Formate dehydrogenase mutant with improved enzyme activity and stability as well as construction method of formate dehydrogenase mutant

A formate dehydrogenase and mutant technology, applied in the field of genetic engineering, can solve the problems of poor operation stability and low specific enzyme activity, and achieve the effects of improving acid resistance, eliminating resistance, increasing specific enzyme activity and catalytic efficiency

Active Publication Date: 2017-03-08
JIANGNAN UNIV
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AI Technical Summary

Problems solved by technology

[0003] Wild-type CboFDH has the disadvantages of low specific enzyme activity and poor operational stability. Therefore, site-directed mutagenesis of CboFDH to improve specific enzyme activity and stability can improve the efficiency of CboFDH to regenerate the coenzyme NADH, which is of great significance to the chiral compound biosynthesis industry.

Method used

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  • Formate dehydrogenase mutant with improved enzyme activity and stability as well as construction method of formate dehydrogenase mutant

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Experimental program
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Effect test

Embodiment 1

[0028] Embodiment 1 Contains the construction of the recombinant vector of formate dehydrogenase mutant

[0029] (1) Obtaining of the A10C 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.

[0030] (2) Combine the recombinant gene with pET28a They were digested with EcoR I and Xho I respectively, and ligated with T4 DNA ligase overnight at 16°C after purification. The ligation product was chemically transformed into E.coli BL21 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 pET28a-A10C. The sequencing work was completed by Shanghai Sangong.

Embodiment 2

[0031] Example 2 Production of Formate Dehydrogenase Mutant Recombinant Escherichia coli Engineering Bacteria Construction

[0032] The strain containing the correct recombinant plasmid pET28a-A10C obtained in Example 1 is the recombinant genetically engineered strain pET28a-A10C / E.coli BL21 of the present invention.

Embodiment 3

[0033] Example 3 Recombinant strain pET28a-A10C / E.coliBL21 expresses formate dehydrogenase and assays its enzyme activity

[0034] The recombinant strain pET28a-A10C / E.coli BL21 constructed in Example 2 and the control strain pET28a-FDH / E.coli BL21 expressing the unmutated wild enzyme CboFDH (amino acid sequence shown in SEQ ID NO: 2) were respectively inoculated in In 10 mL of LB medium containing kanamycin, shake culture at 37°C overnight, transfer to TY fermentation medium at 4% inoculum size the next day, culture at 37°C for 4 hours, add 0.5mM IPTG and induce at 24°C for 16 Hour. The cells were collected by centrifugation and broken, and the cell broken supernatant (crude enzyme solution) was used for the determination of enzyme activity.

[0035] The obtained crude enzyme liquid was purified to obtain the formate dehydrogenase mutant A10C, and the kinetic parameters of the purified recombinant formate dehydrogenase mutant A10C were analyzed, as shown in Table 1. + Affin...

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Abstract

The invention discloses a formate dehydrogenase mutant with improved enzyme activity and stability as well as a construction method of the formate dehydrogenase mutant and belongs to the field of gene engineering technology. According to the mutant, on the basis of amino acid shown in SEQ ID No.2, alanine in the 10th site is mutated into cysteine. The enzyme ratio and the enzyme activity of the obtained mutant are increased by 1.3 times by comparison with those before mutation, the half-life period (t1/2) at the temperature of 60 DEG C is prolonged by 6.8 times by comparison with that before mutation, the tolerance of cupric ions is improved by 30 times than that before mutation, the stability under the condition that pH is equal to 4 is improved by 2.0 times, and the catalytic efficiency is improved by 1.4 times. The formate dehydrogenase mutant and the construction method indicate that an amino acid residue in the 10th site is mutated into cysteine, and then cysteine forms a correct disulfide bond with a 30th cysteine residue of natural formate dehydrogenase, so that the stability and the catalytic efficiency of the enzyme are improved, and the industrial application potential of the enzyme is improved.

Description

technical field [0001] The invention relates to a formate dehydrogenase mutant with improved enzyme activity and stability and a construction method thereof, belonging to the technical field of genetic engineering. Background technique [0002] Formate dehydrogenase (FDH, EC 1.2.1.2) catalyzes formate to carbon dioxide accompanied by NAD + Reduction generates NADH. Since the reaction is irreversible, the substrate is cheap formic acid and the product is CO 2 Formate dehydrogenase is used as a coenzyme cycle system and is often combined with other oxidoreductases for the biotransformation production of important optically active compounds such as hydroxyacids, chiral alcohols, and amino acids. Among them, the wild-type FDH (CboFDH) derived from Candida boidinii is used to regenerate NADH in the production of L-tert-leucine, which is the largest example of the application of formate dehydrogenase in industrial production. With the accurate analysis of the three-dimensional ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12R1/19
CPCC12N9/0008C12Y102/01002
Inventor 饶志明郑俊贤杨套伟周俊平张显徐美娟
Owner JIANGNAN UNIV
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