1,3-methyl glycol oxidoreductase isozyme variant gene and uses thereof

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

Inactive Publication Date: 2008-05-14
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 and its application, to overcome the poor catalytic activity of the existing 1,3-propanediol oxidoreductase in the catalytic process, which seriously restricts the biotransformation Disadvantages of large-scale production of synthetic 1,3-propanediol

Method used

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  • 1,3-methyl glycol oxidoreductase isozyme variant gene and uses thereof

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Embodiment Construction

[0008] Below 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, BamHI and NheI sites were respectively introduced at the 5' end of the primers for the convenience of subsequent operations.

[0011]

[0012] Add 100 pmol of dNTPs to a 500 μl eppendorf tube, 20 pmol each of upstream primer, downstream primer, approximately 1 ng pET28α(+)yqhD as template DNA, and a final concentration of 0.05 mM MnCl 2 , 2.5uTaq DNA 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 at 95°C for 1 min, 54°C for 2 min, and 72°C for 1 min, and then extended at 72°C for 2 min to obtain PCR amplification products.

[0013] 2. Construction of mutant library:

[0014] The error-prone PCR am...

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Abstract

The invention relates to a 1, 3-propanediol oxidoreductase isozyme mutated gene and application thereof. By utilizing error-prone PCR technique-mediated random mutation, the1, 3-propanediol oxidoreductase isozyme mutated gene is obtained by screening. The codon GAC of the No. 99 aspartic acid in the gene is mutated as the codon CAA of glutamine; the codon AAC of the No. 147 asparaginate is mutated as histidine CAC; a nucleotide sequence of the mutated gene is SEQ ID No.1, and is capable of being encoded as 1, 3-propanediol oxidoreductase mutated isozyme. The invention creates 1, 3-propanedioloxidoreductase mutated isozyme that has relatively high catalytic activity to 3-Hydroxypropionaldehyde and can catalyze the 3-hydroxypropionaldehyde to produce chemical raw material 1, 3-propanediol.Compared with the 1, 3-propanediol reducase isozyme, the activity of one of the tested cases is increased by 4 to 4.6 times, which provides broad application prospect for bio-transformed production chemical material 1, 3-propanediol.

Description

technical field [0001] The invention belongs to the technical field of producing 1,3-propanediol chemical raw materials by genetically engineered bacteria in biochemical industry, and specifically relates to a 1,3-propanediol oxidoreductase isoenzyme variant gene and its application. Background technique [0002] 1,3-propanediol is an important solvent and chemical raw material, and it can be used as a monomer to synthesize polycondensates such as polyester and polyurethane with excellent performance. Recently, polytrimethylene terephthalate (PTT) synthesized from 1,3-propanediol and terephthalic acid has very unique properties, so it has been paid attention by many foreign research institutions. At present, the patent of chemical synthesis of 1,3-propanediol is owned by Dutch Shell (Shell) chemical company and German Degussa. The patent for biotransformation and synthesis of 1,3-propanediol was obtained by the U.S. DuPont (DuPont) company and the German National Center for...

Claims

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Application Information

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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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