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Cholesterol oxidase and application thereof

A technology of cholesterol oxidase and cholesterol, applied in the field of enzyme engineering, can solve the problems of low enzyme activity, poor thermal stability of cholesterol oxidase, poor tolerance of organic solvents, failure to meet the needs of industrial production, etc., and achieve good organic solvent Good effect of tolerance and thermal stability

Active Publication Date: 2019-07-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the strains producing the enzyme that have been studied more are mainly Gram-positive bacteria, and the cholesterol oxidase produced has poor thermal stability and organic solvent tolerance and low enzyme activity, which cannot meet the needs of industrial production.

Method used

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  • Cholesterol oxidase and application thereof
  • Cholesterol oxidase and application thereof
  • Cholesterol oxidase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Preparation of cholesterol oxidase using Burkholderia cepacia ZWS15

[0040] Burkholderia cepacia (Burkholderia cepacia) ZWS15 was inoculated into the fermentation medium, and cultured in a shaker at 30° C. with a rotation speed of 200 rpm for 20-28 hours.

[0041] Fermentation medium: add peptone 10g, yeast powder 5g, K 2 HPO 4 2g, KCl 0.5g, NaNO 3 2g, TritonX-100 3.4mL, MgSO 4 100mM, glucose 0.1g, cholesterol 2g, pH 7.0.

[0042] Among them: cholesterol is added in the form of mixing with a certain amount of medium, adding TritonX-100, and then ultrasonically crushing for 10 minutes.

[0043]Centrifuge the obtained fermentation broth at 4° C. at 8000 rpm for 5-10 minutes to remove cells, and take the supernatant as the crude enzyme solution. The activity of cholesterol oxidase in the crude enzyme solution was measured to be 200U·L -1 .

[0044] Precipitate the obtained crude enzyme liquid through 50% ammonium sulfate by mass percentage; collect the...

Embodiment 2

[0045] Embodiment 2: Construction of cholesterol oxidase genetically engineered bacteria

[0046] The bacterial genome kit extracted the genome of Burkholderia cepacia ZWS15 as a template, and designed primers (see Table 1) for PCR amplification. The PCR reaction parameters were: pre-denaturation at 95°C for 4 min, denaturation at 95°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 1.5 min, 30 cycles, and extension at 72°C for 10 min. The gene fragment of cholesterol oxidase whose amino acid sequence is shown in SEQ ID NO.1 (nucleotide sequence is shown in SEQ ID NO.2) is obtained.

[0047] The recovered target fragment was digested by EcoR I and Hind III, and then ligated with the recovered plasmid pET20b(+) digested with the same endonuclease at 16°C under the action of T4 ligase to obtain the recombinant plasmid pET20b(+)-COD , the recombinant plasmid was transformed into Escherichia coli E.coli BL21(DE3), and the genetically engineered bacteria E.coli BL21(...

Embodiment 3

[0050] Embodiment 3: utilize engineering bacteria to prepare cholesterol oxidase

[0051] (1) Preparation of crude enzyme solution

[0052] Seed solution culture conditions: use 250mL shake flask culture, the liquid is 20% LB medium, and add filter-sterilized 100mg·mL to the medium -1 Take 50 μL of kanamycin sulfate, take engineering bacteria E.coli BL21(DE3)-pET20b(+)-COD single colony into the culture medium, and cultivate overnight at 37°C and 200rpm.

[0053] Fermentation 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 100mg·mL of filter-sterilized -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.

[0054] Fermentation medium: Tryptone 10g·L -1 , yeast extract 5g·L -1 ...

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Abstract

The invention discloses a cholesterol oxidase and application thereof, and belongs to the technical field of enzyme engineering. A genetically engineered bacterium E.coli BL21(DE3)-pET20b(+)-COD of the cholesterol oxidase is firstly constructed and obtained, the enzyme activity of the cholesterol oxidase in a crude enzyme solution obtained through fermentation reaches up to 700 U.L-1, the specificenzyme activity of the purified cholesterol oxidase is 18.5 U.mg-1, the molecular weight of the cholesterol oxidase is about 60,000 kDa, and the cholesterol oxidase has good temperature tolerance andorganic solvent tolerance. An engineering bacterium fermentation solution is utilized for preparing cholest-4-alkene-3-ketone and cholest-4-alkene-3,6-diketone, in a two-phase transformation system taking methylbenzene as an organic phase, the transformation rate of preparing the cholest-4-alkene-3-ketone by taking cholesterol as a substrate is 30%, and the transformation rate of preparing the cholest-4-alkene-3,6-diketone is 45%.

Description

technical field [0001] The invention relates to a cholesterol oxidase and its application, belonging to the technical field of enzyme engineering. Background technique [0002] Cholesterol oxidase (Cholesterol oxidase; EC1.1.3.6; COD) belongs to the class of flavin oxidases, which can specifically oxidize steroid substrates containing 3β-OH. Cholesterol oxidase has important medical detection value and can be used to quantify the content of blood cholesterol. For example, in atherosclerotic diseases, it is necessary to evaluate the content of high-density lipoprotein or low-density lipoprotein, as well as assess the risk of thrombosis, etc., and oxidize cholesterol Enzymes immobilized on the membrane and using electrochemical biosensors to measure the content of cholesterol is a recent research upsurge; cholesterol oxidase has a wide range of substrate specificity, and can convert a large amount of 3β-hydroxy steroids, which can be used as industrial steroid drugs, steroid h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12P33/02C12R1/01
CPCC12N9/0006C12P33/02C12Y101/03006
Inventor 张玲王武武迪杨海麟辛瑜
Owner JIANGNAN UNIV
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