Genetically engineered bacterium of bile salt hydrolase, bile salt hydrolase and application of bile salt hydrolase

A technology of bile salt hydrolyzing enzyme and genetically engineered bacteria, applied in the field of bile salt hydrolyzing enzyme and its application, bile salt hydrolyzing enzyme genetically engineered bacteria, can solve the problems of product quality impact, high energy, environmental pollution, etc., and achieve stable enzyme activity Good performance, strong catalytic ability, and good protein expression ability

Pending Publication Date: 2021-06-25
GUANGDONG HEJI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the production of N-acyl amino acid surfactants mostly adopts chemical synthesis. Although it has the advantages of high yield and easy preparation, it needs high energy, strong alkali or highly toxic reagents in the synthesis process, and it needs to be brought in by organic solvents, etc. conditions, which are likely to cause environmental pollution and affect product quality

Method used

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  • Genetically engineered bacterium of bile salt hydrolase, bile salt hydrolase and application of bile salt hydrolase
  • Genetically engineered bacterium of bile salt hydrolase, bile salt hydrolase and application of bile salt hydrolase
  • Genetically engineered bacterium of bile salt hydrolase, bile salt hydrolase and application of bile salt hydrolase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Construction of bile salt hydrolase engineering strain BSH7-BL21:

[0040] (1) The bile salt hydrolase gene was synthesized by conventional PCR. The nucleotide sequence of the bile salt hydrolase gene is shown in SEQ ID NO.1, including 338 amino acids and 1071 nucleotides in full length. Use the SanpGene software infusion to import into the vector plasmid pET-21a(+), and synthesize the recombinant plasmid bsh-pET-21a. The schematic diagram of the nucleotide distribution of the recombinant plasmid is as follows figure 1 shown;

[0041] (2) Transform the recombinant plasmid into the competent cells BL21 (DE3), then spread the transformed competent cells on LB plates containing 0.1 g / ml ampicillin, place them upside down in an incubator and cultivate them overnight for 14 hours to obtain the cloned strain BSH7-BL21 is a bile salt hydrolase engineering strain.

[0042] Preparation of bile salt hydrolyzing enzyme crude enzyme solution:

[0043] In a 250mL Erlenmeyer flask...

Embodiment 2

[0045] Extraction and purification of bile salt hydrolase:

[0046] (1) Take 4ml of the crude bile salt hydrolyzing enzyme solution prepared in Example 1, centrifuge (12000rpm, 1min), discard the supernatant, and resuspend with 2ml of 50mmoL Tri-HCl pH6.4 solution (resuspension ratio 2:1) As for the 5ml centrifuge tube, the cell disruptor is crushed in an ice bath (probe 6, 3min, run for 2s and stop for 4s);

[0047] (2) Take 40μL of the solution obtained in step (1), centrifuge (12000rpm, 1min), take out the supernatant, wash the lower layer with 50mmoL Tri-HCl pH6.4 solution twice, and finally resuspend with 40μL, in the upper and lower layers Add 10 μL of eluent to the liquid, and bathe in boiling water for 10 minutes.

[0048](3) The crude enzyme liquid of bile salt hydrolyzing enzyme obtained in step (2) is purified using an AKTA protein purifier, and the purification column used is a Ni-NTA packing column, and the washing buffer (Tris 50mM, NaCl 300mM, imidazole 30mM, ...

Embodiment 3

[0053] Bile salt hydrolase catalyzes the decomposition of amino acid surfactants:

[0054] Add 10.3 mg of N-lauroyl glycine to a centrifuge tube, add 1 mL of buffer solution (20 mM Tri-HCl, pH 6.0) and 100 μL of the crude bile salt hydrolyzing enzyme solution prepared in Example 1, and react at 37°C for 17 hours. Take 100 μL of the reaction solution, add 400 μL of buffer solution and 500 μL of methanol, mix well, filter, and detect the product by HPLC:

[0055] Inject 20 μL and set the flow rate to 1 mL / min. The mobile phase configuration is as follows: A phase is 0.1% trifluoroacetic acid-water solution, B phase is 0.1% trifluoroacetic acid-methanol solution; 0min, A phase 25%, B phase 75%; 3min, A phase 10%, B phase 90% %; 9min, A phase 10%, B phase 90%; 9.01min, A phase 25%, B phase 75%; 13min end. DVD detector detects at 210nm, column C185μm 4.6×150mm.

[0056] The test results show that the bile salt hydrolyzing enzyme prepared in the example of the present application...

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Abstract

The invention belongs to the technical field of biology. The invention provides a bile salt hydrolase genetically engineered bacterium, a bile salt hydrolase and application of the bile salt hydrolase. The nucleotide sequence of the bile salt hydrolase gene is as shown in SEQ ID NO.1, and the amino acid sequence of the bile salt hydrolase gene is as shown in SEQ ID NO.2. Recombinant vector plasmids for expressing the bile salt hydrolase are constructed through coding genes of the bile salt hydrolase, expression is induced through escherichia coli, the obtained bile salt hydrolase genetically engineered bacteria have good protein expression ability, and the expressed bile salt hydrolase can be used for catalytic decomposition or synthesis of N-acylamino acid surfactants, has the advantages of strong catalytic ability and good enzyme activity stability, and has the potential to be developed into a biocatalyst for synthesizing an amino acid surfactant.

Description

technical field [0001] The application belongs to the field of biotechnology, and in particular relates to a genetically engineered bacterium for bile salt hydrolysis enzyme, bile salt hydrolysis enzyme and application thereof. Background technique [0002] Bile salt hydrolase (BSH) is a very important biological enzyme in the growth and reproduction of microorganisms. Bile salt hydrolase is an intracellular enzyme encoded by the BSH gene, which is insensitive to oxygen and is suitable for production in a slightly acidic environment. It can hydrolyze bound bile salts (taurocholate and glycocholic acid) in the gastrointestinal tract Salt), it is converted into amino acid and unbound cholic acid, thereby reducing the cholesterol content in serum, so it is used for the research on the mechanism and medicine of lowering cholesterol, and there is no application of it in catalytic amino acid surfactant Research. [0003] Amino acid surfactants are surfactants synthesized from am...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N9/80C12N15/70C12P13/02C12P7/64C12R1/19
CPCC12N9/80C12N15/70C12P7/6409C12P13/02C12P7/6436C12Y305/01024
Inventor 胡丽云曾惠羚任杰何廷刚艾勇张炽坚
Owner GUANGDONG HEJI BIOTECH CO LTD
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