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Recombinant escherichia coli for improving enzyme activity of bile salt hydrolase

A technology of recombinant Escherichia coli and bile salt hydrolyzing enzyme, which is applied in the field of bioengineering, can solve the problems of low expression level, unsuitable growth environment and fermentation intensity for large-scale industrial production, easy formation of inclusion bodies, etc., and achieve the effect of simple operation

Inactive Publication Date: 2017-05-31
曹书华
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  • Abstract
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Problems solved by technology

[0004] Since bile salt hydrolase is usually derived from microorganisms such as lactic acid bacteria, its growth environment and fermentation intensity are not suitable for large-scale industrial production
On the other hand, most bile salt hydrolyzing enzymes reported so far have low expression levels and are prone to formation of inclusion bodies

Method used

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  • Recombinant escherichia coli for improving enzyme activity of bile salt hydrolase

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Experimental program
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Effect test

Embodiment 1

[0025] The construction of embodiment 1 recombinant escherichia coli

[0026] Using the plasmid pET-28a(+) as the carrier, the bifidobacterium ATCC 27673 bile salt hydrolase gene (NCBI accession number is CP007522.1) was synthesized to construct the recombinant plasmid pET-28a(+)-bsh, which was transformed into E.coil JM109 Competent cells, pick positive colonies. After culturing overnight on a shaker at 37°C, the plasmid was extracted, and after sequencing verification, the correctly sequenced plasmid pET-28a(+)-bsh was transformed into E. coli E.coil BL21(DE3) to obtain recombinant E.coli BL21(DE3) pET-28a(+)-bsh.

Embodiment 2

[0027] The screening of embodiment 2 bile salt hydrolase gene

[0028] (1) Bifidobacterium ATCC 27672 (purchased from ATCC) was used as the starting strain, inoculated into MRS medium containing 0.5% (mass fraction) cysteine, cultured anaerobically at 37°C until logarithmic phase, and 10 μL The bacterial suspension was dropped on the sterilized and cooled glass slide, and the plasma mutagenesis was carried out with the ARTP mutagenesis breeding instrument (Siqingyuan Biotechnology), and the mutagenesis time was 0s, 5s, 10s, 15s, 20s, 40s, 60s.

[0029] (2) Transfer the mutated bacterial suspension to a sterile tube containing 1 mL of normal saline, and then dilute the sample to 10 -1 ~10 -3 Three concentrations were taken, and the diluted solution was spread on the screening medium (LB solid plate containing 0.1g / L porcine bile salt), placed in a constant temperature incubator at 37°C for anaerobic culture for 24-48h, and each treatment was repeated three times.

[0030] (3...

Embodiment 3

[0032] The construction of embodiment 3 recombinant escherichia coli

[0033] Using the plasmid pET-28a(+) as the vector, construct the recombinant plasmid pET-28a(+)-bsh06, transform it into the competent cells of E.coilJM109, and pick the positive colonies. The plasmid was extracted after overnight culture on a shaker at 37°C. After sequencing verification, the correctly sequenced plasmid pET-28a(+)-bsh06 was transformed into E. coli E.coil BL21(DE3) to obtain recombinant E. coli E.coilBL21(DE3)pET -28a(+)-bsh06.

[0034] Induced expression and enzyme activity determination of enzyme protein in embodiment 3 recombinant bacteria

[0035] (1) Recombinant Escherichia coli E.coil BL21(DE3)pET-28a(+)-bsh and E.coil BL21(DE3)pET-28a(+)-bsh06 expressing bile salt hydrolase were inoculated into Cultivate in LB liquid medium at 37°C and 200rpm for 12h, transfer to TB medium with an inoculum size of 1%, and cultivate at 37°C and 200rpm until the cells grow to OD 600 When the temper...

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Abstract

The invention discloses recombinant escherichia coli for improving the enzyme activity of bile salt hydrolase and belongs to the field of biological engineering. The recombinant escherichia coli disclosed by the invention has the beneficial effects that plasma mutation is carried out on bile salt hydrolase genes coming from bifidobacterium ATCC 27673, and heterologous expression is carried out in the escherichia coli; by induction of IPTG (Isopropyl-beta-d-thiogalactoside), the relative enzyme activity of the bile salt hydrolase produced by the recombinant escherichia coli is increased to 123.2%, and increased by 23.2% compared with a control, and after 32-hour induction, the enzyme activity is increased by 21.6%; and the method is simple in operation and has the potential of industrial application.

Description

technical field [0001] The invention relates to a recombinant Escherichia coli with improved activity of bile salt hydrolyzing enzyme, which belongs to the field of bioengineering. Background technique [0002] Excessive serum cholesterol concentration is an important factor causing coronary heart disease and other cardiovascular diseases. The primary bile salt in humans and animals is mainly synthesized by cholesterol in the liver. At carbon 24, the steroid nucleus and a glycine or taurine are passed through the amide key connection. Conjugated bile salts are stored in the gallbladder and secreted into the small intestine through the bile ducts. This bound bile salts instinctively captures dietary cholesterol and fats, allowing them to be more easily absorbed into the blood through the enterocytes, while about 95% of the bile salts enter the Enterohepatic circulation, 650mg of bile salts can avoid being absorbed by intestinal epithelial cells, therefore, a large amount of ...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N1/21C12N15/70C12N9/80C12R1/19
CPCC12N9/80C12Y305/01024
Inventor 曹书华
Owner 曹书华
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