A kind of γ-glutamyl transpeptidase mutant with improved transpeptide activity and its construction method

A technology of glutamyl transpeptidase and mutants, applied in the field of genetic engineering, can solve existing problems and achieve the effect of improving the potential of industrial application

Active Publication Date: 2020-06-09
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The outstanding problem in the synthesis of L-theanine catalyzed by γ-glutamyl transpeptidase is that there is a hydrolysis reaction, resulting in the synthesis of by-product L-glutamic acid
In general, the reaction is optimized by controlling the dosage ratio between the donor and the acceptor, adjusting pH and other conditions to reduce the synthesis of by-products, but the accumulation of by-product L-glutamic acid still exists

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1 Contains the construction of the recombinant vector of gamma-glutamyl transpeptidase mutant

[0022] (1) Obtaining the T463D mutant: using the nucleotide sequence shown in SEQ ID NO.4 as a template, Fprimer (sequence shown in SEQ ID NO.5) and Rprimer (sequence shown in SEQ ID NO.6) are primers, PCR is performed to obtain the recombinant gene shown in SEQ ID NO.3.

[0023] (2) Digest the recombinant gene and pMA5 with BamHI and MluI, respectively, and ligate with T4 DNA ligase overnight at 16°C after purification. The ligation product was chemically transformed into JM109 competent cells. The transformation solution was applied to an LB plate containing kanamycin (50 mg / L), the plasmid was extracted, and the recombinant plasmid constructed was verified by double enzyme digestion, which was named pMA5-T463D. The sequencing work was completed by Shanghai Sangong.

Embodiment 2

[0024] Example 2 Production of gamma-glutamyl transpeptidase Bacillus subtilis engineering bacteria construction

[0025] The recombinant γ-glutamyl transpeptidase particle pMA5-T463D obtained in Example 1 was chemically transformed into B. subtilis 168 competent cells, and the specific method was as follows:

[0026] (1) The solution required for the transformation experiment is as follows (g / L):

[0027] Sp-A: (NH 4 ) 2 SO 4 4,K 2 HPO 4 28. Sodium citrate 12 Sp-B: MgSO 4 ·7H 2 O 0.4

[0028] 100×CAYE: Casamino acid 20, yeast powder 100Sp I medium: Sp-A49%, Sp-B 49%, 50% glucose 2%, 100×CAYE 2% Sp II medium: Sp I medium 98%, 50mmol / LCaCl 2 1%, 250mmol / LMgCl 2 1%. 115°C damp heat sterilization.

[0029] (2) Inoculate a single colony of B.Subtilis 168 into 2mL Sp I medium (50mL centrifuge tube), and culture overnight at 37°C and 200r / min;

[0030] (3) Take 100 μL of the culture solution into 5 mL of Sp I medium, and culture at 37°C and 200 r / min until the loga...

Embodiment 3

[0032] Example 3 High expression and enzyme activity determination of recombinant bacterium pMA5-T463D / B.subtilis 168γ-glutamyl transpeptidase.

[0033] (1) The recombinant bacteria pMA5-T463D / B.subtilis 168 constructed in Example 2 and the control strain pMA5-ggt / B.subtilis 168 expressing unmutated enzymes were inoculated in 10 mL of LB medium containing kanamycin respectively , shaking culture at 37°C overnight, transfer to Bacillus subtilis fermentation medium at 4% inoculum the next day, culture at 37°C for 24h, take the fermentation broth and centrifuge at 10000r / min for 10min at 4°C, the supernatant is extracellular The crude enzyme solution, the cell broken supernatant is the intracellular crude enzyme solution, which is used for the determination of enzyme activity.

[0034] (2) Bacillus subtilis fermentation medium: sucrose 25g / L, tryptone 5g / L, corn steep liquor 15g / L, MgSO40.3g and K 2 HPO 4 ·3H 2 O 1g / L, add kanamycin to a final concentration of 50 μg / ml, and ad...

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PUM

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Abstract

The invention discloses a gamma-glutamyltranspeptidase mutant with improved transpeptide vitality as well as a construction method thereof and belongs to the field of gene engineering. The mutant is that the 463rd amino acid is mutated from threonine to aspartic acid on the basis of amino acid as shown in SEQ ID NO.3. The mutant is expressed in bacillus subtilis, the transpeptide reaction vitalityof the gamma-glutamyltranspeptidase mutant enzyme is increased by 22.6 percent by compared with that before mutation, and the hydrolytic reaction vitality is reduced by 90 percent. The invention indicates that the 463rd amino acid residue has big influence on the transpeptide reaction vitality of the gamma-glutamyltranspeptidase, certain basis is provided for research on the catalytic mechanism of the enzyme, and the industrial application potential of the enzyme is improved. The gamma-glutamyltranspeptidase mutant provided by the invention can be applied to preparation of L-theanine.

Description

technical field [0001] The invention relates to a gamma-glutamyl transpeptidase mutant with improved transpeptide activity and a construction method thereof, belonging to the technical field of genetic engineering. Background technique [0002] L-theanine is a natural amino acid that exists in tea plants. It determines the quality and flavor of tea and has many health effects on the human body. Its demand as a food component and beverage additive is increasing day by day. . At present, the research on the production of theanine mainly focuses on the enzymatic conversion method, and among them, γ-glutamyl transpeptidase stands out with its unique advantages. [0003] γ-glutamyltranspeptidase (γ-glutamyltranspeptidase, GGT, EC2.3.2.2) is responsible for catalyzing the transfer of γ-glutamyl molecules of γ-glutamyl compounds to other receptor molecules such as amino acids and short peptides ( Transpeptide reaction) or on water molecules (hydrolysis reaction), widely exists in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N1/21C12P13/04C12R1/125
CPCC12N9/104C12P13/04C12Y203/02002
Inventor 杨套伟饶志明杨晨张显徐美娟刘会灵
Owner JIANGNAN UNIV
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