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Esterase BSE01701 and encoding gene and application thereof

A technology of BSE01701 and bse01701, which is applied in the field of esterase BSE01701 and its coding gene and application, can solve the problem of large market gap

Active Publication Date: 2016-05-04
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Methyl D-lactate is the precursor of (R)-2-(4-hydroxyphenoxy)propionic acid, and (R)-2-(4-hydroxyphenoxy)propionic acid is the precursor for the synthesis of aryloxyphenoxy An important intermediate of propionate herbicides, with a large market gap

Method used

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  • Esterase BSE01701 and encoding gene and application thereof
  • Esterase BSE01701 and encoding gene and application thereof
  • Esterase BSE01701 and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Determination of the open reading frame boundary of the esterase gene bse01701 and primer design

[0028] The genomic DNA of Bacillus sp. SCSIO15121 was extracted. After whole genome sequencing, the genome was annotated by bioinformatics, and the esterase gene was analyzed. The open reading frame of the esterase gene bse01701 was determined. The sequence is shown in SEQ ID NO.1, the full length is 768bp (from the start codon to the stop codon), and the amino acid sequence of the encoded esterase BSE01701 is shown in SEQ ID NO.2, with a total of 255 amino acids. According to the sequence of esterase gene bse01701 obtained by analysis, the full-length amplification primers were designed as follows: Upstream primer: 5′-TGCTAGC CATATG ATACAAATTGAGAATCAAGC-3' (the NdeI restriction site is underlined); downstream primer: 5'-AGG AAGCTT TTATAAGTACGTTTCAAACCATT-3' (the HindIII restriction site is underlined).

Embodiment 2

[0029] Example 2: Cloning and vector construction of esterase gene bse01701

[0030] 2.1 PCR amplification

[0031] The primer designed in Example 1 (upstream primer: 5'-TGCTAGC CATATG ATACAAATTGAGAATCAAGC-3'; downstream primer: 5'-AGG AAGCTT TTATAAGTACGTTTCAAACCATT-3′) was sent to Shanghai Bioengineering Co., Ltd. for synthesis. The synthesized primers were dissolved in TE buffer to a final concentration of 10 μM, and the extracted Bacillus sp. SCSIO15121 total DNA was used as the DNA template. The establishment is shown in Table 1. reaction system:

[0032] Table 1PCR reaction system

[0033]

[0034] The esterase gene bse01701 was amplified using the following PCR amplification procedure: denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 62°C for 30 s, extension at 72°C for 1.5 min for 30 cycles; extension at 72°C for 10 min, cooling to 18°C.

[0035]The PCR product was electrophoresed in a 0.8% agarose gel at 120V for 20min, placed in a ...

Embodiment 3

[0046] Example 3: High expression of esterase BSE01701 in Escherichia coli BL21 (DE3)

[0047] 3.1 Preparation of Escherichia coli BL21 (DE3) competent cells

[0048] 1. Put a small amount of Escherichia coli BL21(DE3) into 5mL LB test tube solution, 200rpm, 37℃ overnight culture;

[0049] 2. Inoculate the bacterial liquid in the test tube into a 200mL LB shake flask at an inoculation amount of 1% by volume, and cultivate overnight at 200rpm and 25°C to obtain the original culture;

[0050] 3. Quickly cool the cultured shake flask to 0°C in ice water, distribute the original culture into ice-precooled centrifuge tubes (50 mL), and place on ice for a few minutes;

[0051] 4. Recover cells by centrifugation at 4000 rpm for 15 min at 4°C, and discard the supernatant;

[0052] 5. Pre-chill 10 mL of 0.1 M CaCl with ice 2 Resuspend the cells, centrifuge at 4000rpm for 10-15min at 4°C to recover the cells;

[0053] 6. Repeat 5 with 10 mL of 0.1 M CaCl 2 Resuspend cells in ice ba...

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Abstract

The invention discloses an esterase BSE01701 and an encoding gene and application thereof. A novel esterase gene bse 01701 is obtained from development from Bacillus sp. SCSIO15121, the nucleotide sequence of the esterase gene bse 01701 is shown in SEQ ID No.1, the overall length is 768bp, the nucleotide sequence of the esterase BSE01701 encoded by the esterase gene is shown in SEQ ID No.2, and the esterase contains 255 amino acids. The esterase BSE01701 is used as a catalyst and can catalyze an ester hydrolysis reaction at room temperature, DL-methyl lactate can be split selectively, the optical purity of the obtained DL-methyl lactate can exceed 99%, and the yield can reach 61.79%. The esterase has the good stability, has the good tolerance for some surface active agents and organic solvents, and can be used for the fields of washing agents, biological medicines, cosmetics, fine chemicals and the like.

Description

technical field [0001] The invention belongs to the field of biochemical industry and biotechnology, and in particular relates to an esterase BSE01701 and its encoding gene and application. Background technique [0002] Esterase (EC3.1.1.1) widely exists in animals, plants and microorganisms. It is a class of enzymes that catalyze the hydrolysis or the formation of ester bonds. The substrates are usually esters with less than ten carbon atoms in the aliphatic chain. Esterase belongs to the α / β sheet hydrolase superfamily, the catalytic center is composed of serine, aspartic acid / glutamic acid and histidine, and the conserved sequence is the pentapeptide (GXSXG) sequence near serine. Esterase can catalyze a variety of chemical reactions such as hydrolysis, esterification, and transesterification. It is an important industrial biocatalyst and is widely used in fine chemicals, washing, medicine, food, paper, leather processing, textiles, and wastewater treatment. and feed indu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/70C12N1/21C12P41/00C12P7/62
CPCC12N9/18C12P7/62C12P41/001C12Y301/01001
Inventor 胡云峰黄锦龙梁甲元张云
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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