A formate dehydrogenase mutant with improved enzyme activity and stability and its construction method

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

Active Publication Date: 2019-08-06
JIANGNAN UNIV
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  • Abstract
  • Description
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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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  • A formate dehydrogenase mutant with improved enzyme activity and stability and its construction method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction of recombinant vector containing formate dehydrogenase mutant

[0029] (1) Obtaining of 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 Primer, 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. After purification, they were ligated with T4 DNA ligase at 16°C overnight. The ligation product was chemically transformed into E.coli BL21 competent cells. The transformation solution was coated on an LB plate containing kanamycin (50 mg / L), the plasmid was extracted, and the recombinant plasmid constructed by double enzyme digestion was verified and named pET28a-A10C. The sequencing work was completed by Shanghai Shenggong.

Embodiment 2

[0031] Example 2 Construction of recombinant Escherichia coli engineering bacteria producing formate dehydrogenase mutants

[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.

Embodiment 3

[0033] Example 3 Recombinant strain pET28a-A10C / E.coli BL21 expresses formate dehydrogenase and determination of 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 into 10 mL of kanamycin-containing LB medium, shake culture overnight at 37°C, transfer to TY fermentation medium at a 4% inoculum volume the next day, culture at 37°C for 4 hours, add 0.5mM IPTG to induce at 24°C 16 hour. The cells were collected by centrifugation and crushed, and the supernatant of cell crushing (crude enzyme solution) was used for the determination of enzyme activity.

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

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Abstract

The present invention discloses a formate dehydrogenase mutant with improved enzyme activity and stability and a construction method thereof, which belongs to the technical field of genetic engineering. The mutant of the present invention is obtained by mutating alanine at a 10th site to cysteine based on the amino acid shown in SEQ ID NO. 2. The specific enzyme activity of the mutant enzyme obtained by the present invention is improved by 1.3 times compared with that before the mutation, a half-life period (t1 / 2) at 60° C. is increased by 6.8 times compared with that in the mutation period, the copper ion tolerance is increased by 30 times compared with that before the mutation, and when pH is 4, the stability is improved by 2.0 times, and the catalytic efficiency is increased by 1.4 times. The present invention shows that an amino acid residue at a 10th site is mutated to the cysteine which forms a correct disulfide bond with a cysteine residue at a 30th site of the 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 the formation of carbon dioxide from formic acid and is accompanied by NAD + Reduction to generate NADH. Since the reaction is irreversible, the substrate is cheap formic acid, and the product is CO 2 With many advantages, formate dehydrogenase is used as a coenzyme cycle system and is often combined with other oxidoreductases for the biotransformation of important optically active compounds such as hydroxy acids, chiral alcohols, and amino acids. Among them, wild-type FDH (CboFDH) derived from Candida boidinii is used for NADH regeneration in the production of L-tert-leucine, which is the largest application of formate dehydrogenase in industrial production today. With the accurate analys...

Claims

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Application Information

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Patent Type & Authority Patents(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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