Mutant short-chain dehydrogenase, recombinant expression vector, genetic engineering bacterium and application

A technology of short-chain dehydrogenase and genetically engineered bacteria, which is applied in the application field of preparing optically pure chiral alcohol, can solve the problem that the catalytic activity of short-chain dehydrogenase cannot meet the requirements of industrial production, and achieves good development prospects for industrial application. , easy preparation, wide substrate adaptability

Inactive Publication Date: 2017-05-10
ZHEJIANG UNIV
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  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

However, the catalytic activity of the short-chain dehydrogenase is far from meeting the requirements of industrial production. Therefore, it is necessary to improve the catalytic efficiency of the enzyme through related technologies to fully exploit its application value.

Method used

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  • Mutant short-chain dehydrogenase, recombinant expression vector, genetic engineering bacterium and application
  • Mutant short-chain dehydrogenase, recombinant expression vector, genetic engineering bacterium and application
  • Mutant short-chain dehydrogenase, recombinant expression vector, genetic engineering bacterium and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: the construction of mutant

[0038] The pET-30a recombinant plasmid containing the short-chain dehydrogenase EbSDR8 gene was amplified using the QuickChangeTM method (Stratagene, La Jolla, CA) using the oligonucleotide fragment containing the mutation point as a primer (Table 1).

[0039] Table 1 Mutant construction primers

[0040]

[0041] a Mutation sites are underlined

[0042] PCR reaction system: 5×PrimerSTAR buffer (Mg2+plus), 5 μL; dNTPs (2.5 mM each), 2.0 μL; upstream primer (10 μM), 1.0 μL; downstream primer (10 μM), 1.0 μL; recombinant plasmid template, 15 ng; PrimerSTARpolymeraseTM HS (2.5U / μL), 0.5 μL; add ddH2O to a total volume of 25 μL.

[0043] PCR program: (1) 98°C, 1min; (2) 98°C, 10s; (3) 55°C, 10s; (4) 72°C, 7min. Steps (2)-(4) were cycled 20 times and then cooled to 4°C.

[0044] After the PCR product is washed, it is digested with the restriction endonuclease DpnI that specifically recognizes the methylation site to degrade t...

Embodiment 2

[0046] Example 2: Induced expression of short-chain dehydrogenase mutants

[0047] The engineered bacteria constructed in Example 1 were inoculated into LB liquid medium containing 50 μg / mL kanamycin, cultivated overnight at 37° C., and then inoculated into 50 μg / mL kanamycin containing 50 μg / mL kanamycin with 1% inoculum size (v / v). In 50mL LB medium, culture at 37°C and 200rpm until the cell concentration OD600 to about 0.6, add IPTG with a final concentration of 0.1mM, induce culture at 26°C for 6h, collect the cells by centrifugation at 4°C and 8000rpm for 10min, and store at -80 Store at ℃ for later use.

Embodiment 3

[0048] Embodiment 3: Separation and purification of short-chain dehydrogenase mutants

[0049] The thalli cells that embodiment 2 collects are suspended in 10mL Na 2 HPO 4 -NaH 2 PO 4 In buffer solution (100mM, pH 8.0), shake well and then crush under ultrasonic wave (effective time 8min). The broken liquid was centrifuged at 12,000 rpm for 10 min to remove cell debris, and the supernatant (crude enzyme liquid) was collected for subsequent separation and purification of the enzyme. The purification column is Ni-NTA, and the column volume is 5mL. First equilibrate the Ni-NTA column with loading equilibration buffer (20mM sodium phosphate, 500mM NaCl and 20mM imidazole, pH 7.4), and load the crude enzyme at a rate of 5mL / min. solution, eluted with loading equilibration buffer to remove unadsorbed protein, and finally eluted with elution buffer (20mMT sodium phosphate, 500mM NaCl and 500mM imidazole, pH 7.4) to collect the target protein. The enzyme liquid is desalted with H...

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Abstract

The invention provides a mutant of short-chain dehydrogenase, a recombinant expression vector, a genetic engineering bacterium, a preparation method of the mutant and application of the mutant or the genetic engineering bacterium generating the mutant in asymmetrically reducing a series of prochiral ketones to prepare optical homochiral alcohol. The chiral alcohol prepared by asymmetric reduction of the mutant of the short-chain dehydrogenase or the genetic engineering bacterium containing the mutant has high catalytic activity, and high-optical-purity chiral alcohol (ee>99%) can be synthesized.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering, and specifically relates to a molecular transformation of a short-chain dehydrogenase, and at the same time relates to a recombinant expression vector and a recombinant expression transformant of the corresponding mutant enzyme gene, as well as the above-mentioned mutant enzyme or a recombinant cell containing the mutant enzyme Applications in the asymmetric reduction of a series of latent chiral ketones to prepare optically pure chiral alcohols. Background technique [0002] Chiral alcohols are a class of optically active compounds with hydroxyl groups attached to chiral carbons. The ability of hydroxyl groups to be easily converted into various other functional groups makes chiral alcohols one of the most important chiral building blocks. Optically active chiral alcohols are widely used in the synthesis of chiral drugs, fine chemicals, and agricultural chemicals. The asymmetric red...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12P7/02
CPCC12N9/0006C12P7/02
Inventor 于洪巍李爱朋
Owner ZHEJIANG UNIV
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