CN-121991872-A - Recombinant escherichia coli for producing D-tagatose by cofactor regeneration driving oxidoreductase, construction method and application

Abstract

The invention discloses recombinant escherichia coli for producing D-tagatose by cofactor regeneration driving oxidoreductase, a construction method and application thereof, and belongs to the technical field of bioengineering. The invention introduces xylose reductase xyrB, glucose dehydrogenase Gox2015, galactitol dehydrogenase RlGDH mutant T193G/G98C and water-producing NADH oxidase SpNox into escherichia coli to construct recombinant escherichia coli. The invention designs and constructs a cofactor regeneration driving oxidation-reduction enzyme synthesis D-tagatose pathway, and the pathway combines with beta-galactosidase to completely convert lactose in whey into D-tagatose and sodium gluconate, thereby realizing the high-value utilization of whey, a dairy product processing byproduct.

Inventors

• TANG HONGCHI
• PANG HAO
• WANG ZILONG
• LIN LIHUA
• DU LIQIN
• HUANG RIBO
• LIU YI
• ZHOU LANG

Assignees

• 广西科学院

Dates

Publication Date

20260508

Application Date

20260305

Claims (10)

1. 1. The recombinant escherichia coli for producing the D-tagatose is characterized by comprising recombinant escherichia coli I and recombinant escherichia coli II, wherein the recombinant escherichia coli I simultaneously expresses xylose reductase xyrB, glucose dehydrogenase Gox2015, a galactitol dehydrogenase mutant T193G/G98C and a water-producing NADH oxidase SpNox, and the recombinant escherichia coli II simultaneously expresses the galactitol dehydrogenase mutant T193G/G98C and the water-producing NADH oxidase SpNox; The amino acid sequence of xylose reductase xyrB is shown as SEQ ID NO.1, the amino acid sequence of glucose dehydrogenase Gox2015 is shown as SEQ ID NO.2, the amino acid sequence of galactitol dehydrogenase mutant T193G/G98C is shown as SEQ ID NO.4, and the amino acid sequence of water-producing NADH oxidase SpNox is shown as SEQ ID NO. 3.
2. 2. A method of constructing a recombinant escherichia coli as defined in claim 1, comprising the method of (1) or (2): (1) Coexpression of coding genes of xylose reductase xyrB and glucose dehydrogenase Gox2015 in an expression vector to construct a recombinant plasmid of a reduction module; Coexpression of coding genes of a galactitol dehydrogenase mutant T193G/G98C and a water-producing NADH oxidase SpNox on an expression vector to construct an oxidation module recombinant plasmid; Co-transforming the reduction module recombinant plasmid and the oxidation module recombinant plasmid into competent cells of the escherichia coli T7 to obtain the recombinant escherichia coli I; (2) Coexpression of coding genes of a galactitol dehydrogenase mutant T193G/G98C and a water-producing NADH oxidase SpNox on an expression vector to construct an oxidation module recombinant plasmid; And (3) transforming the recombinant plasmid of the oxidation module into competent cells of the escherichia coli T7 to obtain the recombinant escherichia coli II.
3. 3. Use of the recombinant escherichia coli of claim 1 for the production of D-tagatose.
4. 4. A method for producing D-tagatose, comprising the steps of: (1) Taking hydrolysate of whey powder after beta-galactosidase hydrolysis as a substrate, or taking D-galactose as a substrate and D-glucose as a cofactor NADPH regenerant, adding the cell-breaking supernatant of the recombinant escherichia coli I as a catalyst in claim 1, and reacting under the condition of introducing oxygen to completely convert the D-galactose and the D-glucose into D-tagatose and sodium gluconate; (2) And separating and purifying the reaction liquid to obtain the D-tagatose.
5. 5. The method of claim 4, wherein in the step (1), the final concentration of the cell-destroying supernatant is 10 mg/mL, the reaction condition is that the temperature is 30 ℃, the oscillating rotation speed is 180 rpm, the pH value of a reaction system is 8.0, and the reaction time is 24 h; And/or in the step (2), the separation and purification method comprises the steps of adding CaCl 2 into the reaction solution, converting sodium gluconate into calcium gluconate, precipitating, centrifugally separating the precipitate, and desalting the supernatant by ion exchange to obtain the D-tagatose.
6. 6. A method for producing D-tagatose, comprising the method shown in the following (1) or (2): (1) Taking galactitol as a substrate, adding recombinant escherichia coli II as a catalyst in claim 1, and performing whole-cell reaction on the mixture 24 h to obtain D-tagatose; (2) D-galactose is taken as a substrate, D-glucose is taken as a cofactor NADPH regenerant, the recombinant escherichia coli I as a catalyst in claim 1 is added, the pH value is regulated, the pH value is kept at 8.0,30 ℃, and the whole cell reaction of 180 rpm is carried out for 24 h, so that D-tagatose is obtained.
7. 7. A recombinant escherichia coli for producing galactitol, wherein the recombinant escherichia coli expresses xylose reductase xyrB and glucose dehydrogenase Gox2015 simultaneously; the amino acid sequence of xylose reductase xyrB is shown as SEQ ID NO.1, and the amino acid sequence of glucose dehydrogenase Gox2015 is shown as SEQ ID NO. 2.
8. 8. A method of producing the recombinant escherichia coli of claim 7, comprising the steps of: Coexpression of coding genes of xylose reductase xyrB and glucose dehydrogenase Gox2015 in an expression vector to construct a recombinant plasmid of a reduction module; And transforming the recombinant plasmid of the reduction module into competent cells of the escherichia coli T7 to obtain the recombinant escherichia coli.
9. 9. Use of the recombinant escherichia coli of claim 7 for the production of galactitol.
10. 10. A process for preparing galactitol includes such steps as preparing the regenerated reagent of NADPH as cofactor from D-galactose, regulating pH value to 8.0, and full-cell reaction to 6 h.

Description

Recombinant escherichia coli for producing D-tagatose by cofactor regeneration driving oxidoreductase, construction method and application Technical Field The invention relates to the technical field of bioengineering, in particular to recombinant escherichia coli for producing D-tagatose by cofactor regeneration driving oxidoreductase, a construction method and application thereof. Background The production of dairy products such as cheese, yoghurt and casein produces about 2 hundred million tons of whey per year with an annual growth rate of 4%. In developing countries, whey is still treated as waste, either directly discharged into water resources or sent to landfills, among small and medium cheese manufacturers. If discharged without pretreatment, it may have a negative impact on the environment due to its higher chemical and biological oxygen demand. The past research results pay more attention to recovery of whey proteins, most of lactose is wasted, and disposal thereof also brings serious environmental problems. In recent years, the high-value utilization of lactose, which accounts for about 70% of whey dry matter, has been increasingly emphasized. Lactose or its hydrolysates (D-glucose and D-galactose) in whey are converted by microorganisms or enzymes into valuable biological products like biogas, bioplastic or rare sugars etc. The hydrolysis product D-galactose of lactose in whey is a direct substrate for synthesizing D-tagatose, and the hydrolysis product D-galactose is converted into D-tagatose by adopting a biological method, so that remarkable social and economic benefits are brought. D-tagatose is a natural low calorie functional sweetener with a sweetness of 92% of sucrose but only 38% of the calories. The preparation method has the advantages of reducing blood sugar, resisting oxidation, improving intestinal flora and other biological activities, and has wide application in the fields of foods and medicines. In addition, D-tagatose is listed by the U.S. Food and Drug Administration (FDA) as a material that is Generally Recognized As Safe (GRAS). In the past, whey or lactose was converted to D-tagatose primarily by two-step enzymatic conversion with β -galactosidase and L-arabinose isomerase. However, due to the thermodynamic equilibrium limitations of the L-arabinose isomerase, the equilibrium ratio of D-galactose to D-tagatose is typically between 7:3 and 4:6, which results in a very low conversion rate of D-tagatose from lactose, and no significant whey or lactose processing benefits. The conversion rate of lactose to synthesize D-tagatose is improved, and the method has important significance for realizing the industrialized biosynthesis of the D-tagatose and improving the economic benefit of whey powder processing industry. Disclosure of Invention The invention aims to provide a recombinant escherichia coli for producing D-tagatose by using cofactor regeneration to drive oxidoreductase, a construction method and application thereof, so as to solve the problems in the prior art, and establish a lactose hydrolysis-double enzyme reduction-double enzyme oxidation three-step enzyme method high-efficiency conversion process based on cofactor regeneration circulation, so that lactose is completely converted into D-tagatose and sodium gluconate, and an efficient multienzyme catalytic system is established to be applied to the preparation of D-tagatose, thereby having great significance in improving the economic benefit of whey powder processing industry. In order to achieve the above object, the present invention provides the following solutions: The invention provides a recombinant escherichia coli for producing D-tagatose, which comprises recombinant escherichia coli I and recombinant escherichia coli II, wherein the recombinant escherichia coli I simultaneously expresses xylose reductase xyrB, glucose dehydrogenase Gox2015, a galactitol dehydrogenase mutant T193G/G98C and a water-producing NADH oxidase SpNox, and the recombinant escherichia coli II simultaneously expresses the galactitol dehydrogenase mutant T193G/G98C and the water-producing NADH oxidase SpNox; The amino acid sequence of xylose reductase xyrB is shown as SEQ ID NO.1, the amino acid sequence of glucose dehydrogenase Gox2015 is shown as SEQ ID NO.2, the amino acid sequence of galactitol dehydrogenase mutant T193G/G98C is shown as SEQ ID NO.4, and the amino acid sequence of water-producing NADH oxidase SpNox is shown as SEQ ID NO. 3. The invention also provides a construction method of the recombinant escherichia coli, which comprises the following steps of (1) or (2): (1) Coexpression of coding genes of xylose reductase xyrB and glucose dehydrogenase Gox2015 in an expression vector to construct a recombinant plasmid of a reduction module; Coexpression of coding genes of a galactitol dehydrogenase mutant T193G/G98C and a water-producing NADH oxidase SpNox on an expression vector to construct an oxidation module recombinant pla