Formate dehydrogenase mutant with improved substrate affinity and coenzyme affinity

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

Active Publication Date: 2019-11-05
EAST CHINA UNIV OF SCI & TECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Formate dehydrogenase mutant with improved substrate affinity and coenzyme affinity
  • Formate dehydrogenase mutant 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 spatial structure of BstFDH of the amino acid sequence shown in SEQ ID No. 2 in the sequence table was obtained, and the amino acid residues around the binding site of the coenzyme and the substrate were mutated. Use the recombinant plasmid of parent BstFDH as PCR template for site-directed saturation mutation. The PCR system is: 2×PrimeStar 10μl, upstream primer and downstream primer (10ng / μl) each 1μl, template plasmid (50ng / μl) 1μl, DMSO 1μl and ddH 2 O 6μl. The PCR amplification program is: pre-denaturation at 98°C for 5 minutes and then 30 cycles as follows: denaturation at 98°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 7 minutes; finally, extension at 72°C for 10 minutes. The PCR amplified product was added to DpnI for digestion for 2 hours, and the digested product was transformed into E.coli BL21(DE3...

Embodiment 2B

[0073] Example 2 Combinatorial mutation of BstFDH

[0074] On the basis of the mutations in Example 1, some mutation points were combined to obtain mutants with significantly improved affinity for coenzyme and substrate. The sequences of these mutants and the multiples of the increased affinity of these mutants for coenzyme and substrate are listed in in FIG. 1. In the list of Table 1, the sequence numbers respectively refer to a series of sequences corresponding to the back of Table 1; in the mutant affinity increase fold, a plus sign "+" indicates that the mutant protein ratio is shown by 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; the two plus signs "++" indicate that the mutant protein is compared to 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 is increased by 1-4 times; the...

Embodiment 2

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

[0104] Inoculate recombinant E. coli BL21(DE3) / pET22b-BstFDH into 100mL LB medium containing 50μg / ml ampicillin, shake culture at 37℃ for 12 hours, and then inoculate 1%(v / v) Put the volume into a 500ml Erlenmeyer flask containing 100ml LB medium (containing 50μg / ml ampicillin), and place it on a shaker at 37°C and 180rpm for shaking culture. When the OD of the medium is 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 broth 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 sonicate them in an ice-water bath as follows: 400W power, working 4s, intermittent 6s, 99 cycles, 12000 at 4℃ After centrifugation at ×g for 4...

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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 field of bioengineering technology, and specifically relates 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 co-substrate to reduce NAD + Get NADH, or reduce NADP + Get NADPH, 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 thermal stabili...

Claims

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Application Information

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Patent Type & Authority Applications(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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