Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Genetic engineering bacteria generating bile salt hydrolase as well as construction method and application thereof

A technology of genetically engineered bacteria and bile salt hydrolyzing enzymes, applied in hydrolytic enzymes, methods based on microorganisms, biochemical equipment and methods, etc., can solve the problems of lowering cholesterol concentration, accelerating cholesterol catabolism, etc., and achieve the effect of lowering serum cholesterol

Active Publication Date: 2011-10-19
JIANGNAN UNIV
View PDF3 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some people also believe that because cholesterol is the precursor of bile acid, some cholesterol needs to be converted into bile acid to make up for the part that is decomposed and excreted, which accelerates the catabolism of cholesterol, resulting in a decrease in cholesterol concentration.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Genetic engineering bacteria generating bile salt hydrolase as well as construction method and application thereof
  • Genetic engineering bacteria generating bile salt hydrolase as well as construction method and application thereof
  • Genetic engineering bacteria generating bile salt hydrolase as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Construction and Identification of Embodiment 1 Recombinant Bacteria

[0027] 1) Genomic DNA was extracted from Lactobacillus plantarum BBE7 as a template for PCR amplification, and the primers were:

[0028] bsh1-F:5'-CG GGATCC ATGTGTACTGCCATA ACTTATCAATCTT-3'

[0029] bsh1-R:5'-CCC AAGCTT GTTAACTGCATAGTATTGTGCTTCTGAT-3'

[0030] Lactobacillus plantarum BBE7 has been preserved in the China Center for Type Culture Collection with the preservation number CTCC M 2011116.

[0031] Add the following reagents in sequence to a 0.2mL PCR tube: 10×LA PCR buffer II (Mg 2+ plus) 5 μl; DNTP Mixture 8 μl; template DNA 1 μl; upstream and downstream primers 2 μl each; Taq enzyme 0.5 μl; add double distilled water to a final volume of 50 μl. PCR amplification was performed according to the following procedures: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 57°C for 90 s, extension at 72°C for 1 min (30 cycles); extension at 72°C for 10 min.

[...

Embodiment 2

[0036] Enzyme activity assay and protein electrophoresis of embodiment 2 recombinant bacteria

[0037] Medium: LB medium (1L) for seed and slant medium: tryptone 10g, yeast extract 5g, NaCl 10g, adjust pH to 7.0 with 1N NaOH; slant medium add agar 15g; basic fermentation medium is TB medium (1L): deionized water 900mL, tryptone 12g, yeast extract 24g, glycerol 10mL, autoclave, cool to 60°C, add 100mL sterilized potassium phosphate buffer;

[0038] Cultivation method: Cultivate to OD at 20°C and 200rpm 600 The seeds between 0.6 and 0.7 are transferred to the basic fermentation medium with a 2% inoculation amount, and cultivated at 20°C and 200rpm;

[0039] Induction conditions: the induction OD value is 0.6, the recombinant bacteria are induced at 20° C. for 35 hours, and the IPTG concentration is 1.0 mM.

[0040] Using the empty vector as a control, a protein band with a molecular weight of about 37.5 kDa was obtained by protein electrophoresis (SDS-PAGE) (see image 3 ), t...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses genetic engineering bacteria generating bile salt hydrolase as well as construction method and application thereof and belongs to the technical field of genetic engineering. According to the invention, the bile salt hydrolase (bsh) of Lactobacillus plantarum BBE7 is connected to an escherichia coli expression vector pET28a(+) through gene cloning by utilizing a recombinant DNA (deoxyribonucleic acid) technology, and Escherichia coli BL21(DE3) is transformed, and a strain of recombinant Escherichia coli BL21 (DE3)-pET28a(+)-bsh capable of generating bile salt hydrolase with relatively high activity is obtained by virtue of screening and identification, wherein the collection number is CCTCC No.M2011115. The total enzymatic activity of the bile salt hydrolase expressed by the strain against glycodeoxycholic acid hydrate (GDCA) is 3.7819 U / ml which is nearly 11 times higher than that of wild bacteria. The method lays a good foundation for the large-scale production of bile salt hydrolase and the reduction of serum cholesterol when being used as functional food.

Description

technical field [0001] The invention relates to a genetic engineering bacterium producing bile salt hydrolyzing enzyme, its construction method and application, and belongs to the technical field of genetic engineering. Background technique [0002] Bile acids in bile exist in the form of sodium salt or potassium salt, that is, bile salts, referred to as bile salts. . Bile acids can be divided into two categories: one is primary bile acids, which are synthesized by liver cells, including cholic acid, chenodeoxycholic acid and their products combined with glycine and taurine. The other type is secondary bile acids (secondary bile acids), which are deoxycholic acid and lithocholic acid produced by the action of bacteria in the intestinal tract from primary bile acids and their combined products produced in the liver. Bile acids in human bile are mainly conjugated. [0003] Bile salt hydrolase is a metabolite of lactic acid bacteria, which can hydrolyze bound bile salts and ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N1/21C12N15/70C12N9/14C12R1/19
Inventor 陈坚董自星张娟堵国成李华钟
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com