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Thermostability enhanced formate dehydrogenase mutant and preparation method thereof

A technology for formate dehydrogenase and thermal stability, which is applied in the field of formate dehydrogenase mutants and their preparation, can solve problems such as poor thermal stability of FDH, and achieve the effect of good thermal stability

Active Publication Date: 2015-02-11
常州百奥生物科技有限公司
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Problems solved by technology

However, the FDH found so far has poor thermal stability and rapidly inactivates between 55°C and 60°C.

Method used

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  • Thermostability enhanced formate dehydrogenase mutant and preparation method thereof

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Embodiment 1）

[0021] The polynucleotide (SEQ ID NO: 1) encoding wild-type formate dehydrogenase from Candida boidinii is sequence-optimized and obtained by whole gene synthesis. The optimized polynucleotide encoding formate dehydrogenase was cloned under the control of the promoter of the improved expression vector (SEQ ID NO. 5) to obtain a plasmid capable of expressing wild-type formate dehydrogenase. The resulting plasmid was transformed into E. coli DH1 by standard methods. The cloning method used is homologous recombination, and the amplification primers used are:

[0022] F: 5' TAACTTTTAGGAGGTAAAACATATGAAAATCGTTCTGGTTCTGTACG 3';

[0023] R: 5' AACAGGAGTCCAAGCTCAGCTTATTATTTTTTGTCGTGTTTACCGTAAGC 3'

[0024] Similarly, the polynucleotide (SEQ ID NO: 3) encoding the formate dehydrogenase mutant is cloned under the control of the promoter of the expression vector (SEQ ID NO. 5) to obtain a protein that can express the formate dehydrogenase mutant plasmid. The resulting plasmid was tran...

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Abstract

The invention relates to a thermostability enhanced formate dehydrogenase mutant and a preparation method thereof. The mutant is derived from wild type formate dehydrogenase of Candida boidinii, can catalyze coenzyme NADH cyclic regeneration, shows higher thermostability compared with the wild type formate dehydrogenase, and has one or more mutation characteristics of I98V, V152I, A154D and D158R. The recombinant formate dehydrogenase mutant provided by the invention has better thermostability, and is suitable for cyclic regeneration of NADH under high temperature. Compared with the formate dehydrogenase discovered so far, the formate dehydrogenase mutant has the activity that is not significantly reduced in the range of 50DEG C-55DEG C, and has better thermostability, thus being more suitable for industrial application.

Description

technical field [0001] The invention relates to a formate dehydrogenase mutant, in particular to a formate dehydrogenase mutant with enhanced thermal stability and a preparation method thereof. Background technique [0002] There are more and more applications of oxidoreductases in the preparation of chiral alcohols, amino acids and hydroxy acids. However, 90% of the reactions it catalyzes require nicotinamide adenine dinucleotide (NAD, NADH), or nicotinamide adenine dinucleotide phosphate (NADP, NADPH) as a coenzyme. Because in the application of oxidoreductase, a low-cost and high-efficiency coenzyme regeneration system is also required. Formate Dehydrogenase (FDH) is one of the most successful coenzyme regeneration systems and has been applied in industrial production. Its advantage is that the reaction is irreversible and only produces a by-product - carbon dioxide, which has no effect on the activity of the enzyme. Kula et al. were awarded the German Future Prize in ...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12P19/36C12R1/72
CPCC12N9/0008C12P19/36C12Y102/01002
Inventor 丁雪峰
Owner 常州百奥生物科技有限公司
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