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Engineering saccharomyces cerevisiae and method for preparing artificial bear bile powder

A technology of Saccharomyces cerevisiae and bear bile powder, which is applied in the fields of biotechnology and medicine, can solve problems such as large disputes, endanger the health and protection of bears, and achieve the effects of fast transformation, meeting the needs of the pharmaceutical market, and easy operation.

Pending Publication Date: 2021-05-11
SHANGHAI UNIV OF T C M
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods will seriously endanger the health and protection of bears, so they are extremely controversial

Method used

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  • Engineering saccharomyces cerevisiae and method for preparing artificial bear bile powder
  • Engineering saccharomyces cerevisiae and method for preparing artificial bear bile powder
  • Engineering saccharomyces cerevisiae and method for preparing artificial bear bile powder

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1 Engineering Saccharomyces cerevisiae construction and thalline collection

[0061] (1) Vector construction: two 7α-HSDH and two 7β-HSDH genes (the nucleotide sequences of which are respectively shown in SEQ ID No.1-No.4) were respectively connected to pRS424 by double enzyme digestion and T4 ligase ligation and pRS426 plasmid vectors, respectively named pRS424-α 1 , pRS424-α 2 , pRS426-β 1 , pRS426-β 2 . Its structure is as follows Figure 1-Figure 4 shown.

[0062] (2) Construction of engineering Saccharomyces cerevisiae: pRS424-α 1 , pRS424-α 2 , pRS426-β 1 , pRS426-β 2 Transformed into Saccharomyces cerevisiae CEN.PK2-1C and W303a competent cells respectively to obtain engineering Saccharomyces cerevisiae CEN.PK2-1C-pRS424-α 1 -pRS426-β 1 , CEN.PK2-1C-pRS424-α 1 -pRS426-β 2 , CEN.PK2-1C-pRS424-α 2 -pRS426-β 1 , CEN.PK2-1C-pRS424-α 2 -pRS426-β 2 , W303a-pRS424-α 1 -pRS426-β 1 , W303a-pRS424-α 1 -pRS426-β 2 , W303a-pRS424-α 2 -pRS426-...

Embodiment 3

[0071] Example 3 Preparation of Crude Enzyme Liquid and Crude Enzyme Powder by Pickling Glass Beads Method

[0072] (1) 10 milliliters of engineered Saccharomyces cerevisiae culture fluid according to step (3) of Example 2, centrifuged at 2200 rpm for 5 minutes at 4° C., and discarded the supernatant.

[0073] (2) Resuspend the bacteria in 500 μL of sterile water, transfer to a centrifuge tube, centrifuge at 4°C and 13,000 rpm for 60 seconds at high speed, discard the supernatant, and freeze the cells at -80°C for later use.

[0074] (3) Resuspend the cells with 500 μL of cell disruption buffer (50 mM sodium phosphate buffer, pH 6.5, 5% glycerol), centrifuge at 2200 rpm at 4° C. for 5 minutes, and discard the supernatant.

[0075] (4) Resuspend the cells with 150 μL cell disruption buffer, add the same volume of acid-washed glass beads (424-600 μm), vortex mix-ice bath (vortex mix for 30 seconds, ice bath for 30 seconds, repeat 10 times, total time 10 minutes) to fully lyse t...

Embodiment 4

[0078] Example 4 Homogenizer Crushing Method Prepares Crude Enzyme Liquid and Crude Enzyme Powder

[0079] (1) The 6 liters of engineered Saccharomyces cerevisiae culture solution obtained by the operation in step (4) of Example 2 was centrifuged at 3500 rpm and 4° C. for 10 minutes, and the supernatant was discarded.

[0080] (2) Resuspend the bacterial cells in 200 ml of sterile water, transfer to a centrifuge tube, centrifuge at 12,000 rpm for 60 seconds, discard the supernatant, and freeze the bacterial cells at -80°C for later use.

[0081] (3) Resuspend the cells with 200 ml of cell disruption buffer, centrifuge at 2600 rpm and 4°C for 10 minutes, and discard the supernatant.

[0082] (4) Resuspend the bacteria with 120 ml of cell breaking buffer, and use a homogenizer to break the cells at 600-1000 bar.

[0083] (5) Centrifuge at 12000rpm for 5-10 minutes, transfer the supernatant to a new centrifuge tube, and obtain crude enzyme solution, or dry it in a freeze dryer t...

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Abstract

The invention relates to the field of biotechnology and medicine, in particular to a method for preparing artificial bear bile powder by biocatalysis of active substances expressed by engineering saccharomyces cerevisiae. The method comprises the following steps: performing liquid culture on saccharomyces cerevisiae which simultaneously expresses 7alpha-hydroxysteroid dehydrogenase (7alpha-HSDH) and 7beta-hydroxysteroid dehydrogenase (7beta-HSDH) genes to prepare a crude enzyme extract; directly converting taurochenodeoxycholic acid in poultry bile products such as chicken gall powder into tauroursodeoxycholic acid with a certain proportion. According to the method, in-vitro double-enzyme catalysis is used, NADP+ is added as an auxiliary factor to promote the reaction, the crude enzyme preparation process is simple, the catalytic reaction condition is mild, the result is stable, the whole process is green, safe, rapid, environment-friendly, pollution-free and controllable, and great significance is achieved by utilizing the biotechnology to replace resources with bear bile powder.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medicine, and in particular relates to a method for preparing artificial bear bile powder by using engineering brewer's yeast. Background technique [0002] Bear bile powder is a dry product obtained from the bile drainage of Ursus thibetanus and brown bears (Ursus arctos) after gallbladder surgery. It is clinically used as a bear bile medicine. Efficacy, it is mostly used clinically to treat liver and gallbladder and eye diseases. Bile acids are the active ingredients with the highest content and the most research in bear bile powder, and the main active ingredient is tauroursodeoxycholic acid (TUDCA). [0003] Currently, live drainage of bear bile is the main way to obtain bear bile powder, and has formed an industrial scale. In vivo drainage technology is divided into two types: tubeless and tube. It is usually necessary to select a healthy bear, and after anesthesia, open the abdomen and p...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/53C12P33/06C12R1/865
CPCC12N9/0006C12N15/81C12P33/06C12Y101/01159C12Y101/01201
Inventor 赵淑娟王峥涛金丽娜杨莉周吉燕胡之璧王如锋张金家
Owner SHANGHAI UNIV OF T C M
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