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Beta-glucosidase gene, coding enzyme, engineering bacterium and application thereof

A technology of glucosidase and gene encoding, which is applied in the application field of preparing cucurbitacin B by biotransformation method, and can solve the problems of low efficiency of cucurbitacin B and the like

Pending Publication Date: 2021-09-14
ZHEJIANG UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The object of the present invention is to provide a kind of β-glucosidase gene derived from Streptomyces sp. (Streptomyces sp.) RW-2 bacterial strain, coding enzyme, recombinant expression vector, genetic engineering bacteria, and preparation of cucurbitacin B in biotransformation method The application solves the problem of low efficiency of producing cucurbitacin B in the existing method, and the method of the present invention greatly improves the concentration of the substrate, and the yield of cucurbitacin B is significantly improved

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  • Beta-glucosidase gene, coding enzyme, engineering bacterium and application thereof
  • Beta-glucosidase gene, coding enzyme, engineering bacterium and application thereof
  • Beta-glucosidase gene, coding enzyme, engineering bacterium and application thereof

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Embodiment 1

[0044] The acquisition of embodiment 1β-glucosidase gene, the construction of recombinant vector and recombinant genetically engineered bacteria

[0045] (1) Based on β-glucosidase (such as WP_086868833, WP_078916608, WP_004001391, WP_086870191, WP_086868717) derived from Streptomyces on NCBI (National Center for Biotechnology Information, http: / / www.ncbi.nlm.nih.gov) , WP_037888634, WP_107086935 and WP_107083827) DNA sequences were analyzed for conserved regions, a pair of degenerate primers (primer 1 and primer 2) were designed, using the whole genome DNA of Streptomyces RW-2 as a template, a section of about 836bp was successfully amplified Sequence (SEQ ID No.3), the sequence was BLAST aligned on NCBI, and its sequence homology with the β-glucosidase (from Streptomyces nigra strain 452) with the accession number AWE50172 in Genbank was the highest, reaching 99.4%.

[0046] The sequence of the amalgamative primer 1 is: 5'-AC(G / C)CT(G / C)T(A / T)CCACTGGGACCT-3', and the sequenc...

Embodiment 2

[0062] Expression of embodiment 2 recombinant β-glucosidase

[0063] The genetically engineered bacteria bglS expressing recombinant β-glucosidase can be operated according to the following steps.

[0064](1) According to the inoculation amount of 1% volume concentration, pipette the bacterial liquid of the genetically engineered bacteria bglS glycerol cryopreservation tube, inoculate it into LB liquid medium containing 20mL, and cultivate it in a shaker at 37°C and 150r / min for 12h to obtain OD 600 =2.23 seed liquid; the LB liquid culture medium consists of: yeast extract powder 5g / L, peptone 10g / L, NaCl 10g / L, solvent is deionized water, pH 7.2. 100-mL Erlenmeyer bottle of 20mL Culture medium, tied with 8 layers of gauze, and sterilized by high-pressure steam at 121°C for 20 minutes. The glycerin cryopreservation tube bacterial liquid is Escherichia coli inoculated with LB liquid medium. After culturing at 37°C and 150r / min for 12h, the culture liquid is mixed with 40% glyc...

Embodiment 3

[0075] Example 3 Application of recombinant β-glucosidase in the preparation of cucurbitacin B through biotransformation

[0076] Utilize genetically engineered bacteria bglS to express recombinant β-glucosidase, prepare cell lysate as a catalyst, and apply it to the biotransformation of muskmelon pedicle extract to convert cucurbitacin B glucoside into cucurbitacin B, which can be implemented in the following steps :

[0077] (1) According to the inoculum amount of 1% volume concentration, pipette the bacterial liquid of the genetically engineered bacteria bglS glycerol cryopreservation tube, inoculate it into 20 mL of LB liquid medium, and cultivate it in a shaker at 37°C and 150 r / min for 12 hours to obtain OD 600 =2.54 seed liquid; the LB liquid culture medium is composed of: yeast extract powder 5g / L, peptone 10g / L, NaCl 10g / L, solvent is deionized water, pH 7.2. 250-mL Erlenmeyer bottle of 20mL Culture medium, tied with 8 layers of gauze, and sterilized by high-pressure...

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Abstract

The invention discloses a beta-glucosidase gene, a coding enzyme, an engineering bacterium and application thereof in preparation of cucurbitacin B by biotransformation of cucurbitacin B glucoside. The nucleotide sequence of the gene is shown as SEQ ID No. 1. The engineering bacterium containing the recombinant beta-glucosidase gene is subjected to induced expression culture, thalli are collected and subjected to ultrasonication, the obtained cell lysis solution is used as a catalyst, a muskmelon pedicel ethanol extract methanol solution is used as a substrate, a transformation reaction system is formed in a PBS buffer solution, and the cucurbitacin B is prepared through transformation. The beta-glucosidase gene and the engineering bacterium are derived from a streptomyces RW-2 strain, the cell lysis solution can be used as a biocatalyst, the cucurbitacin B glucoside is transformed into the cucurbitacin B, the concentration of the substrate of the reaction system is increased by 3.89 times, and the transformation rate of the cucurbitacin B glucoside into the cucurbitacin B reaches 90.2%.

Description

[0001] (1) Technical field [0002] The invention belongs to the field of biotechnology, and specifically relates to a β-glucosidase gene derived from Streptomyces sp., an encoding enzyme, a recombinant expression vector, a genetically engineered bacterium, and a β-glucosidase gene in the preparation of cucurbitacin B by a biotransformation method application. [0003] (2) Background technology [0004] β-glucosidase (β-glucosidase, EC 3.2.1.21), also known as β-glucoside glucohydrolase, cellobiase, etc., can act on cellobiose or cellooligosaccharides to assist exoglucanase And endoglucanase, completely hydrolyze cellulose molecules into glucose. In addition, β-glucosidase can also act on the glycosidic bond between the glycosyl atomic group and the aromatic group or hydrocarbon group, degrade the corresponding glycoside compound, and generate aglycone (glycon) and glucose. Therefore, β-glucosidase It can be used in the degradation of glycoside compounds to generate desired a...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/42C12N15/70C12N1/21C12P33/06C12R1/19
CPCC12N9/2445C12Y302/01021C12N15/70C12P33/06
Inventor 梅建凤吴霞陈翔郑素晶吴益春黄朱梁易喻应国清
Owner ZHEJIANG UNIV OF TECH
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