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Engineering bacterium producing 1,3-methyl glycol oxidoreductase and preparation method for the enzyme

A genetically engineered bacteria and purpose technology, applied in the direction of oxidoreductase, biochemical equipment and methods, botanical equipment and methods, etc., can solve the problems of cost and production capacity not reaching industrial production, and achieve the effect of low cost

Inactive Publication Date: 2007-10-31
HUAQIAO UNIVERSITY
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Problems solved by technology

[0005] GDH, GDHt and PDOR are coded by different genes on the dha operon. Some people have used genetic engineering technology to transform the strains. Tong (1991 US "Applied Environmental Microbiology" 57 volumes 3541-46 pages) will come from Klebsiellapneumoniae The dha operon is expressed in Escherichia coli to produce 1,3-PD, Rolf published in 1995 in the American "Bacteriology" (177 volumes 2151-6 pages), and the 1,3-propanediol dehydrogenase gene on Citrobacter freundii Expressed in Escherichia coli, but it does not meet the requirements of industrial production in terms of cost and production capacity

Method used

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  • Engineering bacterium producing 1,3-methyl glycol oxidoreductase and preparation method for the enzyme

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Embodiment 1

[0026] The genetically engineered bacterium BL21(DE3)-pET-dhaT containing 1,3-propanediol oxidoreductase of 10-25% glycerol preserved at -70°C was taken out, and a small amount of bacterial liquid was inoculated into the culture medium of LB containing Amp, 25 Activate at -39°C for 6-12 hours on a shaker.

[0027] Then inoculate the activated bacterial liquid in (1-5% inoculum size) LB medium containing 0-150 μg / mL Amp, culture in a fermenter at 25-39°C for 0.5-2h, add inducer to the final concentration, if When IPTG is used as an inducer, it is 0.05-1mM, and lactose is used as 2-200mM for induction; continue to ferment at 15-28°C for 3-12h.

[0028] The fermented liquid was centrifuged at 6000rpm at 1°C for 10min to collect the thallus, and the binding buffer (20mM Na 3 PO 3 , 0.5M NaCl, pH 7.5-8.5) or Tris buffer (pH 7.3-8.5) to suspend, add 10 mg / mL lysozyme in an amount of 1 / 10 volume of the added binding buffer, ice-bath for 30min, shake at 4°C Shake for 10 minutes; th...

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Abstract

The invention discloses a preparing method of restructuring strain in biological technical domain, which comprises the following steps: adopting PCR technique; cloning dhaT gene from pneumonic klebsiella DSM 2026; constructing bacillus coli expressing carrier pET-dhaT with dhaT gene; transferring into bacillus coli DH5 alpha; proceeding plasmid reproduction; extracting recombinant plasmid; transferring to bacillus coli BL21(DE3); getting recombination gene engineering strain E. coli-pET-dhaT to express 1, 3-propanediol oxidoreductase (PDOR). This strain can get stable PDOR under the condition of non-antibiotic selective pressure.

Description

[0001] invention technical field [0002] The invention relates to a genetically engineered bacterium producing 1,3-propanediol oxidoreductase and a method for preparing the 1,3-propanediol oxidoreductase, that is, a genetically engineered bacterium producing PDOR and a method for preparing the PDOR. The invention belongs to the technical fields of genetic engineering and enzyme engineering. Background technique [0003] 1,3-propanediol (1,3-propanediol, hereinafter referred to as 1,3-PD) is an important environmental-friendly chemical raw material, mainly as a monomer of polyester, polyether and polyurethane, used in carpets, Engineering plastics, clothing fabrics and other fields are widely used, and the supply exceeds demand in the international market. There are two kinds of synthesis of 1,3-PD, chemical method and biological method. At present, although the production of 1,3-PD by the biological method is still in the stage of laboratory research or pilot test, from the...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/53C12N9/02C12R1/19
Inventor 方柏山夏启容
Owner HUAQIAO UNIVERSITY
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