Carbonyl reductase EbSDR8 mutant as well as construction method and application thereof

A construction method and reductase technology, applied in the biological field, can solve the problem of low activity and achieve high catalytic activity and good application and development prospects

Active Publication Date: 2019-03-15
杭州馨海生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this short-chain dehydrogenase is less active in catalyzing aliphatic latent chiral diketones (such as 2,3 butanedione, 2,4-pentanedione), so it

Method used

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  • Carbonyl reductase EbSDR8 mutant as well as construction method and application thereof
  • Carbonyl reductase EbSDR8 mutant as well as construction method and application thereof
  • Carbonyl reductase EbSDR8 mutant as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: the construction of mutant

[0049] Use oligonucleotide fragments containing mutation points as primers

[0050] (A97L-F: GGTCCGCTTGAATTGACAGAAGATTATCC;

[0051] A97L-R: GTCAATTCAAGCGGACCTGCTATTCCGGC;

[0052] K160E-F: CTTCTGCGGAACATGGTGTTGTGGGACTTAC;

[0053] K160E-R: CAACACCATGTTCCGCAGAAGTATAAGC), for PCR amplification reaction. The pET-30a recombinant plasmid containing the carbonyl reductase gene was amplified by the QuickChange™ method (Stratagene, La Jolla, CA).

[0054] Among them, the PCR program: (1) pre-denaturation at 98°C for 1 min; (2) temperature cycle at 98°C for 10s; 55°C for 10s; 72°C for 7min, and cool to 4°C after 20 cycles. After the PCR product is washed, it is digested with the restriction endonuclease DpnI that specifically recognizes the methylation site to degrade the template plasmid. Enzyme digestion reaction system and conditions: 17 μL of washed PCR product, 2.0 μL of 10× buffer, 1.0 μL of restriction endonuclease DpnⅠ, in...

Embodiment 2

[0056] Example 2: Induced expression of carbonyl reductase mutants

[0057] Inoculate the engineered bacteria constructed in Example 1 into LB medium of 50 μg / mL kanamycin, cultivate overnight at 37° C., 200 rpm, and then inoculate to 50 μg / mL kanamycin containing 50 μg / mL kanamycin with 1% inoculum size (v / v). In the LB medium of mycin, 37°C, 200rpm, culture until the bacterial cell concentration OD600 to about 0.6, add isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.1mM, at 26°C After 6 hours of induction culture, the bacterial cells were collected by centrifugation at 4°C and 8000rpm for 10 minutes, and stored at -80°C for later use.

Embodiment 3

[0058] Example 3: Fermentor Cultivation of Carbonyl Reductase Mutants

[0059] Inoculate the engineered bacteria constructed in Example 1 into the LB medium of 50 μg / mL kanamycin, cultivate overnight at 37° C., 200 rpm, and then inoculate to 50 μg / mL kanamycin with 2% inoculum size (v / v). In the culture medium of mycin, 37 ℃, 200rpm culture, inoculate to 15L in the fermentation tank that contains the fermentation medium of 50 μ g / mL kanamycin with 10% inoculum amount (v / v) in logarithmic phase, 37 ℃ , cultivated for about 14 hours (late logarithmic period), added lactose for induction for 20 hours, and centrifuged to collect the bacteria in a tube centrifuge for later use.

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Abstract

The invention discloses a carbonyl reductase EbSDR8 mutant as well as a construction method and application thereof, wherein the amino acid sequence of carbonyl reductase EbSDR8, as shown in SEQ ID NO.2, has single-point mutation or two-point combination mutation in 97th-site alanine or 160th-site lysine, wherein the 97th-site alanine is mutated to leucine and the amino acid sequence of the leucine is as shown in SEQ ID NO.4; the 160th-site lysine is mutated to glutamic acid and the amino acid sequence of the glutamic acid is as shown in SEQ ID NO.6. The invention aims at providing the carbonyl reductase EbSDR8 mutant as well as the construction method and application thereof, and the catalytic activity of the carbonyl reductase EbSDR8 is improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a carbonyl reductase EbSDR8 mutant and its construction method and application. Background technique [0002] Chiral alcohols are optically active compounds with hydroxyl groups attached to chiral carbons. The property that the hydroxyl group can be converted into a variety of other functional groups makes chiral alcohols one of the most important chiral building blocks. Therefore, optically active chiral alcohols are widely used in the synthesis of chiral drugs, fine chemicals and Agrochemicals, etc. The asymmetric reduction of latent chiral ketones is an important method to prepare optically active chiral alcohols. In theory, 100% of substrate ketones can be converted into chiral alcohols of a single enantiomer, which has high industrial application value. Among them, biocatalytic asymmetric reduction of latent chiral ketones to synthesize chiral alcohols has become the preferred...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/70C12P7/02C12R1/19
CPCC12N9/0006C12N15/70C12P7/02C12Y101/01184
Inventor 俞鑫焱张敬鹏马宗杰牛山坡
Owner 杭州馨海生物科技有限公司
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