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1,3-propylene glycol redex enzyme isoenzyme variant gene gln202ala and uses thereof

A technology of propylene glycol and reductase, applied in 1 field, can solve problems such as restriction and poor catalytic activity, and achieve the effects of high catalytic activity and broad application prospects

Inactive Publication Date: 2008-05-07
UNIV OF SHANGHAI FOR SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The purpose of the present invention is to provide a 1,3-propanediol oxidoreductase isoenzyme variant gene Gln202Ala and its use, to overcome the poor catalytic activity of the existing 1,3-propanediol oxidoreductase in the catalytic process, which seriously restricts biological Disadvantages of conversion synthesis of 1,3-propanediol for large-scale production

Method used

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  • 1,3-propylene glycol redex enzyme isoenzyme variant gene gln202ala and uses thereof

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Embodiment Construction

[0008] The following are embodiments of the present invention:

[0009] 1. Design of error-prone PCR primers and acquisition of PCR amplification products:

[0010] According to the gene design of 1,3-propanediol reductase isoenzyme, in order to facilitate subsequent operations, BamHI and NheI sites were respectively introduced into the 5' end of the primers.

[0011]

[0012] To a 500 μl eppendorf tube, add 100 pmol of dNTPs, 20 pmol of each upstream primer, 20 pmol of downstream primer, approximately 1 ng of pET28α(+)yqhD as template DNA, and a final concentration of 0.05 mM MnCl 2 , 2.5uTaqDNA polymerase, 5μl PCR buffer, 12.5μl 25mM MgCl 2 , and then add sterile distilled water to a total volume of 50 μl. The reaction parameters were denaturation at 95°C for 5 minutes, followed by 35 cycles of 1 min at 95°C, 2 min at 54°C, and 1 min at 72°C, and then extended at 72°C for 2 min to obtain PCR amplification products.

[0013] 2. Construction of mutant library:

[0014] ...

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Abstract

The invention discloses a 1, 3-propanediol oxidation reductase isozyme variant gene Gln202Ala and the use. Mediated random mutation of fallible PCR technology is used for screening and getting 1, 3-propanediol oxidation reductase isozyme variant gene. Codon CAG of glutamine of No. 202 in the gene is mutated into a codon GCG of Alanine, thereby getting 1, 3-propanediol oxidation reductase isozyme variant gene Gln202Ala. The nucleotide sequence of the mutated gene is SEQID No.1, which can encode 1, 3-propanediol oxidation reductase isozyme mutations. The variant gene Gln202Ala is reassembled for expressing 1, 3-propanediol oxidation reductase isozyme mutations through gene engineering technology, which is used for catalyzing hydroxy-propionaldehyde and producing 1, 3-propanediol. The invention has the advantages of improving activity by 1.5 to 2 times and providing wide application prospect for chemical raw material 1, 3-propanediol of biotransformation.

Description

technical field [0001] The invention belongs to the technical field of the production of 1,3-propanediol chemical raw materials by genetic engineering bacteria in biochemical industry, and particularly relates to a 1,3-propanediol oxidoreductase isoenzyme variant gene Gln202Ala and uses thereof. Background technique [0002] 1,3-Propanediol is an important solvent and chemical raw material. It can be used as a monomer to synthesize polycondensates such as polyester and polyurethane with excellent performance. Recently, polytrimethylene terephthalate (PTT) synthesized by using 1,3-propanediol and terephthalic acid as monomers has very unique properties, so it has attracted the attention of many foreign research institutions. At present, the patent of chemical synthesis of 1,3-propanediol is owned by the Dutch Shell (Shell) chemical company and Germany's Degussa. The patent for biotransformation and synthesis of 1,3-propanediol was obtained by DuPont of the United States and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12P7/18
Inventor 李红梅李琳陈佳
Owner UNIV OF SHANGHAI FOR SCI & TECH
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