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Bordetellasp and uses for preparations of cholesterol oxidase and cholest-4-en-3-ol thereof

A cholesterol oxidase and Bordetella technology, applied to Bordetella and its application field, can solve the problems of low yield of cholesterol oxidase and the like, and achieve the effect of great application prospect

Inactive Publication Date: 2009-12-02
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the current low yield of cholesterol oxidase and the current situation that various steroid drug intermediates are needed to prepare important steroid drugs, the problem to be solved in the present invention is to provide a strain of Bordetella producing cholesterol oxidase Its application in the preparation of cholesterol oxidase and cholest-4-en-3-one

Method used

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  • Bordetellasp and uses for preparations of cholesterol oxidase and cholest-4-en-3-ol thereof
  • Bordetellasp and uses for preparations of cholesterol oxidase and cholest-4-en-3-ol thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Screening of Bordetella sp. B4 CGMCC No.2229.

[0036] From the soil samples from paddy fields, soybean root soil, corn root soil, and vegetable gardens in the suburbs of Jinan, 48 strains were obtained through enrichment culture, plate streaking and other methods. The strain with strong ability was identified as Bordetella sp. by 16s rDNA identification. Fermentation was carried out at a culture temperature of 37°C, a shaker speed of 260rpm, and an initial pH of the fermentation medium of pH 7.0. Thin-layer chromatography detection, mass spectrometry, and nuclear magnetic analysis showed that Bordetella sp.B4 ) produces cholest-4-en-3 ketone, and the activity of cholesterol oxidase reaches 2000U / L.

[0037] The enrichment culture of the above strains uses a liquid basic inorganic salt medium, and 0.1% by mass of cholesterol is added as the sole carbon source.

[0038] The above-mentioned plate primary screening medium uses a liquid basic inorganic salt med...

Embodiment 2

[0045] Example 2: Identification of 16s rDNA of Bordetella sp. B4 CGMCC No.2229.

[0046] The genome of Bordetella sp. (Bordetella sp.) B4 CGMCC No.2229 thallus described in Example 1 was roughly extracted, and the primers were designed as follows: upstream primer 5'-AGAGTTTGATCCTGGCTCAG-3', downstream primer 5'-AAGGAGGTGATCCAGCCGCA-3' , 16s rDNA was amplified by conventional PCR.

[0047] Sequencing results: the nucleotide sequence of 16srDNA is shown in SEQ ID NO.1.

[0048] Compare with the sequence in NCBI, see the comparison result figure 1 .

[0049] According to the results of 16s rDNA sequence alignment, the screened bacterial strain of the present invention is determined to be Bordetella sp.

Embodiment 3

[0050] Example 3: Preparation of cholesterol oxidase and cholest-4-en-3-one by using Bordetella sp. B4 CGMCC No.2229. Among them, the rotational speed is 220 rpm.

[0051] (1) Strain selection: select Bordetella sp. B4 CGMCC No.2229;

[0052] (2) Slant culture: Inoculate the strains on a basic inorganic salt medium, add agar with a mass volume ratio of 1.8%, and culture statically for 38 hours at 37°C;

[0053] (3) Seed culture: the bacterial strain cultivated in step (2) is inoculated under aseptic conditions with inoculation loops to connect 1 to 2 loops in 100 mL of basic inorganic salt medium containing cholesterol with a mass volume ratio of 0.3%, at 28 ° C. Under the condition of shaking culture for 24 hours, the seed solution was obtained;

[0054] (4) Expanded cultivation: Inoculate the seed solution in 50 mL of basic inorganic salt medium containing 0.3% cholesterol with a volume ratio of 4.0%, and cultivate at 220 rpm for 64 hours at 28°C;

[0055] (5) Collect the...

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Abstract

The invention discloses a Bordetella strain and its application in preparing cholesterol oxidase and cholest-4-en-3-one. The strain is Bordetella sp. B4 CGMCC No.2229, which can synthesize extracellular cholesterol oxidase and convert cholesterol into cholest-4-en-3-one. The strain of the invention has the characteristics of short fermentation time and high enzyme production activity, and the extracellular enzyme activity can reach 2000U / L, which has reached or exceeded the international leading level.

Description

technical field [0001] The present invention relates to a strain of Bordetella and its application, in particular to a strain of Bordetella producing cholesterol oxidase and its application in preparing cholesterol oxidase and cholest-4-en-3-one . Background technique [0002] Cholesterol is an animal sterol. Abnormal metabolism of cholesterol can cause many diseases in the human body, such as atherosclerosis, myocardial infarction, stroke, aneurysm, and gallstones. Therefore, it is necessary to detect cholesterol in plasma and food and reduce food The cholesterol content in plasma has important clinical significance, and cholesterol oxidase can be used as an important tool to detect the cholesterol content in plasma; adding cholesterol oxidase to food can reduce the cholesterol content in food. In addition, cholesterol oxidase can also inhibit the growth of Lepidoptera larvae. Moreover, cholesterol oxidase can oxidize cholesterol into cholest-4-en-3-one, which is an impor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/04C12P7/26
Inventor 宋欣林彦良林建强曲音波
Owner SHANDONG UNIV
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