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Red steroid brevibacterium for highly yielding cholesterol oxidase

A technology of cholesterol oxidase and Brevibacterium sterol, which is applied in the field of Brevibacterium erythroderma, which can solve the problems of fermentation, separation and application of cholesterol oxidase, the inability to meet the requirements of industrial production, and difficulty in dispersing, etc.

Inactive Publication Date: 2008-05-28
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The enzyme-producing ability of wild bacteria is about 300U / L (fermentation liquid), which cannot meet the requirements of industrial production; in addition, the production of cholesterol oxidase by wild bacteria must be induced by substrates, and cholesterol is insoluble in water, so it is difficult to produce in aqueous media. Uniform and stable dispersion also brings difficulties to the fermentation, separation and application of cholesterol oxidase

Method used

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  • Red steroid brevibacterium for highly yielding cholesterol oxidase
  • Red steroid brevibacterium for highly yielding cholesterol oxidase
  • Red steroid brevibacterium for highly yielding cholesterol oxidase

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1: Determination of compound mutagenesis conditions

[0041] Ultrasound, as a physical mutagen, is an auxiliary mutagenesis method. The chemical mutagen NTG is the main mutagenesis method. It is necessary to investigate the bacterial concentration. Under the same conditions of treatment time and temperature, ultrasonic, NTG, and ultrasonic-assisted NTG are lethal to the bacterial species. The impact of the rate. In order to select a mutagenesis treatment program with a strong lethality rate.

[0042] (1) Ultrasonic mutagenesis treatment of the starting strain: Take 30mL of the activated bacterial suspension, and divide it into six test tubes evenly. One is used as a control, and the other five are treated with ultrasound (200w, 50KHz), and each ultrasound is irradiated for 5 minutes. , Stop for 1min in the middle. Intermittent treatment for 2, 3, 4, 5, 6 times, after treatment, diluted and spread in seed culture medium, cultivated at 30°C for 24 hours, counted, and ...

Embodiment 2

[0046] Example 2: Determination of cholesterol oxidase activity

[0047] Cholesterol oxidase enzyme activity is determined by colorimetric method. The principle is: Cholesterol is decomposed into a molecule of cholesterol 4-en-3-one and a molecule of H under the catalysis of COD. 2 O 2 , H 2 O 2 Decomposition under the action of peroxidase can make 4-amino-antipyrine and phenol form a red subquinone compound, which has a maximum absorption peak at 500nm, by measuring the absorbance of the reaction solution at 500nm , Can quantitatively determine the amount of cholesterol oxidation, thereby calculating the enzyme activity unit of cholesterol oxidase.

[0048] 3mL solution A (4-amino-antipyrine 1mmol / L; phenol 6mmol / L; sodium azide 0.2g / L; peroxidase 5000U / L; potassium phosphate buffer 25mmol / L, pH7.5), 150μL solution B (cholesterol 8.26mg / mL; Triton X-100 4.26%; isopropanol as solvent), 50μL enzyme solution, 37°C, react for 5 minutes, boiling water bath for 3 minutes, and measure t...

Embodiment 3

[0049] Example 3: 16s rDNA strain identification

[0050] Take the fresh bacterial liquid in the exponential growth period, collect the bacteria by centrifugation, and extract the genomic DNA according to the literature method. Design and synthesize primers based on the most conserved sequence in bacterial 16S rDNA,

[0051] Forward primer: 5’-AGAGTTTGATCCTGGCTCAG-3’;

[0052] Reverse primer: 5'-AAGGAGGTGATCCAGCCGCA-3'.

[0053] PCR reaction conditions: 50μL system containing 1μg of genomic DNA, 20mmol / L Tris-HCl (pH8.4), 50mmol / L KCl, 1.5mmol / L, MgCl 2 , 200μmol / L dNTP, 500p mol / L of forward and reverse primers, 2.5u Taq enzyme, denatured at 97℃ for 2min, then enter the cycle, 94℃ 1min, 55℃ 1min, 72℃ 10min, a total of 35 cycles, and finally 72℃ extension 10min. The amplification product was purified according to the instructions of Shanghai Huashun Bioengineering Co., Ltd.'s small amount of gel recovery PCR product purification kit. The sequencing was completed by Shanghai Shengg...

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Abstract

Provided is red brevibacterium sp of high-yield cholesterol oxidase, which belongs to the technical field of microorganism bacterial strain. The bacterial strain of the invention is classified and named as brevibacterium sp. DGCDC-82 (choB), and is preserved in the Preservation Center of Chinese Typical Culture, and the preservation number is CCTCC NO: M207182. The bacterial strain of the invention is mutagenized and achieved by the process of combining nitrosoguanidine with ultrasonic on the basis of white brevibacterium sp. The fermentation study finds that the vitality for producing cholesterol oxidase of the red brevibacterium achieves 1.21U per milliliter, which is increased by 140%. The relation of the bacterial strain enzyme production and haematochrome is studied with the process of reverse mutation, finding that the content of the bacterial strain enzyme production has positive correlation with the content of haematochrome, the darker the red color of the bacteria is, the higher the vitality of fermentation and enzyme production is.

Description

Technical field [0001] The present invention relates to a kind of Brevibacterium rubrum erythraea with high production of cholesterol oxidase, belonging to the technical field of microbial strains. Background technique [0002] Cholesterol is an essential component of animal cell membranes. In the human body, it mainly exists in the lipid mixture synthesized by plasma, liver, adrenal glands and cells. It is an important substance in the process of animal lipid metabolism, can regulate the absorption of fat in the digestive tract, and is a bile acid, steroid hormone and The precursor substance of vitamin D biosynthesis. The level of cholesterol in plasma is an important physiological indicator of human health. Many heart, brain, and blood diseases of the human body are related to the cholesterol content in the blood, such as atherosclerosis, coronary heart disease, and cholelithiasis. Therefore, in the past two decades, excessive cholesterol may harm human health has become a hot ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/01C12N9/02C12R1/13
Inventor 王武杨海麟桑吉霍惠芝王龙刚
Owner JIANGNAN UNIV
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