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CN-121975782-A - D-tagatose-3-epimerase mutant and application thereof

CN121975782ACN 121975782 ACN121975782 ACN 121975782ACN-121975782-A

Abstract

The invention discloses a D-tagatose-3-epimerase mutant and application thereof, wherein the D-tagatose-3-epimerase mutant is single-point mutation or multi-point mutation in 45 th, 48 th, 50 th, 80 th, 93 rd, 98 th and 144 th mutations relative to the amino acid sequence SEQ ID NO.1 of wild D-tagatose-3-epimerase. Also provides the corresponding coding gene, recombinant vector, genetically engineered bacterium, biocatalyst and application in preparing rare sugar. The invention develops the D-tagatose-3-epimerase mutant which has stronger adaptability to glucose isomerase and is more stable in cascade reaction, is beneficial to improving the efficiency of the reaction for preparing rare sugar by utilizing xylose, can simultaneously give consideration to higher substrate concentration and D-xylose conversion rate, and reduces the production cost of products.

Inventors

  • LI LIANGZHI
  • JU XIN
  • HU CUIYING
  • CHEN ZHI
  • LONG SI

Assignees

  • 苏州科技大学

Dates

Publication Date
20260505
Application Date
20260309

Claims (10)

  1. 1. A mutant D-tagatose-3-epimerase, characterized in that the mutant D-tagatose-3-epimerase is a single point mutation or a multiple point mutation among mutations at positions 45, 48, 50, 80, 93, 98 and 144 relative to the amino acid sequence of wild-type D-tagatose-3-epimerase SEQ ID No. 1.
  2. 2. The D-tagatose-3-epimerase mutant according to claim 1, wherein the amino acid sequence of the D-tagatose-3-epimerase mutant is shown as SEQ ID No.2 or SEQ ID No. 3.
  3. 3. A coding gene, which is used for coding the D-tagatose-3-epimerase mutant according to claim 2, wherein the nucleotide sequence of the coding gene is shown as SEQ ID NO.4 or SEQ ID NO. 5.
  4. 4. A recombinant vector constructed for the coding gene of claim 3.
  5. 5. A genetically engineered bacterium, characterized in that the genetically engineered bacterium is obtained by transformation of the recombinant vector of claim 4.
  6. 6. A biocatalyst comprising at least one of the D-tagatose-3-epimerase mutants according to claim 1 or 2, transformant cells containing the D-tagatose-3-epimerase mutants, crude enzyme solution, purified enzyme solution and immobilized enzyme.
  7. 7. Use of a D-tagatose-3-epimerase mutant according to claim 1 or 2 for the preparation of rare sugars.
  8. 8. The use according to claim 7, wherein D-xylulose is converted to D-ribulose using the D-tagatose-3-epimerase mutant as a catalyst, or D-xylose is converted to D-ribulose using the D-tagatose-3-epimerase mutant as a catalyst in cascade using a two-enzyme one-pot method.
  9. 9. The application of the double-enzyme one-pot method according to claim 8, wherein the double-enzyme one-pot method cascade process comprises the steps of culturing genetically engineered bacteria containing genes encoding the D-tagatose-3-epimerase mutants to obtain thalli, performing ultrasonic crushing and purification on the thalli to obtain pure enzymes serving as catalysts, respectively adding glucose isomerase and D-tagatose-3-epimerase mutant pure enzymes into phosphate buffer solution with pH value of 7.5 in the presence of magnesium ions and manganese ions, reacting at 50 ℃ for 4-6 hours, and separating and purifying after the complete reaction to obtain D-xylulose and D-ribulose.
  10. 10. The use according to claim 9, characterized in that the D-xylose substrate concentration is greater than or equal to 100mM and the D-tagatose-3-epimerase mutant pure enzyme is used in an amount of less than or equal to 10% by mass.

Description

D-tagatose-3-epimerase mutant and application thereof Technical Field The invention belongs to the field of enzyme engineering, and particularly relates to a D-tagatose-3-epimerase mutant and application thereof. Background The widespread use of rare sugars in the food, pharmaceutical and chemical industries has led to an increasing search for them. Rare sugars such as D-psicose, D-tagatose and D-xylitol are very rare in nature, and are widely used as zero-calorie and low-calorie sweeteners in various foods to prevent diabetes and obesity. Rare saccharides have various health effects, such as D-arabitol and D-xylulose have caries preventing effect, and D-allose has blood sugar and blood lipid reducing effect. Rare sugars are also useful in the fields of chemical synthesis, crop protection and material production. In view of economic and environmental benefits, the preparation of rare sugars mainly relies on biosynthesis, including microbial fermentation and enzymatic catalysis. The commercial production of D-psicose from D-glucose and other inexpensive raw materials has been reported to be successful using the "Izumoring" method. Sugar isomerase is the most important tool of Izumoring process, and can catalyze the reversible conversion of ketoaldoses and C3-oligosaccharides of monosaccharides. However, isomerization between similar structures in the same system generally results in lower gibbs free energy differences, reaction equilibrium constants and final conversions, and thus reaction engineering is critical for the process catalyzed by the sugar isomerase. The actual preparation of rare saccharides requires high reaction efficiency and low process cost, and the single-pot multi-enzyme cascade reaction is an ideal solution. Enzymatic cascade reactions generally combine two or more biocatalysts in one reaction vessel, avoiding laborious separation steps of intermediates and extending the reaction chain to lower cost substrates and higher value added products. At present, a lot of reports on the cascade preparation of rare sugar by isomerase and isomerase exist, and the substrate utilization rate is low (mostly between 40-60%) due to the reasons of no cofactor participation and untimely separation of products. The existing double enzyme cascade catalysis technology for preparing rare sugars such as D-xylulose, D-ribulose and the like by using D-xylose has the problems that the substrate concentration is low (10 mM), the enzyme consumption is high (> 100% w/w), and the high substrate concentration and the D-xylose conversion rate cannot be simultaneously achieved. Disclosure of Invention In view of all or part of the defects in the prior art, the invention aims to provide a D-tagatose-3-epimerase mutant and application thereof, and develop a D-tagatose-3-epimerase (DTE) mutant which has stronger adaptability to Glucose Isomerase (GI) and is more stable in cascade reaction, thereby being beneficial to improving the efficiency of the reaction for preparing rare sugar by utilizing xylose, and being capable of simultaneously achieving higher substrate concentration and D-xylose conversion rate and reducing the production cost of products. In order to achieve the above object, the present invention provides the following technical solutions: The invention provides a D-tagatose-3-epimerase mutant, which is a single-point mutation or multi-point mutation in 45 th, 48 th, 50 th, 80 th, 93 th, 98 th and 144 th mutations relative to the amino acid sequence SEQ ID NO.1 of wild-type D-tagatose-3-epimerase. Wherein serine at position 45, lysine at position 48, arginine at position 50, lysine at position 80, leucine at position 93, histidine at position 98 and aspartic acid at position 144. The invention further provides two specific mutants, but is not limited thereto. The amino acid sequence of the D-tagatose-3-epimerase mutant is shown as SEQ ID NO.2 or SEQ ID NO. 3. The amino acid sequence of SEQ ID NO.2 is a five mutant of D-tagatose-3-epimerase, designated PcDTEmut1. The amino acid sequence of SEQ ID NO.3 is a seven mutant of D-tagatose-3-epimerase, designated PcDTEmut. The invention also provides a coding gene for coding the D-tagatose-3-epimerase mutant, wherein the nucleotide sequence of the coding gene is shown as SEQ ID NO.4 (coding PcDTEmut 1) or SEQ ID NO.5 (coding PcDTEmut). The invention also provides a recombinant vector constructed for the coding gene. The invention also provides a genetic engineering bacterium which is obtained by transforming the recombinant vector, and the genetic engineering bacterium is escherichia coli containing the recombinant vector and expressing the D-tagatose-3-epimerase mutant. Alternatively, the E.coli is E.coli BL21 (DE 3). The invention also provides a biocatalyst comprising at least one of the D-tagatose-3-epimerase mutant, a transformant cell containing the D-tagatose-3-epimerase mutant, crude enzyme liquid, purified enzyme liquid and immobilized enzyme. The invention