Mycobacterium vaccae formic dehydrogenase mutant and its use

A formate dehydrogenase and carrier technology, applied in the fields of application, bacteria, fungi, etc., can solve the problems of increased stability, increased copper stability, and decreased formate dehydrogenase activity

Inactive Publication Date: 2002-10-09
DAICEL CHEM IND LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Slusarczyk et al. have pointed out a mutant of formate dehydrogenase derived from Candida boidinii prepared by site-directed mutagenesis, wherein the cysteine ​​at position 23 is replaced by serine, and the cysteine ​​at position 262 is Cystine is replaced by valine or alanine, and this mutant has increased stability to copper, increased stability to pH in the mildly alkaline pH range, and decreased stability to heat (European Journal of Biochemistry 267, 1280-1289, 2000)
[0008] Despite the efforts of these studies, there is still a problem to be solved, that is, in the process of producing reduction products such as alcohols from oxidized substrates such as ketones and simultaneously regenerating the coenzyme NADH by using the above enzymes, due to formate dehydrogenation Lower enzyme activity resulting in lower yields

Method used

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  • Mycobacterium vaccae formic dehydrogenase mutant and its use
  • Mycobacterium vaccae formic dehydrogenase mutant and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0163] Subcloning of Formate Dehydrogenase from Mycobacterium vaccae

[0164] Formate dehydrogenase was subcloned from the plasmid pMcFDH(E-P) described in a reference (Appl. Microbiol. Biotechnol.), 44, 479-483 (1995). Primers MCF-ATG2 (5'-CTTTCTAGAGGAATTCAACCATGGCAAAAGTTCTGTGTGTTC-3' / SEQ ID NO: 3) and MCF-TAA3 (5'-CAGTCTAGATTAGACCGCTTTTTTGAATTTGGCG-3' / SEQ ID NO: 4) to clone the region containing only the open reading frame of formate dehydrogenase. The plasmid (pMcFDH(E-P)) was used as a template to carry out PCR and cycled 30 times (95°C, 45 seconds, 50°C, 1 minute, 75°C, 7 minutes) to obtain specific amplified DNA. The resulting DNA fragment was double-digested with two restriction endonucleases NcoI and XbaI. Plasmid vector pSE420D (JP-A 2000-189170) was double-digested with NcoI and XbaI, and the PCR-amplified DNA fragment double-digested with these enzymes was ligated thereto with T4 DNA ligase; pSE-MCF15 was thus obtained. The map of the plasmid is shown in the atta...

Embodiment 2

[0166] Construction of co-expression vector pSFR415 of formate dehydrogenase from Mycobacterium vaccae and carbonyl reductase from Kluyveromyces erylieii

[0167] In order to clone the region containing only the open reading frame of carbonyl reductase derived from Kluyveromyces eleminia by PCR, primers KAR-BSG5-3 (5'-TAATCTAGAGGAATTCAATAATGGATCCAACAATGACGTTTC-3' / SEQ ID NO: 5) and KAR - BSG3 (5'-TAGAAGCTTAAGCTATTAAACGCAAGTGTACCCAC-3' / SEQ ID NO: 6). Using pSE-KAR1 (JP-A 2000-236883) as a template, PCR was performed 30 times (95°C, 30 seconds, 50°C, 1 minute, 75°C, 5 minutes) to obtain specifically amplified DNA. The resulting DNA fragment was double-digested with two restriction endonucleases XbaI and HindIII. The plasmid pSE-MCF15 containing the formate dehydrogenase derived from Mycobacterium vaccae constructed in Example 1 was double-digested with XbaI and HindIII two restriction endonucleases, and was combined with T4 DNA ligase A PCR-amplified DNA fragment digested with ...

Embodiment 3

[0176] Introduction of mutations by PCR into a formate dehydrogenase from Mycobacterium vaccae

[0177] On the basis of the co-expression vector pSFR415 derived from the formate dehydrogenase of Mycobacterium vaccae and the carbonyl reductase derived from Kluyveromyces eleminus constructed in Example 2, the Primer MCF-ATG3 (5'-CTTTCTAGAGGAATTCAACCATGGCAAAAGTTCTGTCTGTTC-3' / SEQ ID NO: 7) with serine replacing cysteine ​​at the 6-position of Bacillus formate dehydrogenase and formate dehydrogenase 256 derived from Mycobacterium vaccae Primer McFDH-7mut01 (5'-GTATCCGGTTTGCGACGTCGTGACGCTGAACTCCCCGCTGCACCCCGAA-3' / SEQ ID NO: 8) and primer McFDH-7mut02 (5'-TTCGGGGTGCAGCGGGGAGTTCAGCGTCACGACGTCGCAAANOCCGGATAC-3' / SEQ ID) with cysteine ​​replaced by serine at the position. Substitution of cysteine ​​with serine at position 6 is hereinafter referred to as "C6S". According to this rule, the replacement of cysteine ​​with serine at position 256 is called "C256S".

[0178] The first round o...

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Abstract

An objective of the present invention is to provide polypeptides capable of retaining a strong enzyme activity of formate dehydrogenase in the presence of an organic solvent and to provide the uses thereof. Formate dehydrogenase mutant polypeptides, which are resistant to organic solvents, were constructed by substituting cysteines at position 146 and / or at position 256 in the amino acid sequence of Mycobacterium vaccae-derived formate dehydrogenase by site-directed mutagenesis. The polypeptides have strong activities of formate dehydrogenase in the presence of an organic solvent. The mutants are useful for the production of alcohols using ketones as raw material, etc. <IMAGE>

Description

technical field [0001] The present invention relates to formate dehydrogenase mutants derived from Mycobaterium vaccae, polynucleotides encoding these mutants, and by using them, oxidized β-nicotinamide adenine dinucleotide ( NAD + ) A method for producing reduced β-nicotinamide adenine dinucleotide (NADH). Background technique [0002] There has previously been a known method for preparing optically active (S)-4-halo-3-hydroxybutyrate esters, which is an asymmetric reduction method using microorganisms such as baker's yeast (unexamined publication Japanese Patent Application No. (JP-A) Sho 61-146191; JP-A Hei 6-209782, etc.). However, this method does not have industrial applicability due to the presence of various types of reductases in microbial cells and thus low optical purity and yield of products. Optically active (S)-4-halo-3-hydroxybutyrates can be used as intermediates in pharmaceuticals, etc. Therefore, methods (synthesis or decomposit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N1/15C12N1/19C12N1/21C12N5/10C12N9/02C12N9/04C12N15/53C12P7/02C12P7/62C12P19/36
CPCC12N9/0008C12P7/02C12P19/36
Inventor 三桥和也山本浩明木本训弘
Owner DAICEL CHEM IND LTD
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