Bile salt hydrolase mutant with improved enzyme activity

A bile salt hydrolysis enzyme and mutant technology, applied in the field of enzyme engineering, can solve the problems of easy formation of inclusion bodies and low expression of bile salt hydrolysis enzyme, and achieve the effect of improving enzyme activity and production efficiency

Inactive Publication Date: 2017-04-26
曹书华
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Most bile salt hydrolyzing enzymes reported so far have lo

Method used

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  • Bile salt hydrolase mutant with improved enzyme activity

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preparation example Construction

[0020] Preparation of crude enzyme solution: Centrifuge the fermentation broth at 6000-9000rpm, collect the bacteria, wash the bacteria with 0.1M, pH 6-7 phosphate buffer 2-3 times, and ultrasonically break for 5min, then centrifuge to collect the supernatant That is the crude enzyme solution.

[0021] Bile salt hydrolysis enzyme activity assay method: Take 10 μL enzyme solution and dilute it to 90 μL with 0.1M phosphate buffer (pH 6.0), add 10 μL combined bile salt (200mM) and mix, incubate at 37°C for 30min, add an equal volume of 15% (w / v) trichloroacetic acid to terminate the reaction, after centrifugation, take 10 μL supernatant and mix with 190 μL ninhydrin reagent, react at 100 °C for 15 min, measure the absorbance at 570 nm after cooling, and calculate according to the glycine standard curve. Among them, the composition of ninhydrin reagent is: 0.5mL 1% (w / v) ninhydrin (dissolved in 0.5M, pH5.5 citrate buffer), 1.2mL glycerol, 0.2mL 0.5M, pH5.5 citrate buffer.

Embodiment 1

[0022] Example 1 Construction of Efficient Secretion of Bile Salt Hydrolase Strain

[0023] Using the amino acid shown in SEQ ID NO.1 as a template, the gene bsh shown in SEQ ID NO.2 was synthesized after codon optimization. The gene bsh1 was ligated with the vector pET-22b(+), the ligation system: 4 μL of the target gene bsh, 1 μL of the vector pET-22b(+), 5 μL of solutionI, ligated overnight at 16°C. Transform the ligated recombinant plasmid pET-22b-bsh1 into competent cells E.coil JM109, transform the ampicillin LB plate, pick positive colonies, inoculate them into LB medium, and extract the plasmid after overnight culture on a shaker at 37°C. After the enzyme digestion was verified to be correct, the transformants were sequenced by Shanghai Sangon.

Embodiment 2

[0024] Example 2 Verification of Bile Salt Hydrolase Production Strain with High Secretory Ability

[0025] The plasmid sequenced correctly in Example 1 was transformed into Escherichia coli E.coli BL21(DE3). The selected transformants were inoculated into LB liquid medium, cultured at 37° C. for 12 hours, and then transferred into TB medium with an inoculation amount of 1%. Bacteria grow to OD 600 At 1.0, IPTG was added to induce, and the culture temperature was lowered to 28°C, and cultured for 36h. Centrifuge, collect the bacteria, and detect the intracellular enzyme activity. The results showed that the enzyme activity was 82.68U / mL.

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Abstract

The invention discloses a bile salt hydrolase mutant with improved enzyme activity, and belongs to the technical field of enzyme engineering. The bile salt hydrolase mutant has the advantages that in a site-directed mutation mode, asparagine near active sites inside bile salt hydrolase molecules mutates into phenylalanine, tyrosine and tryptophan with high hydrophobicity; valine mutates into isoleucine; the molecule inside hydrophobicity is changed; the enzyme activity of the bacterial strain expression bile salt hydrolase is obviously improved; the enzyme producing capability of the modified bacterial strain is obviously improved; the enzyme activity is at least improved to 91.32U/mL; the enzyme activity improvement of a compound mutant strain pET-22b(+)-bsh-V59I/N19Y is most obvious and the enzyme activity reaches 141.87 U/mL; the enzyme activity is improved by 71.6 percent through being compared with that of an original strain; the production efficiency can be improved; and the bile salt hydrolase mutant is more suitable for industrial application.

Description

technical field [0001] The invention relates to a bile salt hydrolyzing enzyme mutant with improved enzyme activity, which belongs to the technical field of enzyme engineering. Background technique [0002] Bile salt hydrolase (BSH) is an intracellular enzyme encoded by the bsh gene, widely present in intestinal microorganisms, and is the enzyme required for the first step in degrading the main component of bile—conjugated bile acid. Bile salt hydrolase hydrolyzes conjugated bile acids to taurine or glycine and free bile acids, which can be further degraded in the gut by other gut microbes. The physiological role of bile salt hydrolase is reflected in two aspects: first, it affects the fat metabolism process of the host, reduces the digestion and absorption of fat and lowers cholesterol levels, etc.; second, the degradation of bile reduces the toxicity of bile to intestinal microorganisms, improves The intestinal environment for the survival of microorganisms is guaranteed;...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12N1/21C12N15/70A23L29/00A23L33/00A61K38/50A61P1/16C12R1/19
CPCA61K38/50C12N9/80C12N15/70C12N2800/101C12Y305/01024
Inventor 曹书华
Owner 曹书华
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