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Cladosporium yielding cholesterase and enzyme producing method

A technology for producing sterol esterase and sterol esterase crude enzyme, which is applied in the field of bioengineering, can solve the problems of long operation time, high equipment requirements, and high cost, and achieve good hydrolysis of sterol esters, great application potential, and low fermentation cost low effect

Active Publication Date: 2019-02-15
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It can be mainly classified into two categories. The first category is to use large and medium-sized professional equipment to detect blood samples using the principle of optical absorption. This type of detection method has high requirements for equipment, and the measurement takes a long time and costs high; The second category is to use a special medical hand-held detector for detection, mainly by means of immunological methods, electrochemical methods and some optical methods for detection, which also has the disadvantages of long operation time and high cost.

Method used

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  • Cladosporium yielding cholesterase and enzyme producing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Sterol esterase Cladosporium sp. DZ16 strain screening and identification methods.

[0025] (1) Preliminary screening: Select samples with high content of sterol esters as screening raw materials, and take some samples and dissolve them in sterilized physiological saline to make their tissue distribution even. The sample was inoculated and spread on the primary screening plate medium with sterol ester as the main carbon source through aseptic operation. Inverted culture at 28-30°C for 2-3 days, observe the colony shape and the transparent circle of the medium.

[0026] (2) Re-screening: sort out the strains with a transparent circle in the primary screening medium, and perform a liquid shake flask re-screening. The fermentation conditions are 28-30°C, 150-200rpm, culture for 1-2 days, and then collect the fermentation broth. Determination of enzyme activity.

[0027] (3) Bacteria identification: Bacteria identification is carried out through extraction of ...

Embodiment 2

[0028] Example 2 Cladosporium Sp. DZ16 strain activation and enzyme production culture.

[0029] (1) Activation of bacteria.

[0030] The first step is to preserve and activate the strain, and the Cladosporium The sp. DZ16 strain was transferred from the strain preservation tube frozen at -80°C to the PDA medium in a 100mL Erlenmeyer flask for growth, with a liquid volume of 20mL, and cultured at 28-32°C for 24-48h.

[0031] The second step is to spread the bacteria on the flat plate, pick the vigorously growing seed solution, dilute and separate, and spread it on the PDA solid medium with a coating rod, culture it at 28-32°C for 48 hours, pick the large colony with good growth, and save it for later use .

[0032] (2) Fermentation broth culture.

[0033] The first step is low-salt liquid activation culture. Inoculate the large colonies that grow well in the plate coating into the low-salt liquid activation medium for cultivation. The medium contains 0.2% yeast extract, ...

Embodiment 3

[0035] Example 3 Determination of Enzyme Activity in Crude Enzyme Solution.

[0036]With 0.25mL 0.48mg / mL stigmasterol acetate solution as substrate, add 0.5mL 4-AA phenol working solution, 0.25mL 2U / mL cholesterol oxidase and 0.25mL 12U / mL horseradish peroxidase, at 40 After preheating for 5 minutes at ℃, add 25 µL of the enzyme solution to be tested, react for 30 minutes, stop the reaction with 0.5 mL of 0.1mol / L HCl, measure the absorbance at OD500, use the inactivated enzyme solution as a blank control group, and measure the enzyme activity At 0.5-0.8U / mL.

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Abstract

The invention discloses a cladosporium yielding cholesterase (Cladosporium sp.) DZ16 which is screened, wherein the preservation number is CCTCC M2016685. The bacterial colony of the culture is velvet-like and is activated by means of a first step of preserving and activating the culture and a second step of plate spraying of the culture. In the early stage of fermentation, the biomass of the bacterial strain is activated and cultured in a low salt liquid state in the first step. In the later stage, by means of multifactor compounding action in the second step, the bacterial strain is inducedtop secrete zymoprotein, so that the enzyme activity is improved. The cholesterase produced by fermenting the bacterial strain has a good application potential in the fields of medical detection, food, pulping and paper-making industry, environmental protection and the like.

Description

technical field [0001] The invention relates to a cladosporium strain producing sterol esterase obtained by screening and a method for producing the enzyme, belonging to the technical field of bioengineering. Background technique [0002] Sterols are derivatives of alkylene oxide polyhydrophenanthrene, which are widely distributed in the biological world, play a variety of physiological functions in animals and plants, and play a pivotal role in the body's metabolism. Cholesterol is the most abundant sterol compound in animals. It is mostly found in internal organs such as liver, kidney, intestine, skin, and adipose tissue. It is the precursor of five types of steroid hormones, vitamin D, and cholic acid. substance. Ensuring the supply of cholesterol and clarifying its specific content are necessary to maintain the normal life activities of the body, so the determination of sterol content in the body has important guiding significance in clinical medicine. [0003] Clinica...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N9/18C12R1/645
CPCC12N9/18C12Y301/01013C12R2001/645C12N1/145
Inventor 王鹏江晓路乔乐克杜春影
Owner OCEAN UNIV OF CHINA
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