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Enzyme activity increased gamma-glutamyltranspeptidase mutant, and construction method thereof

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

Active Publication Date: 2018-03-23
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

[0015] Embodiment 1 Contains the construction of the recombinant vector of gamma-glutamyl transpeptidase mutant

[0016] (1) Obtaining the T413C 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.

[0017] (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 (50mg / L), the plasmid was extracted, and the recombinant plasmid constructed was verified by double enzyme digestion, which was named pMA5-T413C. The sequencing work was completed by Shanghai Sangong.

Embodiment 2

[0018] Example 2 Production of gamma-glutamyl transpeptidase Bacillus subtilis engineering bacteria construction

[0019] The recombinant γ-glutamyl transpeptidase particle pMA5-T413C obtained in Example 1 was chemically transformed into B. subtilis 168 competent cells, and the specific method was as follows:

[0020] (1) The solution required for the transformation experiment is as follows (g / L):

[0021] Sp-A: (NH 4 ) 2 SO 4 4,K 2 HPO 4 28. Sodium citrate 12Sp-B: MgSO 4 ·7H 2 O 0.4

[0022] 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.

[0023] (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;

[0024] (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 logarithm...

Embodiment 3

[0026] Example 3 High expression and enzyme activity determination of recombinant bacterium pMA5-T413C / B.subtilis 168γ-glutamyl transpeptidase.

[0027] (1) The recombinant bacteria pMA5-T413C / 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 24 hours, 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.

[0028] (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, a...

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Abstract

The invention discloses an enzyme activity increased gamma-glutamyltranspeptidase mutant, and a construction method thereof, and belongs to the field of gene engineering. According to the constructionmethod, an amino acid represented by SEQ ID NO.2 is taken as a base, at the 413th site, mutation of threonine into cysteine is realized. The expression of the obtained enzyme activity increased gamma-glutamyltranspeptidase mutant is carried out in bacillus subtilis, the transpeptidation reaction activity of the gamma-glutamyltranspeptidase mutant enzyme is increased by 12% of that before mutation, and hydrolysis reaction activity is reduced by 50%. It is shown that the amino acid residue on the 413th site possesses relatively high influences on the transpeptidation reaction activity of gamma-glutamyltranspeptidase, certain foundation is provided for study on the catalytic mechanisms of gamma-glutamyltranspeptidase, the industrial application potential value of gamma-glutamyltranspeptidaseis increased; and the enzyme activity increased gamma-glutamyltranspeptidase mutant can be used for preparing L-theanine.

Description

technical field [0001] The invention relates to a gamma-glutamyl transpeptidase mutant with improved enzyme 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 mamma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/75C12N1/21C12P13/04C12R1/125
CPCC12N9/104C12N15/75C12P13/04C12Y203/02002
Inventor 饶志明杨套伟张显徐美娟刘会灵
Owner JIANGNAN UNIV
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