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Glutamate decarboxylase mutants with improved thermostability and their applications

A technology of glutamic acid decarboxylase and thermal stability, applied in the field of bioengineering, can solve problems such as unfavorable GAD application, and achieve the effect of improving the stability of enzyme activity

Active Publication Date: 2019-05-17
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The half-life of Lactobacillus breve GAD enzyme is 30.9min at 55°C, and the half-life of Escherichia coli GAD enzyme is 24min at 50°C, which is very unfavorable for the application of GAD in the biological preparation of GABA

Method used

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  • Glutamate decarboxylase mutants with improved thermostability and their applications
  • Glutamate decarboxylase mutants with improved thermostability and their applications
  • Glutamate decarboxylase mutants with improved thermostability and their applications

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Embodiment 1

[0039] Embodiment 1 Construction of glutamic acid decarboxylase GAD recombinant bacteria

[0040] Using Escherichia coli DH5α genomic DNA as a template, the gadB gene sequence was amplified by PCR using primers 5'-AATTGGATCCATGGATAAGAAGCAAG-3' and 5'-AAGGCTCGAGGGTATGTTTAAAGCTG-3' ( figure 1 ). figure 1 It was shown that the target gene of about 1400bp was successfully obtained. The PCR product and the vector pET28a(+) were digested with restriction endonucleases Nco I and Xho I. After the digested product was purified, T4 ligase was ligated overnight at 16°C, transformed into E.coli DH5α competent cells, picked out a single clone and cultured, extracted the plasmid, and identified the presence of gadB with BamH I and Xho I double restriction plasmids ( figure 2 ). figure 2 It showed that the two recombinants picked had a gadB target gene band of about 1400bp and a pET28a(+) carrier band of about 5300bp, both of which were positive clones. The 1# recombinant was selected ...

Embodiment 2G

[0041] Construction of embodiment 2GAD mutant recombinant bacteria

[0042] Using the plasmid pET28-GADB as a template, the primers 5'-ACCATGGATAAGAAGNNNNNNNNNGATTTAAGGTCGGAAC-3' and 5'-NNNNNNNNNNCTTCTTATCCATGGTATATCTCCTTC-3' were used to amplify the entire plasmid sequence containing the gadB gene by reverse PCR. After digesting with Dpn I, it was transformed into E.coli BL21( DE3) Competent cells, selected monoclonal bacteria, cultured overnight in a 96-well deep-well plate in LB medium containing kanamycin.

Embodiment 3

[0043] The screening of the glutamic acid decarboxylase recombinant bacterium that embodiment 3 thermostability improves

[0044] Transfer the bacterium in Example 2 to a new 96-well deep-well plate containing kanamycin-containing LB medium, culture at 37°C for 2-4h until the OD600 is about 0.6, and add IPTG with a final concentration of 0.2mM , induced overnight at 16°C. Centrifuge to remove the culture medium, add 200ul of 10mM acetic acid-sodium acetate solution containing 20mM bromocresol green, 10mM sodium glutamate, 0.1mMPLP, 10mM pH4.8, incubate at 45°C for 1h, and screen the recombinant strains with dark blue color in the wells. The nucleotide and amino acid sequences of the corresponding plasmid mutation sites were analyzed by sequencing.

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Abstract

The invention relates to a glutamate decarboxylase mutant with improved thermal stability and an application thereof, belonging to the field of bioengineering. The mutant is prepared primarily by the following steps: performing saturated mutation of glutamine Q, valine V and threonine T on the sites 5-7 of the amino acid sequence of glutamate decarboxylase, and screening to obtain high-stability mutants Q5E / V6S / T7V, Q5Y / V6R / T7K and Q5N / V6Y / T7V. In the glutamate decarboxylase mutant prepared in the invention, the half-inactivation temperature is 45-50.5 DEG C which is 4.9-10.2 DEG C higher than that of wild type mutant; and the half-life period at 45 DEG C is 76-129min, which is 3.2-4.3 times higher than 24min of wild type mutant. By transforming glutamic acid for 12h with the whole cell of glutamate decarboxylase mutant synthesized from recombinant escherichia coli, 260-350g / L gamma-aminobutyric acid (GABA) can be obtained, and the molar yield is 76.6-97.8%. In the glutamate decarboxylase mutant prepared in the invention, the thermal stability is obviously improved, the production of gamma-aminobutyric acid is facilitated, and a foundation is laid for efficient synthesis of gamma-aminobutyric acid.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a glutamic acid decarboxylase (GAD) mutant with improved thermostability and application thereof. Background technique [0002] γ-Aminobutyric acid (GABA) is a natural functional non-protein amino acid, which widely exists in animals, plants and microorganisms. It has a wide range of applications in medicine, food health care, chemical industry, agriculture and other fields due to its functions of hormone secretion, liver protection and kidney benefit. At present, γ-aminobutyric acid is mainly prepared by chemical synthesis method, microbial fermentation method and biotransformation enzyme method at home and abroad. In the process of chemically synthesizing GABA, strong corrosive solvents such as strong acid or strong base are needed, the reaction conditions are severe, the raw materials are highly toxic, the price is expensive, and there are many safety hazards; the microbial fermen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12P13/00
CPCC12N9/88C12P13/005C12Y401/01015
Inventor 赵黎明范立强李明伟秦臻陈启明邱勇隽
Owner EAST CHINA UNIV OF SCI & TECH
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