Genetically engineered bacterium expressing microorganism cholesterol esterase and construction method thereof

A technology of cholesterol esterase and microorganisms, applied in the field of enzyme engineering, can solve the problem that compatible carriers are not easy to obtain, and achieve high-efficiency expression

Active Publication Date: 2016-05-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, such compatible vectors are not readily available because most commercial E. coli expression vectors are ColE1 / pMB1 replicons

Method used

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  • Genetically engineered bacterium expressing microorganism cholesterol esterase and construction method thereof
  • Genetically engineered bacterium expressing microorganism cholesterol esterase and construction method thereof
  • Genetically engineered bacterium expressing microorganism cholesterol esterase and construction method thereof

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Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1: the construction of recombinant vector and genetically engineered bacteria

[0024] (1) Cholesterol esterase cheS gene fragment, whose sequence is GenBank accession number AB175022.1 (cholesterol esterase from Burkholderiacepacia.). The nucleotide sequence of the SD gene fragment is: gatccataaggaggcaatctt. Cholesterol esterase molecular chaperone limS gene fragment, the sequence of which is GenBank accession number is AB175022.1 (cholesterol esterase molecular chaperone from Burkholderiacepacia.).

[0025] (2) chemically synthesize the cal gene fragment of the sequence shown in SEQ ID NO.2, or optimize the cheS gene and limS gene in the previous step, and connect them with SD to obtain the fragment of the sequence shown in SEQ ID NO.2.

[0026] (3) Perform PCR with the fragment of the sequence shown in SEQIDNO.2 as a template, and sequences such as SEQIDNO.3 and SEQIDNO.4 as upstream and downstream primers respectively, digest the PCR product with quickcu...

Embodiment 2

[0028] Embodiment 2: Enzyme production by genetic engineering bacteria fermentation

[0029] Preparation of crude enzyme solution

[0030] The culture conditions of the seed liquid: use 250mL shake flask culture, the filling liquid is 20% LB medium, and add filter-sterilized 100mg·mL in the medium -1 Take 50 μL of kanamycin sulfate, take a single colony into the culture medium, and culture overnight at 37°C and 180rpm.

[0031] Fermentation broth culture conditions: use 500mL shake flask culture, the liquid is 20% fermentation medium, the MgSO 4 ·7H 2 O, glucose, and glycerin were made into mother liquors, sterilized separately, and the corresponding amount was added when used, and 100 mg·mL was added for filter sterilization -1 100 μL of kanamycin sulfate was added to 5% seed solution, 37°C, 200rpm, cultured for 8h, added 20% lactose induction solution, 28°C, 200rpm, induced for 20h.

[0032] Collection of bacteria and acquisition of crude enzyme solution: centrifuge the ...

Embodiment 3

[0036] Example 3: Purification of CAL

[0037] Affinity purification was performed using agarose as matrix, 2-hydroxy-1,3-propanediamine linking arm, and 8-chloroalloxazine as ligand synthesis medium.

[0038] 1. Sample preparation: adjust the conductivity of the loaded crude enzyme solution to be consistent with the conductivity of 0.11M potassium phosphate buffer solution (pH7.0).

[0039] 2. Column packing: 15mL plastic small column is fixed vertically, the tube at the lower end of the column is opened, and 3mL of medium is loaded, and allowed to settle, so that the ethanol in the column flows out to about a little higher than the medium interface.

[0040] 3. Equilibration: Equilibrate 10 column volumes with 0.11 M potassium phosphate buffer (pH 7.0).

[0041] 4. Sample loading: Take 20mL of crude enzyme solution and add it to the well-balanced affinity medium.

[0042] 5. Washing: Wash 10 column volumes with 0.11M potassium phosphate buffer (pH7.0) to remove unbound pro...

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Abstract

The invention discloses a genetically engineered bacterium expressing microorganism cholesterol esterase and a construction method thereof, and belongs to the technical field of enzyme engineering. The method comprises the steps that Burkholderia cepacia. source cholesterol esterase and cholesterol esterase molecular chaperone genes are connected in series to achieve coexpression, wherein cholesterol esterase genes and the molecular chaperone genes are connected through a SD sequence. According to the method, the Burkholderia cepacia. source cholesterol esterase is successively expressed, and active expression and efficient expression of the Burkholderia cepacia. source cholesterol esterase are achieved. The cholesterol esterase activity in a crude enzyme solution fermented by the constructed genetically engineered bacterium can reach 640 U / L.

Description

technical field [0001] The invention relates to a genetically engineered bacterium expressing microbial cholesterol esterase and a construction method thereof, belonging to the technical field of enzyme engineering. Background technique [0002] Cholesterol esterase is an enzyme that catalyzes the hydrolysis of cholesterol esters into cholesterol and fatty acids. Studies in recent years have shown that many human frequently-occurring diseases, such as cardiovascular and cerebrovascular diseases, liver and gallbladder diseases, kidney diseases, diabetes, etc., are related to abnormal cholesterol metabolism in the human body. Therefore, the determination of plasma total cholesterol has become a clinical practice. An important reference index in diagnosis. As far as TC detection is concerned at the present stage, cholesterol esterase is one of the key enzymes, which can quickly and accurately detect the concentration of total cholesterol in serum, which can be used as a diagno...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12N15/70C12N1/21
CPCC12N9/18C12Y301/01013
Inventor 杨海麟张玲王武辛瑜张亚慧
Owner JIANGNAN UNIV
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