CN-121991244-A - Recombinant domain fusion enzyme and application thereof

Abstract

The invention discloses a recombinant domain fusion enzyme and application thereof in preparation of D-psicose, wherein the recombinant domain fusion enzyme is formed by splicing and recombining a 5 'domain and a part of a 3' domain of D-tagatose 3-differential enzyme with an active center domain of xylose isomerase. The invention takes the domain fusion enzyme of the 5 'domain and part of the 3' domain of the D-tagatose 3-epimerase and the active center domain of xylose isomerase as a biocatalyst, and the yield of catalyzing 260g/L glucose isomerization reaction to produce D-psicose is 35.9%. The method has the advantages of high speed, high efficiency, simple reaction condition, high substrate conversion rate and few byproducts, reduces the pressure of separation and purification, and is suitable for large-scale industrial production.

Inventors

• LI JINSHAN
• TAN MING
• CHEN LU
• XU JIANYONG

Assignees

• 天工生物科技（天津）有限公司

Dates

Publication Date

20260508

Application Date

20241104

Claims (5)

1. 1. A recombinant domain fusion enzyme, which is characterized in that the recombinant domain fusion enzyme is formed by splicing and recombining a5 'domain and a part of a 3' domain of D-tagatose 3-epimerase with an active center domain of xylose isomerase.
2. 2. The D-tagatose 3-epimerase (DTE) is derived from rhodobacter sphaeroides, the amino acid sequence is shown as SEQ ID No. 2, the nucleotide sequence of a coding gene is shown as SEQ ID No. 3, the xylose isomerase (XYI) is derived from Thermotoga maritima, the amino acid sequence is shown as SEQ ID No. 4, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 5.
3. 3. The nucleotide sequence of the recombinant domain fusion enzyme (DTE-5 '-XYI-VE-DTE-3') coding gene is shown as SEQ ID No. 9.
4. 4. The construction of the recombinant gene engineering bacteria and the vector containing the recombinant fusion enzyme gene comprises the following steps of constructing a nucleotide sequence of a recombinant domain fusion enzyme (DTE-5 '-XYI-VE-DTE-3') encoding gene, amplifying encoding genes of a 5 'domain (DTE-5'), a xylose isomerase active center domain (XYI-VE) and a D-tagatose 3-differential enzyme part 3 'domain (DTE-3') respectively in sequence, splicing the three encoding genes into a fusion gene segment by using overlap PCR, and connecting the fusion gene segment to NdeI and NotI restriction enzyme cleavage sites of a vector pET22b by a one-step cloning method to obtain a recombinant plasmid. And transforming the recombinant plasmid into escherichia coli E.coli BL21 (DE 3) to obtain the recombinant genetically engineered bacterium containing the recombinant fusion enzyme gene.
5. 5. The application of recombinant domain fusion enzyme (DTE-5 '-XYI-VE-DTE-3') in preparing D-psicose comprises the steps of fermenting and culturing engineering bacteria containing fusion enzyme (DTE-5 '-XYI-VE-DTE-3') coding genes to obtain crude enzyme liquid, resting cells, purifying pure enzyme or immobilized enzyme serving as a biological catalyst, adding substrate glucose, forming a conversion system by taking 50mM buffer solution with pH value of 6.0-10.0 as a reaction medium, reacting at 50-65 ℃ for 8-24 hours, performing ion exchange and decolorization on reaction supernatant, and separating by macroporous resin to obtain D-psicose and unreacted substrate glucose.

Description

Recombinant domain fusion enzyme and application thereof Technical Field The invention belongs to the technical field of bioengineering, and particularly relates to a recombinant domain fusion enzyme and application thereof in preparation of D-psicose. Background D-psicose is a very low content of hexose in nature, and is an epimer of the C-3 site of D-fructose. D-psicose is hardly digested and absorbed and hardly provides energy for the life activities, and thus is a very useful low-calorie sweetener. The D-psicose 3-epimerase or D-tagatose 3-epimerase can achieve the interconversion between D-fructose and D-psicose. D-fructose can be produced by catalyzing glucose to generate epimerization reaction through glucose isomerase or xylose isomerase. Therefore, by using a double enzyme system composed of D-psicose 3-epimerase or D-tagatose 3-epimerase and glucose isomerase or xylose isomerase, D-psicose can be produced in one step from glucose in the reaction system, but a large amount of intermediate D-fructose exists in the process, which greatly affects the conversion efficiency, which is unavoidable in the conventional double enzyme reaction system or the fusion enzyme reaction system composed of double enzymes through connecting peptides. The functional domains of the enzyme molecules are constructed into brand new enzyme molecules by utilizing gene fusion, and thus the recombinant domain fusion enzyme can be obtained. Different from the simple way of combining two enzyme molecules into one macromolecule through a connecting peptide chain, the recombinant domain fusion enzyme has more similar functional domains in structure, stronger generated 'proximity effect', more obvious promotion effect on promoting forward direction of enzymatic reaction and reducing free energy of reaction, and lower enzyme molecular weight than the fusion enzyme simply connected through the connecting peptide chain, and can avoid the problems of insoluble expression and difficult correct folding of macromolecular proteins caused by the connection of different enzyme molecules. Chinese patent 202010805264. X discloses a recombinant fusion enzyme and application thereof in synthesizing methyl glyoxylate, wherein macromolecular recombinant enzyme constructed by connecting glycolytic acid oxidase, catalase and hemoglobin through connecting peptide is used as a biocatalyst to catalyze the oxidation of 200mM methyl glyoxylate, and the yield of the methyl glyoxylate after 6 hours of reaction reaches 95.4%. Chinese patent 202111188472.0 discloses a fusion enzyme for producing nicotinamide mononucleotide and application thereof, which connects nicotinamide ribophosphate transferase and ribophosphate pyrophosphatase through connecting peptide to form macromolecular recombinase, and converts the macromolecular recombinase into escherichia coli or bacillus subtilis to be used as a cell catalyst or purified, and nicotinamide is used as a substrate to ferment and produce the nicotinamide mononucleotide, wherein the yield of the nicotinamide mononucleotide can be improved by more than 50%. Chinese patent 202210352448.4 discloses a fusion protein for catalyzing glucose to synthesize D-psicose and a construction method thereof, which utilizes flexible connecting peptides and rigid connecting peptides with different lengths to connect glucose isomerase genes and D-psicose 3-epimerase, uses macromolecule recombinase with the best screening effect as a catalyst, uses 100g/L glucose as a substrate, and has the yield of D-psicose not lower than 5.69 g/L. Although improved as compared to the reaction system of the two enzymes, the conversion efficiency is still lower. Disclosure of Invention The invention discloses a recombinant domain fusion enzyme and application thereof in preparation of D-psicose, wherein the recombinant domain fusion enzyme is formed by splicing and recombining a 5 'domain and a part of a 3' domain of D-tagatose 3-epimerase with an active center domain of xylose isomerase. The invention takes the domain fusion enzyme of the 5 'domain and part of the 3' domain of the D-tagatose 3-epimerase and the active center domain of xylose isomerase as a biocatalyst, and the yield of catalyzing 260g/L glucose isomerization reaction to produce D-psicose is 35.9%. The method has the advantages of high speed, high efficiency, simple reaction condition, high substrate conversion rate and few byproducts, reduces the pressure of separation and purification, and is suitable for large-scale industrial production. The technical scheme adopted by the invention is as follows: The invention provides a recombinant fusion enzyme, which is formed by splicing and recombining a 5 'domain (DTE-5') and a part of 3 'domain (DTE-3') of D-tagatose 3-differential enzyme, and a xylose isomerase active center domain (XYI-VE), wherein the amino acid sequence of the recombinant domain fusion enzyme (DTE-5 '-XYI-VE-DTE-3') is shown as SEQ ID No. 1; Further, the D-tagatose