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Mutants of formate dehydrogenase with improved substrate affinity and coenzyme affinity

A formate dehydrogenase and mutant technology, applied in the field of bioengineering, can solve problems such as unsuitable for practical application, high concentration of formate substrate and coenzyme, increased cost of coenzyme regeneration and reaction liquid treatment cost, etc.

Active Publication Date: 2020-11-27
EAST CHINA UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Afterwards, Q197N was introduced on the basis of the double-point mutant D195Q / Y196R to obtain the triple-point mutant D195Q / Y196R / Q197N, which has an effect on NADP + K m down to 0.029mM, which is by far the most effective for NADP + The FDH mutant with the highest affinity, but the specific activity is only 0.12U / mg, which is not suitable for practical application (J.Mol.Catal.B:Enzym.,2009,61:157–161)
[0004] In 2010, formate dehydrogenase BstFDH from Burkholderia stabilis 15516 was reported, which is the first reported natural NADP + Dependent on FDH, on NADP + the k cat / K m up to 30mM -1 the s -1 , is the highest among the reported FDHs, but due to its K for substrates and coenzymes m They are 55.5mM and 0.16mM respectively, which makes the concentration of formate substrate and coenzyme added in the actual coenzyme regeneration reaction higher, which increases the cost of coenzyme regeneration and the processing cost of the reaction solution

Method used

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  • Mutants of formate dehydrogenase with improved substrate affinity and coenzyme affinity
  • Mutants of formate dehydrogenase with improved substrate affinity and coenzyme affinity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Site-directed mutation of BstFDH

[0071] Through Uniprot, NCBI BLAST and spatial structure modeling, the three-dimensional structure of BstFDH with the amino acid sequence shown in the sequence table SEQ ID No.2 was obtained, and the amino acid residues around the binding site of the coenzyme and the substrate were mutated. Site-directed saturation mutation was performed using the recombinant plasmid of the female parent BstFDH as a PCR template. The PCR system was: 10 μl of 2×PrimeStar, 1 μl of upstream primer and downstream primer (10ng / μl), 1 μl of template plasmid (50ng / μl), 1 μl of DMSO and ddH 2 O 6 μl. The PCR amplification program was as follows: 30 cycles of denaturation at 98°C for 5 minutes followed by denaturation at 98°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 7 minutes, and finally extension at 72°C for 10 minutes. The PCR amplified product was digested with DpnI for 2 hours, the digested product was transformed ...

Embodiment 2B

[0073] Combination mutation of embodiment 2BstFDH

[0074] On the basis of the mutation in Example 1, some mutation points were combined to obtain mutants with significantly improved affinity for coenzyme and substrate. in FIG. 1. In the list in Table 1, the sequence numbers refer to the corresponding series of sequences at the back of Table 1; in the mutant affinity improvement multiple, a plus sign "+" indicates that the ratio of the mutant protein is shown in SEQ ID No.2 in the sequence table The affinity of the protein composed of the amino acid sequence to the substrate or coenzyme is increased by 0.1-1 times; two plus signs "++" indicate that the mutant protein is more sensitive to the substrate than the protein composed of the amino acid sequence shown in SEQ ID No.2 in the sequence table The affinity of the substance or coenzyme has been improved by 1-4 times; three plus signs "+++" indicate that the affinity of the mutant protein to the substrate or coenzyme is impro...

Embodiment 2

[0103] Example 2 Expression and catalytic efficiency of recombinant BstFDH k cat / K m Determination of

[0104] Inoculate the recombinant Escherichia coli E. coli BL21(DE3) / pET22b-BstFDH into 100mL LB medium containing 50μg / ml ampicillin, culture on a shaker at 37°C for 12 hours, and then inoculate at 1% (v / v) The amount was added to a 500ml Erlenmeyer flask equipped with 100ml LB medium (containing 50μg / ml ampicillin), placed in a shaker at 37°C and 180rpm for shaking culture, when the OD of the culture solution 600 When it reached 0.6, IPTG with a final concentration of 0.2mmol / L was added as an inducer and induced at 16°C for 24h. The culture solution was centrifuged at 8000×g for 10 min, the cells were collected, and washed twice with saline to obtain resting cells. Suspend the cells obtained in 100ml of culture medium in 10ml of potassium phosphate buffer (100mM, pH 7.0), and perform the following ultrasonic disruption in an ice-water bath: 400W power, working for 4s, ...

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Abstract

The invention relates to an NADP+ dependent formate dehydrogenase mutant with significantly improved substrate affinity and coenzyme affinity and a coding nucleic acid of the mutant, and a recombinantexpression vector and a recombinant expression transformant containing a nucleic acid sequence. The formate dehydrogenase mutant is a derived protein constituted by a new amino acid sequence formed by substituting one or more amino acid residues of 30th arginine, 124th isoleucine, 146th glycine, 216th glycine, 261st proline, 262nd serine, 287th alanine, 361st arginine or 381st glutamine in an amino acid sequence shown in SEQ ID No.2 with other amino acid residues; the substrate affinity and the coenzyme affinity of the derived protein are higher than those of formate dehydrogenase constitutedby the amino acid sequence shown in SEQ ID No.2. Compared with the prior art, the mutant has the advantages of high substrate affinity and coenzyme affinity, and has a good application prospect.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a NADP + A formate dehydrogenase mutant with significantly improved substrate affinity and coenzyme affinity, its encoding nucleic acid, a recombinant expression vector containing the nucleic acid sequence, and a recombinant expression transformant. Background technique [0002] Formate dehydrogenase (FDH) is an important coenzyme regeneration tool enzyme, which uses formate as a cosubstrate to reduce NAD + Get NADH, or reduce NADP + NADPH is obtained, and the by-product CO 2 Overflow from the reaction system, thereby pushing the reaction toward the direction of generating NADH or NADPH. The coenzyme regeneration system catalyzed by formate dehydrogenase has the following advantages: (1) the molecular weight of formate is relatively small and the price is cheap; (2) the reaction product is CO 2 , easy to separate; (3) The reaction is almost irreversible; (4) The therma...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12P19/36
CPCC12N9/0008C12P19/36C12Y102/01002
Inventor 郑高伟江和文陈琦郁惠蕾潘江许建和钱小龙
Owner EAST CHINA UNIV OF SCI & TECH
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