage of alcohol oxidase is 10U/mL, the dosage of catalase is 10U/mL, the dosage of xylose isomerase is 10U/mL, the dosage of xylulokinase is 10U/mL, the dosage of ribulose phosphate 3-epimerase is 10U/mL, the dosage of 6-phosphohexulose synthase is 10U/mL, the dosage of 6-phosphohexuloisomerase is 10U/mL, the dosage of polyphosphate kinase is 10U/mL, the dosage of psicose 6-phosphate 3-epimerase is 10U/mL, and the dosage of phosphatase is 10U/mL.

(17) Provides a method for preparing mannose by catalyzing xylose and methanol through in vitro multi-enzyme reaction:

using Xylose and methanol as substrates, Alcohol oxidase (Alcohol oxidase, EC 1.1.3.13), Catalase (Catalase, EC 1.11.1.6), Xylose isomerase (Xylose isomerase, EC 5.3.1.5), Xylulokinase (Xylulokinase, EC2.7.1.17), Ribulose phosphate 3-epimerase (Ribulose 5-phosphoestere 3-epimerase, EC 5.1.3.1), 6-phosphohexulose synthase (3-phosphohexulose 6-phosphoestersynthase, EC4.1.2.43), 6-phosphohexuloisomerase (6-phosphoester 3-hexolosomerase, EC 5.3.1.27), Polyphosphate kinase (Polyphosphate, EC2.7.4.1), Mannose 6-phosphoisomerase (Mannose 6-phosphoesterase, EC 5.3.1.8), and a multi-enzyme catalytic reaction were added to carry out.

In the experiment for converting xylose and methanol into mannose by in vitro multi-enzyme catalysis, in a reaction system, the concentrations of xylose and methanol are respectively 1-1000mM, the dosage of alcohol oxidase is 0.1-10000U/mL, the dosage of catalase is 0.1-10000U/mL, the dosage of xylose isomerase is 0.1-10000U/mL, the dosage of xylulokinase is 0.1-10000U/mL, the dosage of ribulose phosphate 3-epimerase is 0.1-10000U/mL, the dosage of 6-phosphohexulose synthase is 0.1-10000U/mL, the dosage of 6-phosphohexulose isomerase is 0.1-10000U/mL, the dosage of polyphosphate kinase is 0.1-10000U/mL, the dosage of mannose 6-phosphoisomerase is 0.1-10000U/mL, the amount of phosphatase is 0.1-10000U/mL.

Preferably, the concentration of xylose is 50mM, the concentration of methanol is 60mM, the dosage of alcohol oxidase is 10U/mL, the dosage of catalase is 10U/mL, the dosage of xylose isomerase is 10U/mL, the dosage of xylulokinase is 10U/mL, the dosage of ribulose phosphate 3-epimerase is 10U/mL, the dosage of 6-phosphohexulose synthase is 10U/mL, the dosage of 6-phosphohexuloisomerase is 10U/mL, the dosage of polyphosphate kinase is 10U/mL, the dosage of mannose 6-phosphate isomerase is 10U/mL, and the dosage of phosphatase is 10U/mL.

(18) Provides a method for preparing tagatose by catalyzing xylose and methanol through in vitro multi-enzyme reaction:

using Xylose and methanol as substrates, Alcohol oxidase (Alcohol oxidase, EC 1.1.3.13), Catalase (Catalase, EC 1.11.1.6), Xylose isomerase (Xylose isomerase, EC 5.3.1.5), Xylulokinase (Xylulokinase, EC2.7.1.17), Ribulose phosphate 3-epimerase (Ribulose 5-phosphoesterase, EC 5.1.3.1), 6-phosphohexulose synthase (3-phosphohexulose 6-phosphosynthase, EC4.1.2.43), 6-phosphohexulose isomerase (6-phospho 3-hexokinase, EC 5.3.1.27), Polyphosphate kinase (Polyphosphate, EC2.7.4.1), tagatose6-phosphate 4-epimerase (tagatose 6-phosphoisomerase, EC 5.1.3.40) were added to carry out a reaction catalyzed by enzyme system.

In the experiment for converting xylose and methanol into tagatose by in vitro multi-enzyme catalysis, in a reaction system, the concentrations of xylose and methanol are respectively 1-1000mM, the dosage of alcohol oxidase is 0.1-10000U/mL, the dosage of catalase is 0.1-10000U/mL, the dosage of xylose isomerase is 0.1-10000U/mL, the dosage of xylulokinase is 0.1-10000U/mL, the dosage of ribulose phosphate 3-epimerase is 0.1-10000U/mL, the dosage of 6-phosphohexulose synthase is 0.1-10000U/mL, the dosage of 6-phosphohexulose isomerase is 0.1-10000U/mL, the dosage of polyphosphate kinase is 0.1-10000U/mL, the dosage of tagatose6-phosphate 4-epimerase is 0.1-10000U/mL, the amount of phosphatase is 0.1-10000U/mL.

Preferably, the concentration of xylose is 50mM, the concentration of methanol is 60mM, the dosage of alcohol oxidase is 10U/mL, the dosage of catalase is 10U/mL, the dosage of xylose isomerase is 10U/mL, the dosage of xylulokinase is 10U/mL, the dosage of ribulose phosphate 3-epimerase is 10U/mL, the dosage of 6-phosphohexulose synthase is 10U/mL, the dosage of 6-phosphohexuloisomerase is 10U/mL, the dosage of polyphosphate kinase is 10U/mL, the dosage of tagatose6-phosphate 4-epimerase is 10U/mL, and the dosage of phosphatase is 10U/mL.

In summary, according to the embodiments of the present invention, the xylose isomerase of the present invention may be replaced by any enzyme having a function of converting xylose into xylulose, or may be a mutant enzyme having an equivalent function obtained by protein engineering. For example, sources of the xylose isomerase include, but are not limited to: thermus thermophilus HB8, Thermoanaerobacter ethanolicus, Thermoanaerobacterium saccharolyticum, and the like. Preferably xylose isomerase originating from Thermus thermophilus HB 8.

The xylulokinase can be replaced by any enzyme with the function of converting xylulose and ATP into xylulose 5-phosphate and ADP, and can also be mutant enzyme with the same function obtained by protein engineering. For example, sources of the xylulose isomerase include, but are not limited to: parageobacillus caldoxylolyticus, Thermotoga maritima MSB8, Thermoanaerobacterium thermosaccharolyticum, and the like. Preferably a xylose isomerase derived from Thermotoga maritima MSB 8.

The ribulose phosphate 3-epimerase can be replaced by any enzyme with the function of converting xylulose 5-phosphate into ribulose5-phosphate, and can also be a mutant enzyme with the same function obtained by protein engineering modification. For example, sources of the ribulose phosphate 3-epimerase include, but are not limited to: thermotoga maritima MSB8, Escherichia coli, Thermoanaerobacterium thermosaccharolyticum DSM 571, and the like. Preferably a ribulose phosphate 3-epimerase from Thermotoga maritima MSB 8.

The hexulose-6-phosphate synthase of the invention can be replaced by any enzyme with the function of condensing ribulose-5-phosphate and formaldehyde into hexulose-6-phosphate, and can also be a mutant enzyme with the same function obtained by protein engineering modification. For example, sources of the hexulose-6-phosphate synthase include, but are not limited to: bacillus methanolica, Mycobacterium consortium, Thermococcus kodakarensis. Preferred is a hexulose-6-phosphate synthase from Bacillus methanolicus.

The hexulose-6-phosphate isomerase provided by the invention can be replaced by any enzyme with the function of converting hexulose-6-phosphate into fructose-6-phosphate, and can also be a mutant enzyme with the same function obtained by protein engineering. For example, sources of the hexulose-6-phosphate isomerase include, but are not limited to: bacillus methanolica, Mycobacterium consortium, Thermococcus kodakarensis, and the like. Preferred is hexulose6-phosphate isomerase from Bacillus methanolica.

The polyphosphate kinase can be replaced by any enzyme with the function of converting polyphosphate and ADP into ATP, and can also be mutant enzyme with the same function obtained by protein engineering modification. For example, sources of the polyphosphate kinase include, but are not limited to: rhodobacter sphaeroides, Thermosynechococcus elongatus BP-1, Mycobacterium tuberculosis, and the like. Polyphosphate kinases from Rhodobacter sphaeroides are preferred.

The phosphoglucose isomerase can be replaced by any enzyme with the function of converting fructose 6-phosphate into glucose 6-phosphate, and can also be mutant enzyme with the same function obtained by protein engineering modification. Sources of the phosphoglucose isomerase include, but are not limited to, Thermus thermophilus, Pyrococcus furiosus, Clostridium thermocellum, and the like. Preferably a phosphoglucose isomerase originating from Thermus thermophilus.

The inositol 1-phosphate synthase of the present invention may be replaced with any enzyme having a function of converting glucose 6-phosphate into inositol 1-phosphate, or may be a mutant enzyme having an equivalent function obtained by protein engineering. Sources of such inositol 1-phosphate synthases include, but are not limited to, Thermococcus kodakanensis, Archaeoglobus fulgidus, Saccharomyces cerevisiae, and the like. Preferably an inositol 1-phosphate synthase derived from Archaeoglobus fulgidus.

The inositol monophosphatase of the invention can be replaced by any enzyme with the function of dephosphorylating inositol 1-phosphate to generate inositol, and can also be a mutant enzyme with the same function obtained by protein engineering modification. Sources of such phytases include, but are not limited to, Thermotoga maritima MSB8, Escherichia coli, Mycobacterium smegmatis, and the like. Preferably a myo-inositol mono-phosphatase derived from Thermotoga maritima MSB 8.

The 6-phosphofructokinase can be replaced by any enzyme with the function of generating fructose 1,6-diphosphate and ADP from fructose 6-phosphate and ATP, and can also be mutant enzyme with the same function obtained by protein engineering modification. Sources of the 6-phosphofructokinase include, but are not limited to, Thermus thermophilus, Saccharomyces cerevisiae, Escherichia coli, and the like. Preferably 6-phosphofructokinase derived from Thermus thermophilus.

The psicose 6-phosphate 3-epimerase of the present invention may be replaced by any enzyme having a function of converting fructose 6-phosphate into psicose 6-phosphate, or a mutant enzyme having the same function obtained by protein engineering. Sources of the psicose 6-phosphate 3-epimerase include, but are not limited to, Escherichia coli, Thermoanaerobacterium thermosaccharolyticum DSM 571, and the like. Preferably psicose 6-phosphate 3-epimerase from Thermoanaerobacterium thermosaccharolyticum DSM 571.

The mannose 6-phosphate isomerase can be replaced by any enzyme with the function of converting fructose 6-phosphate into mannose 6-phosphate, and can also be a mutant enzyme with the same function obtained by protein engineering modification. Sources of the mannose-6-phosphate isomerase include, but are not limited to, Thermus thermophilus, Thermotoga maritima MSB8, Archaeoglobus fulgidus, and the like. Preferably mannose-6-phosphate isomerase originating from Thermus thermophilus.

The tagatose6-phosphate 4-epimerase can be replaced by any enzyme with the function of converting fructose 6-phosphate into tagatose6-phosphate, and can also be a mutant enzyme with the same function obtained by protein engineering modification. Sources of the tagatose 4-phosphate epimerase include, but are not limited to, Agrobacterium tumefaciens C58, Dictyoglomonus thermophilum, Thermoanaerobacter indeensis, and the like. Preferably tagatose6-phosphate 4-epimerase derived from Dictyoglomonus thermophilum.

The phosphatase of the invention can be phytase, acid phosphatase, alkaline phosphatase and specific phosphatase. Phosphatases with good substrate specificity are preferred. When psicose 6-phosphate is used as a substrate, preferred is psicose 6-phosphate phosphatase derived from Acidothermus cellulolyticus, Escherichia coli, Clostridium thermocellum, and Bacteroides fragilis NCTC 9343, the gene numbers of which are Acel _0099(KEGG), b3399(KEGG), Cth _0261(KEGG), and BF9343_ 0892. More preferably an psicose 6-phosphate phosphatase corresponding to the gene number Cthe _0261 (KEGG). When mannose 6-phosphate is used as a substrate, phosphatases derived from Thermotoga maritima MSB8, Pseudomonas putida, and Pseudomonas syringae, whose gene numbers are TM0651(KEGG), PP 1764(KEGG), and PSPPH 2719, respectively, are preferable. More preferably mannose-6-phosphate phosphatase corresponding to the gene number TM0651 (KEGG). When tagatose6-phosphate is used as a substrate, tagatose6-phosphate phosphatase derived from Archaeoglobus fulgidus is preferred, and the gene number thereof is AF _0444 (KEGG).

The alcohol oxidase can be replaced by any enzyme with the function of converting methanol and oxygen into formaldehyde and hydrogen peroxide, and can also be mutant enzyme with the same function obtained by protein engineering modification. For example, sources of the alcohol oxidase include, but are not limited to: ogataea angusta, Pichia pastoris, Thermoascus aurantiacus, and the like. Preferably an alcohol oxidase derived from Pichia pastoris.

The catalase of the present invention may be replaced with any enzyme having the function of decomposing hydrogen peroxide into oxygen and water, or may be a mutant enzyme having the same function obtained by protein engineering. Sources of such catalase include, but are not limited to Aspergillus niger, Bacillus subtilis, Escherichia coli, and the like. Preferred is catalase derived from Bacillus subtilis.

The alcohol dehydrogenase of the present invention may be any alcohol dehydrogenase having the ability to convert methanol and NAD⁺The enzyme converted into formaldehyde and NADH can be replaced by mutant enzyme with the same function obtained by protein engineering. Sources of the alcohol dehydrogenase include, but are not limited to, Geobacillus thermomodenitificans, Clostridium thermocellum, and the like,Geobacillus stearothermophilus DSM2334 and the like. Preferably an alcohol dehydrogenase derived from Geobacillus stearothermophilus DSM 2334.

The NADH oxidase of the invention can be any one enzyme capable of converting NADH and oxygen into NAD⁺In place of the enzyme having water function, a mutant enzyme having equivalent function obtained by protein engineering may be used. For example, sources of NADH oxidase include, but are not limited to, Lactococcus lactis, Clostridium aminovaliericum, Streptococcus mutans, and the like. NADH oxidase derived from Streptococcus mutans d is preferred.

Drawings

FIG. 1 is a schematic diagram of the in vitro multi-enzyme catalytic pathway for xylose and formaldehyde to make fructose 6-phosphate, fructose 1,6-diphosphate, inositol, psicose, mannose and tagatose. Xi (xylose isomerase): a xylose isomerase; xk (xylulokinase): xylulokinase; RPE (ribolose 5-phosphate 3-epimerase): ribulose5-phosphate 3-epimerase; HPS (3-hexulose6-phosphate synthsase): 6-hexulose phosphate synthase; PHI (6-phospho 3-hexulosomerase): 6-phosphohexulose isomerase; ppk (polyphosphate kinase) polyphosphate kinase; pfk (phosphokinase): 6-phosphofructokinase; pgi (phosphoglucose isomerase): glucose phosphate isomerase; IPS (inositol 1-phophate synthsase): inositol 1-phosphate synthase; imp (inositol monophosphatase): a phytase; APE (D-allolose 6-phosphate 3-epimerase): psicose 6-phosphate 3-epimerase; a6PP (allolose 6-phosphate phosphatase): psicose 6-phosphate phosphatase; MPI (mannose 6-phosphate isomerase): mannose-6-phosphate isomerase; m6PP (mannose 6-phosphate phosphatase), mannose 6-phosphate phosphatase; TPE (tagatose6-phosphate 4-epimerase), tagatose6-phosphate 4-epimerase; t6PP (agatase 6-phosphate phosphatase), tagatose6-phosphate phosphatase; poly P_n: polyphosphoric acid; ATP: adenosine triphosphate; ADP: adenosine diphosphate; p_i: inorganic phosphorus.

FIG. 2 is a schematic diagram of an in vitro multi-enzyme catalytic pathway for the production of fructose 6-phosphate, fructose 1,6-diphosphate, inositol, psicose, mannose and tagatose from xylose and methanol dependent on methanol dehydrogenase. Mdh (metallic dehydrogenase): methanol dehydrogenase; nox (nadh oxidase): NADH oxidase.

FIG. 3 is a schematic diagram of the in vitro multi-enzyme catalytic pathway for the production of fructose 6-phosphate, fructose 1,6-diphosphate, inositol, psicose, mannose and tagatose from xylose and methanol dependent on alcohol oxidase. Mox (lateral oxidase): a methanol oxidase; cat (catalase): a catalase.

FIG. 4 is a graph showing the reaction process for preparing fructose 6-phosphate from xylose and formaldehyde.

FIG. 5 is a graph of the reaction process for preparing fructose 6-phosphate using two catalytic pathways to catalyze xylose and methanol.

FIG. 6 is a graph showing the reaction process for preparing fructose-1, 6-bisphosphate from xylose and methanol.

FIG. 7 is a graph of the reaction process for the preparation of inositol from xylose and methanol.

FIG. 8 is a graph showing the reaction process of preparing fructose-1, 6-bisphosphate from corn cob hydrolysate and methanol.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

EXAMPLE 1 preparation of fructose 6-phosphate from xylose and Formaldehyde

The catalytic pathways for the conversion of xylose and formaldehyde to fructose 6-phosphate by an in vitro multi-enzyme catalytic system are shown in FIG. 1. Key enzymes involved in this pathway are: (1) xylose isomerase (Xylose isomerase, EC 5.3.1.5), catalyzing the conversion of Xylose to xylulose; (2) xylulokinase (EC 2.7.1.17), which catalyzes the conversion of xylulose and ATP to xylulose 5-phosphate and ADP; (3) ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1) catalyzes the isomerization of xylulose 5-phosphate and Ribulose 5-phosphate; (4) hexulose6-phosphate synthase (EC 4.1.2.43), which catalyzes the condensation of formaldehyde and ribulose5-phosphate, the product being hexulose 6-phosphate; (5) hexulose6-phosphate isomerase (6-phospho 3-hexulosomerase, EC 5.3.1.27), which catalyzes the isomerization of hexulose6-phosphate to fructose 6-phosphate; (6) polyphosphate kinase (EC 2.7.4.1) catalyzes polyphosphoric acid and ADP to regenerate ATP.

In this example, xylose isomerase was derived from Thermus thermophilus HB8, with gene number D90256 (Genebank); xylulokinase and ribulose phosphate 3-epimerase are derived from Thermotoga maritima and have the gene numbers of TM0116(KEGG) and TM1718(KEGG), respectively; the 6-hexulose phosphate synthase and the 6-hexulose phosphate isomerase are derived from Bacillus methanolicus and have the gene numbers of BMMGA 3-06845 (KEGG) and BMMGA 3-06840 (KEGG), respectively; polyphosphate kinase was derived from Rhodobacter sphaeroides, and the gene number was RSP _0766 (KEGG). These genomic DNAs are all available from the ATCC's official website (www.atcc.org). The genome sequence is obtained by adopting a gene synthesis mode after codon optimization. The target gene was subsequently cloned into pET vector (Novagen, Madison, Wis.) to obtain the corresponding expression vectors pET20b-ttcxi, pET20b-tmxk, pET20b-tmrpe, pET21a-bmhps, pET28 a-bmchi, pET28 a-rsppk. Then, these plasmids were transformed into E.coli BL21(DE3) (Invitrogen, Carlsbad, Calif.) respectively, and protein expression and purification were performed.

A1.0 mL reaction system contained 50mM xylose, 60mM formaldehyde, 50mM Tris-HCl buffer (pH8.0), 10mM magnesium sulfate, 0.5mM manganese chloride, 2mM ADP, 20mM sodium hexametaphosphate, 2U/mL xylose isomerase, 2U/mL xylulokinase, 2U/mL ribulose phosphate 3-epimerase, 2U/mL 6-phosphohexulosynthase, 2U/mL 6-phosphohexuloisomerase, 2U/mL polyphosphate kinase. The catalytic reaction was carried out at 37 ℃ for 7 hours. The concentration of fructose 6-phosphate was determined enzymatically as follows: fructose 6-phosphate is converted to glucose 6-phosphate catalyzed by excess Phosphoglucose isomerase (EC 5.3.1.9) and subsequently assayed using the glucose 6-phosphate assay kit (Sigma-Aldrich, cat # MAK 014).

As shown in FIG. 4, the concentration of fructose 6-phosphate gradually increased with time to reach 38.5mM after 7 hours, and the molar conversion of fructose 6-phosphate to xylose was 77.0%.

EXAMPLE 2 preparation of fructose 1, 6-bisphosphate from xylose and Formaldehyde

The catalytic pathway for the conversion of xylose and formaldehyde to fructose 1, 6-bisphosphate by an in vitro multienzyme catalytic system is shown in FIG. 1. Key enzymes involved in this pathway are: (1) xylose isomerase (Xylose isomerase, EC 5.3.1.5), catalyzing the conversion of Xylose to xylulose; (2) xylulokinase (EC 2.7.1.17), which catalyzes the conversion of xylulose and ATP to xylulose 5-phosphate and ADP; (3) ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1) catalyzes the isomerization of xylulose 5-phosphate and Ribulose 5-phosphate; (4) hexulose6-phosphate synthase (EC 4.1.2.43), which catalyzes the condensation of formaldehyde and ribulose5-phosphate, the product being hexulose 6-phosphate; (5) hexulose6-phosphate isomerase (6-phospho 3-hexulosomerase, EC 5.3.1.27), which catalyzes the isomerization of hexulose6-phosphate to fructose 6-phosphate; (6) 6-phosphofructokinase (EC 2.7.1.11), which catalyzes fructose 6-phosphate and ATP to produce fructose 1,6-diphosphate and ADP; (7) polyphosphate kinase (EC 2.7.4.1) catalyzes polyphosphoric acid and ADP to regenerate ATP.

In this example, the 6-phosphofructokinase is derived from Thermus thermophilus HB8 with the gene number TTHA1962 (KEGG). The gene is obtained by gene synthesis. Cloning to pET28a to obtain corresponding expression plasmid pET28 a-ttcpfk. Then transferred into Escherichia coli BL21(DE3) for protein expression and purification. Other enzymes were expressed and purified as in the above examples.

A1.0 mL reaction system contained 50mM xylose, 60mM formaldehyde, 50mM Tris-HCl buffer (pH8.0), 10mM magnesium sulfate, 0.5mM manganese chloride, 2mM ADP, 20mM sodium hexametaphosphate, 2U/mL xylose isomerase, 2U/mL xylulokinase, 2U/mL ribulose phosphate 3-epimerase, 2U/mL 6-phosphohexulosynthase, 2U/mL 6-phosphohexuloisomerase, 2U/mL polyphosphate kinase, 2U/mL 6-phosphofructokinase. The catalytic reaction was carried out at 37 ℃ for 7 hours. The concentration of fructose 1, 6-bisphosphate was determined enzymatically (Wang W, Liu M, You C, Li Z, Zhang YP.2017.ATP-free biosynthesis of a high-energy phosphate free 1,6-diphosphate by in vitro metabolism engineering. Metal Eng 42: 168. 174.).

The experimental results are as follows: the concentration of fructose 1,6-diphosphate gradually increased with time to reach 40.1mM after 7 hours, with a molar conversion of fructose 1,6-diphosphate to xylose of 80.2%.

EXAMPLE 3 preparation of inositol from xylose and Formaldehyde

The catalytic pathways for the conversion of xylose and formaldehyde to inositol by an in vitro multi-enzyme catalytic system are shown in figure 1. Key enzymes involved in this pathway are: (1) xylose isomerase (Xylose isomerase, EC 5.3.1.5), catalyzing the conversion of Xylose to xylulose; (2) xylulokinase (EC 2.7.1.17), which catalyzes the conversion of xylulose and ATP to xylulose 5-phosphate and ADP; (3) ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1) catalyzes the isomerization of xylulose 5-phosphate and Ribulose 5-phosphate; (4) hexulose6-phosphate synthase (EC4.1.2.43), catalyzing the condensation of formaldehyde and ribulose5-phosphate, the product being hexulose 6-phosphate; (5) hexulose6-phosphate isomerase (6-phospho 3-hexulosomerase, EC 5.3.1.27), which catalyzes the isomerization of hexulose6-phosphate to fructose 6-phosphate; (6) phosphoglucose isomerase (EC 5.3.1.9), which catalyzes the conversion of fructose 6-phosphate to glucose 6-phosphate; (7) inositol 1-phosphate synthase (EC 5.5.1.4), which catalyzes the conversion of glucose 6-phosphate to Inositol 1-phosphate; (8) inositol monophosphatase (Inositol monophosphatase, EC3.1.3.25), catalyzes the dephosphorylation of Inositol 1-phosphate to myo-Inositol; (9) polyphosphate kinase (EC 2.7.4.1) catalyzes polyphosphoric acid and ADP to regenerate ATP.

In this example, phosphoglucose isomerase was derived from Thermus thermophilus HB8, with gene number TTC1710 (KEGG); inositol-1-phosphate synthase is derived from Archaeoglobus fulgidus, and has gene number AF-1794 (KEGG); the phytase was derived from Thermotoga maritima with the gene number TM1415 (KEGG). These genes were obtained by gene synthesis and cloned into pET vectors to obtain the corresponding expression plasmids pET28a-ttcpgi, pET20b-afips and pET20 b-tmimp. Then transferred into Escherichia coli BL21(DE3) for protein expression and purification. Other enzymes were expressed and purified as in the above examples.

A1.0 mL reaction system contained 50mM xylose, 60mM formaldehyde, 50mM Tris-HCl buffer (pH8.0), 10mM magnesium sulfate, 0.5mM manganese chloride, 2mM ADP, 20mM sodium hexametaphosphate, 2U/mL xylose isomerase, 2U/mL xylulokinase, 2U/mL ribulose phosphate 3-epimerase, 2U/mL 6-phosphohexulose synthase, 2U/mL 6-phosphohexulose isomerase, 2U/mL polyphosphate kinase, 2U/mL glucose phosphate isomerase, 2U/mL inositol 1-phosphate synthase, 2U/mL phytase. The enzyme-catalyzed reaction is first reacted at 37 ℃ for 8 hours, and then the temperature is raised to 70 ℃ for reaction to 30 hours. The concentration of inositol was determined by HPLC. The conditions for HPLC were: HPX-87H chromatographic column (Bio-Rad) and 5mM sulfuric acid solution as mobile phase, with flow rate of 0.6mL/min, column temperature of 60 deg.C, differential refractometer.

The experimental results are as follows: the reaction time was 30 hours, the inositol concentration reached 36mM, and the molar conversion of inositol to xylose was 72%.

EXAMPLE 4 preparation of psicose from xylose and Formaldehyde

The catalytic pathway for the conversion of xylose and formaldehyde to psicose by an in vitro multienzyme catalytic system is shown in figure 1. Key enzymes involved in this pathway are: (1) xylose isomerase (Xylose isomerase, EC 5.3.1.5), catalyzing the conversion of Xylose to xylulose; (2) xylulokinase (EC 2.7.1.17), which catalyzes the conversion of xylulose and ATP to xylulose 5-phosphate and ADP; (3) ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1) catalyzes the isomerization of xylulose 5-phosphate and Ribulose 5-phosphate; (4) hexulose6-phosphate synthase (EC 4.1.2.43), which catalyzes the condensation of formaldehyde and ribulose5-phosphate, the product being hexulose 6-phosphate; (5) hexulose6-phosphate isomerase (6-phospho 3-hexulosomerase, EC 5.3.1.27), which catalyzes the isomerization of hexulose6-phosphate to fructose 6-phosphate; (6) psicose 6-phosphate 3-epimerase (D-allolose 6-phosphate 3-epimerase, EC 5.1.3.-) catalyzes the isomerization of fructose 6-phosphate to psicose 6-phosphate; (7) a phosphatase catalyzing the dephosphorylation of psicose 6-phosphate to form psicose; (8) polyphosphate kinase (EC 2.7.4.1) catalyzes polyphosphoric acid and ADP to regenerate ATP.

In this example, the psicose 6-phosphate 3-epimerase is derived from Thermoanaerobacterium thermosaccharolyticum DSM 571, which has the gene number Tth-1731 (KEGG); the phosphatase was derived from Clostridium thermocellum and has the gene number Cth _0261 (KEGG). After obtaining the genes through gene synthesis, cloning the genes to pET20b vector to obtain corresponding expression plasmids pET20b-ttape and pET20b-cta6 pp. Then transferred into Escherichia coli BL21(DE3) for protein expression and purification. Other enzymes were expressed and purified as in the above examples.

A1.0 mL reaction system contained 50mM xylose, 60mM formaldehyde, 50mM Tris-HCl buffer (pH8.0), 10mM magnesium sulfate, 0.5mM manganese chloride, 2mM ADP, 20mM sodium hexametaphosphate, 2U/mL xylose isomerase, 2U/mL xylulokinase, 2U/mL ribulose phosphate 3-epimerase, 2U/mL 6-phosphohexulose synthase, 2U/mL 6-phosphohexuloisomerase, 2U/mL polyphosphate kinase, 2U/mL psicose 6-phosphate 3-epimerase, 2U/mL phosphatase. The enzyme-catalyzed reaction is carried out at 45 ℃. The concentration of psicose was determined by HPLC. The conditions for HPLC were: HPX-87H column (Bio-Rad) with 5mM sulfuric acid solution as mobile phase, flow rate 0.6mL/min, column temperature 60 deg.C, differential refractometer detector.

The experimental results are as follows: after 30 hours of reaction, the concentration of the psicose reaches 41mM, and the molar conversion rate of the psicose to xylose is 82%.

EXAMPLE 5 preparation of mannose from xylose and Formaldehyde

The catalytic pathway for the conversion of xylose and formaldehyde to mannose by an in vitro multi-enzyme catalytic system is shown in figure 1. Key enzymes involved in this pathway are: (1) xylose isomerase (Xylose isomerase, EC 5.3.1.5), catalyzing the conversion of Xylose to xylulose; (2) xylulokinase (EC 2.7.1.17), which catalyzes the conversion of xylulose and ATP to xylulose 5-phosphate and ADP; (3) ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1) catalyzes the isomerization of xylulose 5-phosphate and Ribulose 5-phosphate; (4) hexulose6-phosphate synthase (EC4.1.2.43), catalyzing the condensation of formaldehyde and ribulose5-phosphate, the product being hexulose 6-phosphate; (5) hexulose6-phosphate isomerase (6-phospho 3-hexulosomerase, EC 5.3.1.27), which catalyzes the isomerization of hexulose6-phosphate to fructose 6-phosphate; (6) mannose 6-phosphate isomerase (Mannose-6-phosphate isomerase, EC 5.3.1.8), which catalyzes the isomerization of fructose 6-phosphate to Mannose 6-phosphate; (7) a phosphatase catalyzing the dephosphorylation of mannose 6-phosphate to produce mannose; (8) polyphosphate kinase (EC 2.7.4.1) catalyzes polyphosphoric acid and ADP to regenerate ATP.

In this example, mannose-6-phosphate isomerase is derived from Thermus thermophilus HB27, with the gene number TTHA 1345; the phosphatase is derived from Thermotoga maritima MSB8, and the gene number is TM 0651. After obtaining these genes by gene synthesis, the genes were cloned into a PET vector to obtain the corresponding expression plasmids pET20b-tthmpi and pET21a-tmm6 pp. Then transferred into Escherichia coli BL21(DE3) for protein expression and purification. Other enzymes were expressed and purified as in the above examples.

A1.0 mL reaction system contained 50mM xylose, 60mM formaldehyde, 50mM Tris-HCl buffer (pH8.0), 10mM magnesium sulfate, 0.5mM manganese chloride, 2mM ADP, 20mM sodium hexametaphosphate, 2U/mL xylose isomerase, 2U/mL xylulokinase, 2U/mL ribulose phosphate 3-epimerase, 2U/mL 6-phosphohexulosynthase, 2U/mL 6-phosphohexuloisomerase, 2U/mL polyphosphate kinase, 2U/mL mannose 6-phosphate isomerase, 2U/mL phosphatase. The enzyme-catalyzed reaction is carried out at 45 ℃. The concentration of mannose was determined by HPLC. The conditions for HPLC were: HPX-87H column (Bio-Rad) with 5mM sulfuric acid solution as mobile phase, flow rate 0.6mL/min, column temperature 60 deg.C, differential refractometer detector.

The experimental results are as follows: after 48 hours of reaction, the concentration of mannose reached 35mM and the molar conversion of mannose to xylose was 70%.

Example 6 preparation of tagatose from xylose and Formaldehyde

The catalytic pathway for the conversion of xylose and formaldehyde to tagatose by an in vitro multi-enzyme catalytic system is shown in figure 1. Key enzymes involved in this pathway are: (1) xylose isomerase (Xylose isomerase, EC 5.3.1.5), catalyzing the conversion of Xylose to xylulose; (2) xylulokinase (EC 2.7.1.17), which catalyzes the conversion of xylulose and ATP to xylulose 5-phosphate and ADP; (3) ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1) catalyzes the isomerization of xylulose 5-phosphate and Ribulose 5-phosphate; (4) hexulose6-phosphate synthase (EC4.1.2.43), catalyzing the condensation of formaldehyde and ribulose5-phosphate, the product being hexulose 6-phosphate; (5) hexulose6-phosphate isomerase (6-phospho 3-hexulosomerase, EC 5.3.1.27), which catalyzes the isomerization of hexulose6-phosphate to fructose 6-phosphate; (6) tagatose6-phosphate epimerase (tagatose-6-phosphate epimerase) that catalyzes epimerization of fructose 6-phosphate to tagatose 6-phosphate; (7) phosphatase catalyzing tagatose6-phosphate to remove phosphate group to generate tagatose; (8) polyphosphate kinase (EC 2.7.4.1) catalyzes polyphosphoric acid and ADP to regenerate ATP.

In this example, tagatose 4-phosphate epimerase was derived from Dictyoglycous thermophilum, gene number DICTH _0118 (KEGG); the phosphatase was derived from Archaeoglobus fulgidus and its gene number was AF _0444 (KEGG). After obtaining these genes by gene synthesis, they were cloned into pET vector to obtain the corresponding expression plasmids pET20b-atutpe, pET21a-aft6 pp. Then transferred into Escherichia coli BL21(DE3) for protein expression and purification. Other enzymes were expressed and purified as in the above examples.

A1.0 mL reaction system contained 50mM xylose, 60mM formaldehyde, 50mM Tris-HCl buffer (pH8.0), 10mM magnesium sulfate, 0.5mM manganese chloride, 2mM ADP, 20mM sodium hexametaphosphate, 2U/mL xylose isomerase, 2U/mL xylulokinase, 2U/mL ribulose phosphate 3-epimerase, 2U/mL 6-phosphohexulose synthase, 2U/mL 6-phosphohexuloisomerase, 2U/mL polyphosphate kinase, 2U/mL tagatose6-phosphate 4-epimerase, 2U/mL phosphatase. The enzyme-catalyzed reaction is carried out at 45 ℃. The concentration of tagatose was determined by HPLC. The conditions for HPLC were: HPX-87H column (Bio-Rad) with 5mM sulfuric acid solution as mobile phase, flow rate 0.6mL/min, column temperature 60 deg.C, differential refractometer detector.

The experimental results are as follows: after 48 hours of reaction, the concentration of tagatose reached 37.6mM, and the molar conversion rate of allulose to xylose was 75.2%.

EXAMPLE 7 preparation of fructose 6-phosphate from xylose and methanol

The catalytic pathway for the conversion of xylose and methanol to fructose 6-phosphate by an in vitro multi-enzyme catalytic system is shown in FIG. 2. Key enzymes involved in this pathway are: (1) xylose isomerase (Xylose isomerase, EC 5.3.1.5), catalyzing the conversion of Xylose to xylulose; (2) xylulokinase (EC 2.7.1.17), which catalyzes the conversion of xylulose and ATP to xylulose 5-phosphate and ADP; (3) ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1) catalyzes the isomerization of xylulose 5-phosphate and Ribulose 5-phosphate; (4) methanol dehydrogenase (EC 1.1.1.1) catalyzes the reduction of Methanol to formaldehyde with the concomitant production of NADH; (5) NADH oxidase (NADH oxidase, EC1.6.3.4), catalyzing NADH and oxygen to produce NAD⁺And H₂O; (6) hexulose6-phosphate synthase (3-hexulose6-phosphate synthase, EC4.1.2.43), catalyzes the condensation of formaldehyde and ribulose5-phosphate, with the product hexulose 6-phosphate; (7) hexulose6-phosphate isomerase (6-phospho 3-hexulosomerase, EC 5.3.1.27), which catalyzes the isomerization of hexulose6-phosphate to fructose 6-phosphate; (8) polyphosphate kinase (EC 2.7.4.1) catalyzes polyphosphoric acid and ADP to regenerate ATP.

In this example, the alcohol dehydrogenase is derived from Geobacillus stearothermophilus DSM2334, and the gene number is Z25544.1 (Genebank); NADH oxidase is derived from Streptococcus mutans, and its gene is numbered SMU _ 1117. The gene is obtained by gene synthesis and cloned into a pET20b vector to obtain corresponding expression vectors pET20b-gsmdh and pET20 b-smnox. Then the plasmid is transformed into an escherichia coli expression bacterium BL21(DE3) for protein expression and purification. Other enzymes were expressed and purified as in the above examples.

A1.0 mL reaction system contained 50mM xylose, 60mM methanol, 50mM Tris-HCl buffer (pH8.0), 10mM magnesium sulfate, 0.5mM chlorideManganese, 2mM ADP, 20mM sodium hexametaphosphate, 0.01mM FAD, 1mM DTT, 5mM NAD⁺2U/mL alcohol dehydrogenase, 2U/mL NADH oxidase, 2U/mL xylose isomerase, 2U/mL xylulokinase, 2U/mL ribulose phosphate 3-epimerase, 2U/mL hexulose6-phosphate synthase, 2U/mL 6-hexulose phosphate isomerase, 2U/mL polyphosphate kinase. The catalytic reaction was carried out at 37 ℃ for 7 hours. The fructose 6-phosphate concentration was measured as in example 1.

The results of the experiment are shown in FIG. 5: the reaction was carried out at 37 ℃ for 7 hours, the concentration of fructose 6-phosphate reached 14.9mM, and the conversion of fructose 6-phosphate to xylose was 29.9%.

EXAMPLE 8 preparation of fructose 1, 6-bisphosphate from xylose and methanol

The catalytic pathways for the conversion of xylose and methanol to fructose 1, 6-bisphosphate by an in vitro multi-enzyme catalytic system are shown in FIG. 2. Key enzymes involved in this pathway are: (1) xylose isomerase (Xylose isomerase, EC 5.3.1.5), catalyzing the conversion of Xylose to xylulose; (2) xylulokinase (EC 2.7.1.17), which catalyzes the conversion of xylulose and ATP to xylulose 5-phosphate and ADP; (3) ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1) catalyzes the isomerization of xylulose 5-phosphate and Ribulose 5-phosphate; (4) methanol dehydrogenase (ec 1.1.1.1) catalyzes the reduction of Methanol to formaldehyde with the concomitant production of NADH; (5) NADH oxidase (NADH oxidase, EC1.6.3.4), catalyzes the production of NAD from NADH and oxygen⁺And H₂O; (6) hexulose6-phosphate synthase (EC 4.1.2.43), which catalyzes the condensation of formaldehyde and ribulose5-phosphate, the product being hexulose 6-phosphate; (7) hexulose6-phosphate isomerase (6-phospho 3-hexulosomerase, EC 5.3.1.27), which catalyzes the isomerization of hexulose6-phosphate to fructose 6-phosphate; (8) 6-phosphofructokinase (EC 2.7.1.11), which catalyzes fructose 6-phosphate and ATP to produce fructose 1,6-diphosphate and ADP; (9) polyphosphate kinase (EC 2.7.4.1) catalyzes polyphosphoric acid and ADP to regenerate ATP.

In this example, the expression of the enzyme was purified as in the above example.

In a 1.0mL reaction system containingThere were 50mM xylose, 60mM methanol, 50mM Tris-HCl buffer (pH8.0), 10mM magnesium sulfate, 0.5mM manganese chloride, 2mM ADP, 20mM sodium hexametaphosphate, 0.01mM FAD, 1mM DTT, 5mM NAD⁺2U/mL alcohol dehydrogenase, 2U/mL NADH oxidase, 2U/mL xylose isomerase, 2U/mL xylulokinase, 2U/mL ribulose phosphate 3-epimerase, 2U/mL hexulose6-phosphate synthase, 2U/mL 6-hexulose phosphate isomerase, 2U/mL polyphosphate kinase, 2U/mL fructose 6-phosphate kinase. The catalytic reaction was carried out at 37 ℃ for 7 hours. The concentration of fructose-1, 6-bisphosphate was determined as in example 2.

The experimental results are as follows: after 12 hours of reaction, the concentration of the fructose 1,6-diphosphate reaches 15.1mM, and the molar conversion rate of the product fructose 1,6-diphosphate to xylose is 30.2%.

EXAMPLE 9 preparation of inositol from xylose and methanol

The catalytic pathway for the conversion of xylose and methanol to inositol by an in vitro multi-enzyme catalytic system is shown in figure 2. Key enzymes involved in this pathway are: (1) xylose isomerase (Xylose isomerase, EC 5.3.1.5), catalyzing the conversion of Xylose to xylulose; (2) xylulokinase (EC 2.7.1.17), which catalyzes the conversion of xylulose and ATP to xylulose 5-phosphate and ADP; (3) ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1) catalyzes the isomerization of xylulose 5-phosphate and Ribulose 5-phosphate; (4) methanol dehydrogenase (EC 1.1.1.1) catalyzes the reduction of Methanol to formaldehyde with the concomitant production of NADH; (5) NADH oxidase (NADH oxidase, EC1.6.3.4), catalyzing NADH and oxygen to produce NAD⁺And H₂O; (6) hexulose6-phosphate synthase (3-hexulose6-phosphate synthase, EC4.1.2.43), catalyzes the condensation of formaldehyde and ribulose5-phosphate, with the product hexulose 6-phosphate; (7) hexulose6-phosphate isomerase (6-phospho 3-hexulosomerase, EC 5.3.1.27), which catalyzes the isomerization of hexulose6-phosphate to fructose 6-phosphate; (8) phosphoglucose isomerase (EC 5.3.1.9), which catalyzes the conversion of fructose 6-phosphate to glucose 6-phosphate; (9) inositol 1-phosphate synthase (EC5.5.1.4), which catalyzes the conversion of glucose 6-phosphate to Inositol 1-phosphate; (10) inositol monophosphatase (Inositol mon)ophosphatase, EC3.1.3.25), catalyzing the dephosphorylation of inositol 1-phosphate to inositol; (11) polyphosphate kinase (EC 2.7.4.1) catalyzes polyphosphoric acid and ADP to regenerate ATP.

A1.0 mL reaction system contained 50mM xylose, 60mM methanol, 50mM Tris-HCl buffer (pH8.0), 10mM magnesium sulfate, 0.5mM manganese chloride, 2mM ADP, 20mM sodium hexametaphosphate, 0.01mM FAD, 1mM DTT, 5mM NAD⁺2U/mL alcohol dehydrogenase, 2U/mL NADH oxidase, 2U/mL xylose isomerase, 2U/mL xylulokinase, 2U/mL ribulose phosphate 3-epimerase, 2U/mL 6-phosphohexulose synthase, 2U/mL 6-phosphohexuloisomerase, 2U/mL polyphosphate kinase, 2U/mL phosphoglucose isomerase, 2U/mL inositol-1-phosphate synthase, 2U/mL phytase. The enzyme-catalyzed reaction is first reacted at 37 ℃ for 8 hours, and then the temperature is raised to 70 ℃ for reaction to 30 hours. The concentration of inositol was determined as in example 3.

The experimental results are as follows: the reaction time is 48 hours, the concentration of the inositol reaches 10.8mM, and the molar conversion rate of the inositol to the xylose is 21.6%.

EXAMPLE 10 preparation of psicose from xylose and methanol

The catalytic pathway for the conversion of xylose and methanol to psicose by an in vitro multienzyme catalytic system is shown in figure 2. Key enzymes involved in this pathway are: (1) xylose isomerase (Xylose isomerase, EC 5.3.1.5), catalyzing the conversion of Xylose to xylulose; (2) xylulokinase (EC 2.7.1.17), which catalyzes the conversion of xylulose and ATP to xylulose 5-phosphate and ADP; (3) ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1) catalyzes the isomerization of xylulose 5-phosphate and Ribulose 5-phosphate; (4) methanol dehydrogenase (EC 1.1.1.1) catalyzes the reduction of Methanol to formaldehyde with the concomitant production of NADH; (5) NADH oxidase (NADH oxidase, EC1.6.3.4), catalyzes the production of NAD from NADH and oxygen⁺And H₂O; (6) hexulose6-phosphate synthase (3-hexulose6-phosphate synthase, EC4.1.2.43), catalyzes the condensation of formaldehyde and ribulose5-phosphate, with the product hexulose 6-phosphate; (7) 6-phosphorusAcid hexulose isomerase (6-phospho3-hexuloisomerase, EC 5.3.1.27), catalyzes the isomerization of hexulose6-phosphate to fructose 6-phosphate; (8) psicose 6-phosphate 3-epimerase (D-allolose 6-phosphate 3-epimerase, EC 5.1.3.-) catalyzes the isomerization of fructose 6-phosphate to psicose 6-phosphate; (9) a phosphatase catalyzing the dephosphorylation of psicose 6-phosphate to form psicose; (10) polyphosphate kinase (EC 2.7.4.1) catalyzes polyphosphoric acid and ADP to regenerate ATP.

A1.0 mL reaction system contained 50mM xylose, 60mM methanol, 50mM Tris-HCl buffer (pH8.0), 10mM magnesium sulfate, 0.5mM manganese chloride, 2mM ADP, 20mM sodium hexametaphosphate, 0.01mM FAD, 1mM DTT, 5mM NAD⁺2U/mL alcohol dehydrogenase, 2U/mL NADH oxidase, 2U/mL xylose isomerase, 2U/mL xylulokinase, 2U/mL ribulose phosphate 3-epimerase, 2U/mL hexulose6-phosphate synthase, 2U/mL 6-phosphohexuloisomerase, 2U/mL polyphosphate kinase, 2U/mL psicose 6-phosphate 3-epimerase, 2U/mL phosphatase. The enzyme-catalyzed reaction is carried out at 37 ℃. The allulose concentration was determined as in example 4.

The experimental results are as follows: after 48 hours of reaction, the concentration of the psicose reached 13.7mM, and the molar conversion rate of the psicose to xylose was 27.4%.

EXAMPLE 11 preparation of mannose from xylose and methanol

The catalytic pathway for the conversion of xylose and methanol to mannose by an in vitro multi-enzyme catalytic system is shown in figure 2. Key enzymes involved in this pathway are: (1) xylose isomerase (Xylose isomerase, EC 5.3.1.5), catalyzing the conversion of Xylose to xylulose; (2) xylulokinase (EC 2.7.1.17), which catalyzes the conversion of xylulose and ATP to xylulose 5-phosphate and ADP; (3) ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1) catalyzes the isomerization of xylulose 5-phosphate and Ribulose 5-phosphate; (4) methanol dehydrogenase (EC 1.1.1.1) catalyzes the reduction of Methanol to formaldehyde with the concomitant production of NADH; (5) NADH oxidase (NADH oxidase, EC1.6.3.4) catalyzing generation of NADH and oxygenTo NAD⁺And H₂O; (6) hexulose6-phosphate synthase (3-hexulose6-phosphate synthase, EC4.1.2.43), catalyzes the condensation of formaldehyde and ribulose5-phosphate, with the product hexulose 6-phosphate; (7) hexulose6-phosphate isomerase (6-phospho 3-hexulosomerase, EC 5.3.1.27), which catalyzes the isomerization of hexulose6-phosphate to fructose 6-phosphate; (8) mannose 6-phosphate isomerase (Mannose 6-phosphate isomerase, EC 5.3.1.8), which catalyzes the isomerization of fructose 6-phosphate to Mannose 6-phosphate; (9) a phosphatase catalyzing the dephosphorylation of mannose 6-phosphate to produce mannose; (10) polyphosphate kinase (EC 2.7.4.1) catalyzes polyphosphoric acid and ADP to regenerate ATP.

A1.0 mL reaction system contained 50mM xylose, 60mM methanol, 50mM Tris-HCl buffer (pH8.0), 10mM magnesium sulfate, 0.5mM manganese chloride, 2mM ADP, 20mM sodium hexametaphosphate, 0.01mM FAD, 1mM DTT, 5mM NAD⁺2U/mL alcohol dehydrogenase, 2U/mL NADH oxidase, 2U/mL xylose isomerase, 2U/mL xylulokinase, 2U/mL ribulose phosphate 3-epimerase, 2U/mL hexulose6-phosphate synthase, 2U/mL 6-phosphohexuloisomerase, 2U/mL polyphosphate kinase, 2U/mL mannose 6-phosphate isomerase, 2U/mL phosphatase. The enzyme-catalyzed reaction is carried out at 37 ℃. The concentration of mannose was measured as in example 5.

The experimental results are as follows: after 48 hours of reaction, the concentration of mannose reached 14.8mM and the molar conversion of mannose to xylose was 29.6%.

Example 12 preparation of tagatose from xylose and methanol

The catalytic pathway for xylose and methanol conversion to tagatose by an in vitro multi-enzyme catalytic system is shown in figure 2. Key enzymes involved in this pathway are: (1) xylose isomerase (Xylose isomerase, EC 5.3.1.5), catalyzing the conversion of Xylose to xylulose; (2) xylulokinase (EC 2.7.1.17), which catalyzes the conversion of xylulose and ATP to xylulose 5-phosphate and ADP; (3) ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1) catalyzes the isomerization of xylulose 5-phosphate and Ribulose 5-phosphate; (4) methanol dehydrogenase (Methanol dehydrogenase)enase, EC1.1.1.1), catalyzing the reduction of methanol to formaldehyde with the concomitant generation of NADH; (5) NADH oxidase (NADH oxidase, EC1.6.3.4), catalyzing NADH and oxygen to produce NAD⁺And H₂O; (6) hexulose6-phosphate synthase (3-hexulose6-phosphate synthase, EC4.1.2.43), catalyzes the condensation of formaldehyde and ribulose5-phosphate, with the product hexulose 6-phosphate; (7) hexulose6-phosphate isomerase (6-phospho 3-hexulosomerase, EC 5.3.1.27), which catalyzes the isomerization of hexulose6-phosphate to fructose 6-phosphate; (8) tagatose6-phosphate epimerase (tagatose6-phosphate epimerase) which catalyzes epimerization of fructose 6-phosphate into tagatose 6-phosphate; (9) phosphatase catalyzing tagatose6-phosphate to remove phosphate group to generate tagatose; (10) polyphosphate kinase (EC 2.7.4.1) catalyzes polyphosphoric acid and ADP to regenerate ATP.

A1.0 mL reaction system contained 50mM xylose, 60mM methanol, 50mM Tris-HCl buffer (pH8.0), 10mM magnesium sulfate, 0.5mM manganese chloride, 2mM ADP, 20mM sodium hexametaphosphate, 0.01mM FAD, 1mM DTT, 5mM NAD⁺2U/mL alcohol dehydrogenase, 2U/mL NADH oxidase, 2U/mL xylose isomerase, 2U/mL xylulokinase, 2U/mL ribulose phosphate 3-epimerase, 2U/mL hexulose6-phosphate synthase, 2U/mL 6-phosphohexuloisomerase, 2U/mL polyphosphate kinase, 2U/mL tagatose6-phosphate 4-epimerase, 2U/mL phosphatase. The enzyme-catalyzed reaction is carried out at 37 ℃. The tagatose concentration was measured in the same manner as in example 6.

The experimental results are as follows: after 48 hours of reaction, the concentration of tagatose reached 14.5mM, and the molar conversion rate of mannose to xylose was 29.0%.

EXAMPLE 13 preparation of fructose 6-phosphate from xylose and methanol

The catalytic pathway for the conversion of xylose and methanol to fructose 6-phosphate by an in vitro multi-enzyme catalytic system is shown in FIG. 3. Key enzymes involved in this pathway are: (1) xylose isomerase (Xylose isomerase, EC 5.3.1.5), catalyzing the conversion of Xylose to xylulose; (2) xylulokinase (EC 2.7.1.17), which catalyzes the conversion of xylulose and ATP to xylulose 5-phosphate and ADP; (3) ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1) catalyzes the isomerization of xylulose 5-phosphate and Ribulose 5-phosphate; (4) alcohol oxidase (EC 1.1.3.13), which catalyzes the reduction of methanol to formaldehyde while consuming oxygen to produce hydrogen peroxide; (5) catalase (Catalase, EC 1.11.1.6) for hydrogen peroxide generated as a by-product during the hydrolysis reaction; (6) hexulose6-phosphate synthase (EC 4.1.2.43), which catalyzes the condensation of formaldehyde and ribulose5-phosphate, the product being hexulose 6-phosphate; (7) hexulose6-phosphate isomerase (6-phospho 3-hexulosomerase, EC 5.3.1.27), which catalyzes the isomerization of hexulose6-phosphate to fructose 6-phosphate; (8) polyphosphate kinase (EC 2.7.4.1) catalyzes polyphosphoric acid and ADP to regenerate ATP.

In this example, alcohol oxidase was derived from Pichia pastoris, purchased from Sigma-Aldrich, under the accession number A2404. The catalase is derived from Bacillus subtilis, and the gene number of the catalase is BSU 08820. The gene is obtained by PCR, and is cloned into a pET20b vector by a method of Simple Cloning (You C, Zhang XZ, Zhang Y-HP.2012.Simple Cloning via direct transformation of PCR product (DNA Multimer) to Escherichia coli and Bacillus subtilis. appl.Environ.Microbiol.78(5):1593-5.), so as to obtain a corresponding expression vector pET20 b-bskatA. Then, the plasmid is transformed into an escherichia coli expression bacterium BL21(DE3) for protein expression and purification. Other enzymes were expressed and purified as in the above examples.

A1.0 mL reaction system contained 50mM xylose, 60mM methanol, 50mM Tris-HCl buffer (pH8.0), 10mM magnesium sulfate, 0.5mM manganese chloride, 2mM ADP, 20mM sodium hexametaphosphate, 0.01mM FAD, 2U/mL alcohol oxidase, 200U/mL catalase, 2U/mL xylose isomerase, 2U/mL xylulokinase, 2U/mL ribulose phosphate 3-epimerase, 2U/mL 6-hexulose phosphate synthase, 2U/mL 6-hexulose phosphate isomerase, 2U/mL polyphosphate kinase. The catalytic reaction was carried out at 37 ℃ for 7 hours. The concentration of fructose 6-phosphate was determined enzymatically, and fructose 6-phosphate was converted to glucose 6-phosphate catalyzed by Phosphoglucose Isomerase (EC 5.3.1.9), which was then determined using a glucose 6-phosphate assay kit (Sigma-Aldrich, cat # MAK 014).

As shown in FIG. 5, the concentration of fructose 6-phosphate gradually increased with time to reach 41.5mM after 7 hours, and the molar conversion of fructose 6-phosphate to xylose was 83.1%.

EXAMPLE 14 preparation of fructose 1, 6-bisphosphate from xylose and methanol

The catalytic pathway for the conversion of xylose and methanol to fructose 1, 6-bisphosphate by an in vitro multienzyme catalytic system is shown in FIG. 3. Key enzymes involved in this pathway are: (1) xylose isomerase (Xylose isomerase, EC 5.3.1.5), catalyzing the conversion of Xylose to xylulose; (2) xylulokinase (EC 2.7.1.17), which catalyzes the conversion of xylulose and ATP to xylulose 5-phosphate and ADP; (3) ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1) catalyzes the isomerization of xylulose 5-phosphate and Ribulose 5-phosphate; (4) alcohol oxidase (EC 1.1.3.13), which catalyzes the reduction of methanol to formaldehyde while consuming oxygen to produce hydrogen peroxide; (5) catalase (Catalase, EC 1.11.1.6) for hydrogen peroxide generated as a by-product during the hydrolysis reaction; (6) hexulose6-phosphate synthase (3-hexulose6-phosphate synthase, EC4.1.2.43), catalyzes the condensation of formaldehyde and ribulose5-phosphate, with the product hexulose 6-phosphate; (7) hexulose6-phosphate isomerase (6-phospho 3-hexulosomerase, EC 5.3.1.27), which catalyzes the isomerization of hexulose6-phosphate to fructose 6-phosphate; (8) 6-phosphofructokinase (EC 2.7.1.11), which catalyzes fructose 6-phosphate and ATP to produce fructose 1,6-diphosphate and ADP; (9) polyphosphate kinase (EC 2.7.4.1) catalyzes polyphosphoric acid and ADP to regenerate ATP.

A1.0 mL reaction system contained 50mM xylose, 60mM methanol, 50mM Tris-HCl buffer (pH8.0), 10mM magnesium sulfate, 0.5mM manganese chloride, 2mM ADP, 20mM sodium hexametaphosphate, 0.01mM FAD, 2U/mL alcohol oxidase, 200U/mL catalase, 2U/mL xylose isomerase, 2U/mL xylulokinase, 2U/mL ribulose phosphate 3-epimerase, 2U/mL 6-hexulose phosphate synthase, 2U/mL 6-hexulose phosphate isomerase, 2U/mL polyphosphate kinase, 2U/mL 6-phosphofructokinase. The catalytic reaction was carried out at 37 ℃ for 8 hours. The concentration of fructose-1, 6-bisphosphate was determined as in example 2.

The experimental results are shown in fig. 6: the concentration of fructose 1,6-diphosphate gradually increased with time to reach 42.9mM after 7 hours, with a molar conversion of fructose 1,6-diphosphate to xylose of 84.8%.

EXAMPLE 15 preparation of inositol from xylose and methanol

The catalytic pathway for the conversion of xylose and methanol to inositol by an in vitro multi-enzyme catalytic system is shown in figure 3. Key enzymes involved in this pathway are: (1) xylose isomerase (Xylose isomerase, EC 5.3.1.5), catalyzing the conversion of Xylose to xylulose; (2) xylulokinase (EC 2.7.1.17), which catalyzes the conversion of xylulose and ATP to xylulose 5-phosphate and ADP; (3) ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1) catalyzes the isomerization of xylulose 5-phosphate and Ribulose 5-phosphate; (4) alcohol oxidase (EC 1.1.3.13), which catalyzes the reduction of methanol to formaldehyde while consuming oxygen to produce hydrogen peroxide; (5) catalase (Catalase, EC 1.11.1.6) for hydrogen peroxide generated as a by-product during the hydrolysis reaction; (6) hexulose6-phosphate synthase (EC 4.1.2.43), which catalyzes the condensation of formaldehyde and ribulose5-phosphate, the product being hexulose 6-phosphate; (7) hexulose6-phosphate isomerase (6-phospho 3-hexulosomerase, EC 5.3.1.27), which catalyzes the isomerization of hexulose6-phosphate to fructose 6-phosphate; (8) phosphoglucose isomerase (EC 5.3.1.9), which catalyzes the conversion of fructose 6-phosphate to glucose 6-phosphate; (9) inositol 1-phosphate synthase (EC 5.5.1.4), which catalyzes the conversion of glucose 6-phosphate to Inositol 1-phosphate; (10) inositol monophosphatase (Inositol monophophase, EC3.1.3.25), which catalyzes the dephosphorylation of Inositol 1-phosphate to produce Inositol; (11) polyphosphate kinase (EC2.7.4.1) catalyzes polyphosphoric acid and ADP to regenerate ATP.

A1.0 mL reaction system contained 50mM xylose, 60mM methanol, 50mM Tris-HCl buffer (pH8.0), 10mM magnesium sulfate, 0.5mM manganese chloride, 2mM ADP, 20mM sodium hexametaphosphate, 0.01mM FAD, 2U/mL alcohol oxidase, 200U/mL catalase, 2U/mL xylose isomerase, 2U/mL xylulokinase, 2U/mL ribulose phosphate 3-epimerase, 2U/mL 6-hexulose phosphate synthase, 2U/mL 6-hexulose phosphate isomerase, 2U/mL polyphosphate kinase, 2U/mL glucose phosphate isomerase, 2U/mL inositol 1-phosphate synthase, 2U/mL inositol monophosphatase. The enzyme-catalyzed reaction is first reacted at 37 ℃ for 8 hours, and then the temperature is raised to 70 ℃ for reaction to 30 hours. The concentration of inositol was determined as in example 3.

The results of the experiment are shown in FIG. 7: the reaction time was 30 hours, the concentration of inositol reached 38.5mM, and the molar conversion of inositol to xylose was 77%.

EXAMPLE 16 preparation of psicose from xylose and methanol

The catalytic pathway for the conversion of xylose and methanol to psicose by an in vitro multienzyme catalytic system is shown in figure 3. Key enzymes involved in this pathway are: (1) xylose isomerase (Xylose isomerase, EC 5.3.1.5), catalyzing the conversion of Xylose to xylulose; (2) xylulokinase (EC 2.7.1.17), which catalyzes the conversion of xylulose and ATP to xylulose 5-phosphate and ADP; (3) ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1) catalyzes the isomerization of xylulose 5-phosphate and Ribulose 5-phosphate; (4) alcohol oxidase (EC 1.1.3.13), which catalyzes the reduction of methanol to formaldehyde while consuming oxygen to produce hydrogen peroxide; (5) catalase (Catalase, EC 1.11.1.6) for hydrogen peroxide generated as a by-product during the hydrolysis reaction; (6) hexulose6-phosphate synthase (EC 4.1.2.43), which catalyzes the condensation of formaldehyde and ribulose5-phosphate, the product being hexulose 6-phosphate; (7) hexulose6-phosphate isomerase (6-phospho 3-hexulosomerase, EC 5.3.1.27), which catalyzes the isomerization of hexulose6-phosphate to fructose 6-phosphate; (8) psicose 6-phosphate 3-epimerase (D-allolose 6-phosphate 3-epimerase, EC 5.1.3.-) catalyzes the isomerization of fructose 6-phosphate to psicose 6-phosphate; (9) a phosphatase catalyzing the dephosphorylation of psicose 6-phosphate to form psicose; (10) polyphosphate kinase (EC 2.7.4.1) catalyzes polyphosphoric acid and ADP to regenerate ATP.

A1.0 mL reaction system contained 50mM xylose, 60mM methanol, 50mM Tris-HCl buffer (pH8.0), 10mM magnesium sulfate, 0.5mM manganese chloride, 2mM ADP, 20mM sodium hexametaphosphate, 0.01mM FAD, 2U/mL alcohol oxidase, 200U/mL catalase, 2U/mL xylose isomerase, 2U/mL xylulokinase, 2U/mL ribulose phosphate 3-epimerase, 2U/mL 6-hexulose phosphate synthase, 2U/mL 6-hexulose phosphate isomerase, 2U/mL polyphosphate kinase, 2U/mL psicose 6-phosphate 3-epimerase, 2U/mL phosphatase. The enzyme-catalyzed reaction is carried out at 37 ℃. The allulose concentration was determined as in example 4.

The experimental results are as follows: after 48 hours of reaction, the concentration of the psicose reached 40.5mM, and the molar conversion rate of the psicose to xylose was 81.0%.

Example 17 preparation of mannose from xylose and methanol

The catalytic pathway for the conversion of xylose and methanol to mannose by an in vitro multi-enzyme catalytic system is shown in figure 3. Key enzymes involved in this pathway are: (1) xylose isomerase (Xylose isomerase, EC 5.3.1.5), catalyzing the conversion of Xylose to xylulose; (2) xylulokinase (EC 2.7.1.17), which catalyzes the conversion of xylulose and ATP to xylulose 5-phosphate and ADP; (3) ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1) catalyzes the isomerization of xylulose 5-phosphate and Ribulose 5-phosphate; (4) alcohol oxidase (EC 1.1.3.13), which catalyzes the reduction of methanol to formaldehyde while consuming oxygen to produce hydrogen peroxide; (5) catalase (Catalase, EC 1.11.1.6) for hydrogen peroxide generated as a by-product during the hydrolysis reaction; (6) hexulose6-phosphate synthase (EC 4.1.2.43), which catalyzes the condensation of formaldehyde and ribulose5-phosphate, the product being hexulose 6-phosphate; (7) hexulose6-phosphate isomerase (6-phospho 3-hexulosomerase, EC 5.3.1.27), which catalyzes the isomerization of hexulose6-phosphate to fructose 6-phosphate; (8) mannose 6-phosphate isomerase (Mannose 6-phosphate isomerase, EC 5.3.1.8), which catalyzes the isomerization of fructose 6-phosphate to Mannose 6-phosphate; (9) a phosphatase catalyzing the dephosphorylation of mannose 6-phosphate to produce mannose; (10) polyphosphate kinase (EC 2.7.4.1) catalyzes polyphosphoric acid and ADP to regenerate ATP.

A1.0 mL reaction system contained 50mM xylose, 60mM methanol, 50mM Tris-HCl buffer (pH8.0), 10mM magnesium sulfate, 0.5mM manganese chloride, 2mM ADP, 20mM sodium hexametaphosphate, 0.01mM FAD, 2U/mL alcohol oxidase, 200U/mL catalase, 2U/mL xylose isomerase, 2U/mL xylulokinase, 2U/mL ribulose phosphate 3-epimerase, 2U/mL 6-hexulose phosphate synthase, 2U/mL 6-hexulose phosphate isomerase, 2U/mL polyphosphate kinase, 2U/mL mannose 6-phosphate isomerase, 2U/mL phosphatase. The enzyme-catalyzed reaction is carried out at 37 ℃. The allulose concentration was determined as in example 5.

The experimental results are as follows: after 48 hours of reaction, the concentration of mannose reached 42.1mM and the molar conversion of mannose to xylose was 84.2%.

Example 18 preparation of tagatose from xylose and methanol

The catalytic pathway for xylose and methanol conversion to tagatose by an in vitro multi-enzyme catalytic system is shown in figure 3. Key enzymes involved in this pathway are: (1) xylose isomerase (Xylose isomerase, EC 5.3.1.5), catalyzing the conversion of Xylose to xylulose; (2) xylulokinase (EC 2.7.1.17), which catalyzes the conversion of xylulose and ATP to xylulose 5-phosphate and ADP; (3) ribulose phosphate 3-epimerase (Ribulose5-phosphate 3-epimerase, EC 5.1.3.1) catalyzes the isomerization of xylulose 5-phosphate and Ribulose 5-phosphate; 4) alcohol oxidase (EC 1.1.3.13), which catalyzes the reduction of methanol to formaldehyde while consuming oxygen to produce hydrogen peroxide; (5) catalase (Catalase, EC 1.11.1.6) for hydrogen peroxide generated as a by-product during the hydrolysis reaction; (6) hexulose6-phosphate synthase (EC 4.1.2.43), which catalyzes the condensation of formaldehyde and ribulose5-phosphate, the product being hexulose 6-phosphate; (7) hexulose6-phosphate isomerase (6-phospho 3-hexulosomerase, EC 5.3.1.27), which catalyzes the isomerization of hexulose6-phosphate to fructose 6-phosphate; (8) tagatose6-phosphate epimerase (tagatose6-phosphate epimerase) which catalyzes epimerization of fructose 6-phosphate into tagatose 6-phosphate; (9) the phosphatase catalyzes dephosphorylation of tagatose6-phosphate to produce tagatose. (10) Polyphosphate kinase (EC 2.7.4.1) catalyzes polyphosphoric acid and ADP to regenerate ATP.

A1.0 mL reaction system contained 50mM xylose, 60mM methanol, 50mM Tris-HCl buffer (pH8.0), 10mM magnesium sulfate, 0.5mM manganese chloride, 2mM ADP, 20mM sodium hexametaphosphate, 0.01mM FAD, 2U/mL alcohol oxidase, 200U/mL catalase, 2U/mL xylose isomerase, 2U/mL xylulokinase, 2U/mL ribulose phosphate 3-epimerase, 2U/mL 6-hexulose phosphate synthase, 2U/mL 6-hexulose phosphate isomerase, 2U/mL polyphosphate kinase, 2U/mL tagatose6-phosphate 4-epimerase, 2U/mL phosphatase. The enzyme-catalyzed reaction is carried out at 37 ℃. The tagatose concentration was measured in the same manner as in example 6.

The experimental results are as follows: after 48 hours of reaction, the concentration of tagatose reached 36.8mM, and the molar conversion rate of mannose to xylose was 73.6%.

Example 19 preparation of fructose 6-phosphate from xylose solution and methanol obtained from Biomass pretreatment

The biological hyaluronic acid treatment hydrolysate or the gas explosion treatment hydrolysate contains a large amount of xylose, and can replace pure xylose to be used in the invention.

In this example, the corncobs were treated with dilute acid under 2% sulfuric acid at 175 ℃ for 5 minutes. Then extracting with water, concentrating, decoloring and neutralizing with pH to obtain biomass hydrolysate containing high-concentration xylose.

A1.0 mL reaction system contained biomass hydrolysate in an amount equivalent to 50mM xylose, 60mM methanol, 50mM Tris-HCl buffer (pH8.0), 10mM magnesium sulfate, 0.5mM manganese chloride, 2mM ADP, 20mM sodium hexametaphosphate, 0.01mM FAD, 2U/mL alcohol oxidase, 200U/mL catalase, 2U/mL xylose isomerase, 2U/mL xylulokinase, 2U/mL ribulose phosphate 3-epimerase, 2U/mL hexulose6-phosphate synthase, 2U/mL hexulose6-phosphate isomerase, 2U/mL polyphosphate kinase. The catalytic reaction was carried out at 37 ℃ for 12 hours. The fructose 6-phosphate concentration was measured as in example 1.

As a result of the experiment, the concentration of fructose 6-phosphate at the end of the reaction was 39.5mM, and the conversion of xylose by the product was 79.0%.

Example 20 preparation of fructose 1, 6-bisphosphate from xylose solution and methanol obtained from Biomass pretreatment

In this example, the expression of the enzyme was purified as in the above example. The biomass hydrolysate was prepared as in the above example.

A1.0 mL reaction system contained biomass hydrolysate in an amount equivalent to 50mM xylose, 60mM methanol, 50mM Tris-HCl buffer (pH8.0), 10mM magnesium sulfate, 0.5mM manganese chloride, 2mM ADP, 20mM sodium hexametaphosphate, 0.01mM FAD, 2U/mL alcohol oxidase, 200U/mL catalase, 2U/mL xylose isomerase, 2U/mL xylulokinase, 2U/mL ribulose phosphate 3-epimerase, 2U/mL hexulose6-phosphate synthase, 2U/mL hexulose6-phosphate isomerase, 2U/mL polyphosphate kinase, 2U/mL fructokinase 6-phosphate. The catalytic reaction was carried out at 37 ℃ for 12 hours. The concentration of fructose-1, 6-bisphosphate was determined as in example 2.

As shown in FIG. 8, the concentration of fructose-1, 6-bisphosphate was 40.1mM and the conversion of xylose by the product was 80.2% after the completion of the reaction.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for immobilizing a carbon compound using xylose, the method comprising the steps of:

2. The method of claim 1, further comprising the step of converting methanol to formaldehyde, wherein the converting step is catalyzed by methanol dehydrogenase, NADH oxidase.

3. The process of claim 1, further comprising the step of converting the methanol to formaldehyde, wherein the converting step is catalyzed by an alcohol oxidase, a catalase.

4. The method according to claim 1, wherein the concentrations of xylose and formaldehyde are 1 to 1000mM, respectively, the amount of xylose isomerase used is 0.1 to 10000U/mL, the amount of xylulokinase used is 0.1 to 10000U/mL, the amount of ribulose phosphate 3-epimerase used is 0.1 to 10000U/mL, the amount of 6-phosphohexulose synthase used is 0.1 to 10000U/mL, and the amount of polyphosphate kinase used is 0.1 to 10000U/mL;

preferably, the concentration of xylose is 50mM, the concentration of formaldehyde or methanol is 60mM, the dosage of xylose isomerase is 10U/mL, the dosage of xylulokinase is 10U/mL, the dosage of ribulose phosphate 3-epimerase is 10U/mL, the dosage of 6-hexulose phosphate synthase is 10U/mL, and the dosage of polyphosphate kinase is 10U/mL;

the enzyme catalysis reaction system is carried out at the temperature of 20-90 ℃ and the reaction time is 1-100 hours;

the multi-enzyme reaction system also contains buffer solution, magnesium salt, manganese salt, polyphosphoric acid and Adenosine Diphosphate (ADP);

preferably, the buffer solution is Tris-HCl buffer solution, the pH value of the buffer solution is 4.0-9.0, and the concentration of the buffer solution is 20-500 mM; magnesium salt is magnesium sulfate with concentration of 0.01-500 mM; manganese salt is manganese sulfate, and the concentration is 0.01-500 mM; the polyphosphate is sodium polyphosphate with the concentration of 5-500 mM; the concentration of Adenosine Diphosphate (ADP) is 0.01-500 mM.

5. The method according to claim 2, wherein the concentrations of xylose and methanol are 1 to 1000mM, respectively, the amount of xylose isomerase is 0.1 to 10000U/mL, the amount of xylulokinase is 0.1 to 10000U/mL, the amount of ribulose phosphate 3-epimerase is 0.1 to 10000U/mL, the amount of 6-hexulose phosphate synthase is 0.1 to 10000U/mL, the amount of polyphosphate kinase is 0.1 to 10000U/mL, the amount of methanol dehydrogenase is 0.1 to 10000U/mL, and the amount of NADH oxidase is 0.1 to 10000U/mL;

preferably, the concentration of xylose is 50mM, the concentration of methanol is 60mM, the dosage of xylose isomerase is 10U/mL, the dosage of xylulokinase is 10U/mL, the dosage of ribulose phosphate 3-epimerase is 10U/mL, the dosage of 6-hexulose phosphate synthase is 10U/mL, the dosage of polyphosphate kinase is 10U/mL, the dosage of methanol dehydrogenase is 10U/mL, and the dosage of NADH oxidase is 10U/mL;

the enzyme catalysis reaction system is carried out at the temperature of 30-90 ℃ and the reaction time is 1-100 hours;

the multi-enzyme reaction system also comprises buffer solution, magnesium salt, manganese salt, polyphosphoric acid, Adenosine Diphosphate (ADP), nicotinamide adenine dinucleotide (NAD +) and reducing agent;

preferably, the buffer solution is Tris-HCl buffer solution, the pH value of the buffer solution is 4.0-9.0, and the concentration of the buffer solution is 20-500 mM; magnesium salt is magnesium sulfate with concentration of 0.01-500 mM; manganese salt is manganese sulfate, and the concentration is 0.01-500 mM; the polyphosphate is sodium polyphosphate with the concentration of 5-500 mM; adenosine Diphosphate (ADP) at a concentration of 0.01-500 mM; nicotinamide adenine dinucleotide (NAD +) at a concentration of 0.01 to 500 mM; the reducing agent is Dithiothreitol (DTT) with concentration of 0.001-100 mM.

6. The method according to claim 3, wherein the concentrations of xylose and methanol are 1 to 1000mM, xylose isomerase is 0.1 to 10000U/mL, xylulokinase is 0.1 to 10000U/mL, ribulose phosphate 3-epimerase is 0.1 to 10000U/mL, hexulose6-phosphate synthase is 0.1 to 10000U/mL, polyphosphate kinase is 0.1 to 10000U/mL, alcohol oxidase is 0.1 to 10000U/mL, and catalase is 0.1 to 10000U/mL, respectively;

preferably, the concentration of xylose is 50mM, the concentration of methanol is 60mM, the dosage of xylose isomerase is 10U/mL, the dosage of xylulokinase is 10U/mL, the dosage of ribulose phosphate 3-epimerase is 10U/mL, the dosage of 6-hexulose phosphate synthase is 10U/mL, the dosage of polyphosphate kinase is 10U/mL, the dosage of alcohol oxidase is 10U/mL, and the dosage of catalase is 10U/mL;

The multi-enzyme reaction system also comprises buffer solution, magnesium salt, manganese salt, polyphosphoric acid, Adenosine Diphosphate (ADP) and Flavin Adenine Dinucleotide (FAD);

preferably, the buffer is Tris-HCl buffer, the pH value of the buffer is 4.0-9.0, and the concentration of the buffer is 20-500 mM; magnesium salt is magnesium sulfate with concentration of 0.01-500 mM; manganese salt is manganese sulfate, and the concentration is 0.01-500 mM; the polyphosphate is sodium polyphosphate with the concentration of 5-500 mM; adenosine Diphosphate (ADP) at a concentration of 0.01-500 mM; the concentration of Flavin Adenine Dinucleotide (FAD) is 0.00001-10 mM.

7. The method of any of claims 1-6, further comprising the step of converting hexulose6-phosphate to fructose 6-phosphate, the step being catalyzed by hexulose6-phosphate isomerase.

8. The method of claim 7, wherein the hexulose-6-phosphate isomerase is present in an amount of 0.1 to 10000U/mL, preferably 10U/mL.

9. A process for producing inositol, which comprises the step of converting fructose-6-phosphate into inositol by the process according to claim 7 or 8:

converting fructose-6-phosphate into inositol-1-phosphate by using inositol-1-phosphate synthetase;

inositol 1-phosphate is converted to inositol using an inositol monophosphatase.

10. The method of claim 9, wherein the amount of inositol 1-phosphate synthase is 0.1-10000U/mL, and the amount of phytase is 0.1-10000U/mL;

preferably, the amount of inositol 1-phosphate synthase is 10U/mL and the amount of inositol monophosphatase is 10U/mL.