1,3-propylene glycol oxidoreductase isozyme mutant, and gene and application thereof

A technology of propylene glycol and reductase, applied in 1 field, can solve problems such as difficulty in large-scale industrialization, limited improvement, and achieve the effects of improved activity, high catalytic activity, and broad application prospects

Inactive Publication Date: 2012-07-25
UNIV OF SHANGHAI FOR SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the previous patent applications CN20071017175812 and CN200710171759.6 of the inventor of this patent, it was also disclosed that on the basis of the 1,3-propanediol oxidoreductase isoenzyme gene yqhD, the catalytic 3-hydroxypropanal activity was improved by mutation directed evolution technology. High variant enzyme, but the degree of improvement is limited, and there are still some difficulties in the large-scale industrialization of biotransformation and synthesis of 1,3-propanediol

Method used

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  • 1,3-propylene glycol oxidoreductase isozyme mutant, and gene and application thereof
  • 1,3-propylene glycol oxidoreductase isozyme mutant, and gene and application thereof
  • 1,3-propylene glycol oxidoreductase isozyme mutant, and gene and application thereof

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Experimental program
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Effect test

Embodiment 1

[0015] Design of Saturation Mutation Primers and Acquisition of PCR Amplified Products

[0016] Upstream primer 5'-ACACCGTGGAANNNTATGTTACCAAACC-3' (SEQ ID NO: 2)

[0017] Downstream primer 5'-GGTTTGGTAACATANNNTTCCACGGTGT-3' (SEQ ID NO: 3)

[0018] Add 100pmol of dNTPs to a 500μL eppendorf tube, 15pmol of each upstream primer and downstream primer, about 2ng pET28α(+)yqhD (obtained for cloning yqhD into pET28α(+) vector, has been reported in many literatures) as template DNA , 2.5U PfuDNA polymerase, 5 μL of MgCl containing 25mmol / L 2 The Pfu PCR buffer was then added to a total volume of 50 μL with sterile distilled water. The reaction parameters were denaturation at 95°C for 1min, followed by 20 cycles at 95°C for 30s, 52°C for 1min, and 72°C for 1min, and then extended at 72°C for 2min to obtain PCR amplification products.

[0019] Mutation library construction

[0020] The above-mentioned PCR amplified PCR product was digested with DpnI for 2 hours, then transformed int...

Embodiment 2

[0024] Fermentative Production of 1,3-Propanediol by Recombinant Escherichia coli

[0025] a) The positive clone containing the 1,3-propanediol reductase isoenzyme variant gene Gln202His obtained in picking Example 1 was inserted into the seed medium containing 40 μg / ml kanamycin, 180r / min, 37 Cultivate overnight on a shaker for 12 hours. Insert the seed solution into the fermentation medium containing 40 μg / mL kanamycin at an inoculum size of 2% (v / v), culture at 180 r / min, shaker at 37°C until the OD of the bacteria 600 About 0.7-0.9, add IPTG to the final concentration of 0.4mmol / L, and add 25g / L substrate 3-hydroxypropanal, at 25°C, 120r / min fermentation culture for 10 hours.

[0026] b) Collect the fermentation broth, centrifuge at 3000-5000r / min for 15-30 minutes, and collect the supernatant. Add an equal volume of organic solvent chloroform to extract 1,3-propanediol, and then use rotary hair growth to concentrate the extract for gas chromatography analysis .

[0027...

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Abstract

The invention discloses a 1,3-propylene glycol oxidoreductase isozyme mutant, and a gene and application thereof. The 1,3-propylene glycol oxidoreductase isozyme mutant, provided by the invention, has activity for catalyzing 3-hydroxypropionaldehyde to generate 1,3-propylene glycol, and is 1,3-propylene glycol oxidoreductase isozyme containing Q202H mutation. A variant gene is measured by an enzyme kinetics curve; in comparison with the 1,3-propylene glycol oxidoreductase isozyme mutant, the catalysis efficiency is remarkably improved, and the wide application prospect is provided for biological production of 1,3-propylene glycol.

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 in particular relates to a 1,3-propanediol oxidoreductase isozyme that catalyzes 3-hydroxypropionaldehyde to generate 1,3-propanediol Mutants and their uses. 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. ...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12P7/18
Inventor 李红梅王伟洁邓龙华
Owner UNIV OF SHANGHAI FOR SCI & TECH
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