Method for increasing secretion efficiency of bile salt hydrolase

A bile salt hydrolase and efficient technology, applied in the field of genetic engineering, can solve the problem that the original nucleotide sequence of exogenous genes is not very suitable, the high-efficiency expression of alkaline pectinase and the influence of its properties, and the limited high-efficiency expression of bile salt hydrolase, etc. problem, to achieve the effect of improving secretion efficiency

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

[0004] Although the Pichia host has the advantages of expressing protein and being easy to purify, when the current bile salt hydrolase is expressed in Pichia, the expression level is not high or the original nucleotide sequence of the foreign gene is not very suitable for Pichia. The problem of the yeast host, which limits the high-efficiency expression of bile salt hydrolase in Pichia pastoris, the glycosylation phenomenon in the yeast eukaryotic expression system has a great impact on the high-efficiency expression and properties of alkaline pectinase

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  • Method for increasing secretion efficiency of bile salt hydrolase
  • Method for increasing secretion efficiency of bile salt hydrolase

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Embodiment 1

[0023] Construction and identification of embodiment 1 recombinant plasmid

[0024] The bsh gene (NCBI accession number ) derived from Lactobacillus plantarum was code freed, and the sequence shown in SEQ ID NO.1 was obtained by chemical synthesis, primers were designed, and the sequence shown in SEQ ID NO.1 was synthesized by fusion PCR Linked with the signal peptide α-MF, the column recovered the product to obtain the fusion fragment. The fusion fragment αMF-bsh and pPIC9K plasmid were ligated overnight at 16°C. The ligation product pPIC9K-αMF-bsh was transformed into Escherichia coli JM109 competent cells by chemical method. The transformation solution was spread on an LB plate containing 50 mg / L kanamycin, and the plasmid was extracted and sequenced to verify the constructed recombinant plasmid.

Embodiment 2

[0025] Example 2 Construction of recombinant Pichia pastoris producing bile salt hydrolase

[0026] The recombinant plasmid pPIC9K-αMF-bsh was linearized and transformed into pichia pastoris GS115 competent cells by electric shock, the specific method is as follows:

[0027] (1) Inoculate pichia pastoris GS115 activated on YPD plate in 25mL / 250mL Erlenmeyer flask, culture overnight at 30°C; inoculate the above culture solution in Erlenmeyer flask containing 50mL / 500mL medium with 1% (volume ratio), and cultivate the bacterial cell concentration OD 600 1.3~1.5;

[0028] (2) 5000r / min, centrifuge at 4°C for 10min to collect the bacteria, and suspend the cells with 50mL and 25mL sterile water respectively;

[0029] (3) Resuspend the above cells in 5mL of 1M sorbitol, centrifuge at 5000r / min, 4°C for 10min to collect the cells;

[0030] (4) Resuspend the above cells in 500 μL of 1M sorbitol, aliquot into 80 μL / 1.5 mL EP tubes for electrotransformation of competent cells; 5) Mix...

Embodiment 3

[0035] The engineered bacterium constructed in the present invention is used as a production strain and activated on a YPD plate. For seed liquid culture, inoculate 50mL / 250mL seed medium, and cultivate at 30°C and 220r / min for 24h. Centrifuge the seed solution, according to the final concentration of seeds in the fermentation medium as OD 600 =1 to inoculate the fermentation medium, culture at 28°C, 220r / min for 3 days. The extracellular enzyme activity of bile salt hydrolase was determined to be 5.46U / mL.

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Abstract

The invention discloses a method for increasing the secretion efficiency of bile salt hydrolase, belonging to the technical field of genetic engineering. In the invention, by adopting the recombinant DNA technology, the bile salt hydrolase gene bsh from lactobacillus plantarum is fused with signal peptide alpha-MF and connected with a carrier pPIC9K and transformed into pichia pastoris GS115 to obtain bile salt hydrolase producing recombinant pichia pastoris. By exogenously adding castanospermine and Fe<2+>, the secretion efficiency of pichia pastoris bile salt hydrolase is increased, the activity of the fermentation 3d bile salt hydrolase reaches 14.96U / mL which is 1.74 times higher than that without exogenous substance.

Description

technical field [0001] The invention relates to a method for improving the secretion efficiency of bile salt hydrolyzing enzyme, which belongs to the technical field of genetic 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 i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80C12N1/19C12N15/81C12R1/84
CPCC12N9/80C12Y305/01024
Inventor 曹书华
Owner 曹书华
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