Gene engineering bacteria capable of producing bile salt hydrolase and construction method and application thereof

A technology of genetically engineered bacteria and bile salt hydrolyzing enzymes, applied in genetic engineering, hydrolyzing enzymes, and methods based on microorganisms, can solve the problems of enzymatic substrate hydrolysis ability and low enzymatic properties

Inactive Publication Date: 2015-10-28
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Studies have reported the cloning and expression of bile salt hydrolysis enzyme genes from various sources in Escherichia coli, lactic acid bacteria, bifidobacte...

Method used

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  • Gene engineering bacteria capable of producing bile salt hydrolase and construction method and application thereof
  • Gene engineering bacteria capable of producing bile salt hydrolase and construction method and application thereof
  • Gene engineering bacteria capable of producing bile salt hydrolase and construction method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1: Construction and identification of recombinant bacteria

[0034] 1) Synthesize the nucleotide sequence shown in SEQ ID NO.2; use the synthesized sequence as a template to design primers for PCR:

[0035] bsh-F: 5'-GGGAATTC CATATG ATGTGCACTGCAGTACGTTTCGATG-3'

[0036] bsh-R: 5'-CCG CTCGAG TTTGGACTGCAGCTGGTTGCTAGACG-3"

[0037] 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. The PCR reaction program was as follows: pre-denaturation at 95°C for 5 min, denaturation at 98°C for 10 s, annealing at 57°C for 30 s, extension at 72°C for 1 min, and finally extension at 68°C for 10 min, 30 cycles.

[0038] 2) Verify with 1% agarose gel electrophoresis and recover the PCR amplified product with 0.8% agarose gel electrophoresis, as a result, a bsh gene fragmen...

Embodiment 2

[0042] Embodiment 2: Enzyme activity assay and protein electrophoresis of recombinant bacteria

[0043] Culture method: Pick a single colony of Escherichia coli BL21(DE3)(pET-22b(+)-bsh) and inoculate it into LB liquid medium, inoculate at 37°C, 200r min -1 conditions overnight. The seeds were transferred to the basic fermentation medium with a 2% inoculation amount, and were incubated at 37°C and 200r min -1 cultivated under conditions;

[0044] Induction conditions: fermented broth cultured to OD 600 When it is 0.6, add IPTG (final concentration 1mmol L -1 ), induced for 10h. Samples were taken for the activity determination of recombinant bile salt hydrolase and SDS-PAGE detection.

[0045] 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 figure 2 ), indicating that the recombinant bacteria successfully expressed bile salt hydrolase.

Embodiment 3

[0046] Embodiment 3: the influence of culture condition on producing enzyme

[0047] (1) The genetically engineered bacteria seed liquid is transferred to the basic fermentation medium with an inoculum of 1%, and cultivated to OD 600 value is 1.5, then add the final concentration of 0.8mmol L -1 Induced by IPTG, cultured at 20°C for 26h. The activity of the obtained recombinant bile salt hydrolase on the two substrates of GDCA and TDCA was 105.3 U·mL -1 , 120.3U·mL -1 .

[0048] (2) Transfer the genetically engineered bacteria seed solution to the basic fermentation medium with an inoculum of 3%, and cultivate it to OD at 36-38°C 600 value is 2.0, then add the final concentration of 1.0mmol L -1 Induced by IPTG, cultured at 20°C for 36h. The activity of the recombinant bile salt hydrolase on the two substrates of GDCA and TDCA reached the highest, respectively 123.1U·mL -1 , 115.2U·mL -1 .

[0049] (3) compared different induced OD 600 The effect on enzyme production...

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Abstract

The invention discloses a gene engineering bacteria capable of producing bile salt hydrolase and a construction method and application thereof, and belongs to the technical field of gene engineering. According to the gene engineering bacteria capable of producing the bile salt hydrolase and the construction method and application thereof, by adopting a recombinant DNA technology, the bile salt hydrolase (bsh) of Bifidobacterium infantis KL412 is connected to a prokaryotic expression vector pET-22b(+) in a gene-cloning mode and converted into Escherichia coli BL21(DE3), and recombinant Escherichia coli BL21(DE3)-pET-22b(+)-bsh capable of producing the high-activity bile salt hydrolase is obtained through screening and identifying. 2% inoculation amount of seed liquid is inoculated to a fermentation medium, the recombinant bacteria is induced for 32 h to reach the highest enzyme activity on the condition that the temperature is 20 DEG C, OD<600> is 2.0, and IPTG is 1 mmol/L, and the enzyme activities of the recombinant bacteria for glycodeoxycholic acid sodium (GDCA) and taurodeoxycholic acid sodium (TDCA) are 135.2 U/mL and 121.3 U/mL respectively. Therefore, a good foundation is laid for large-scale production of the bile salt hydrolase and using the bile salt hydrolase as functional food to decrease serum cholesterol.

Description

technical field [0001] The invention relates to a genetic engineering bacterium producing bile salt hydrolyzing enzyme and its construction method and application, belonging to the technical field of genetic engineering. Background technique [0002] Bile salt hydrolyzing enzyme can hydrolyze the bound bile salt in vertebrates into free bile salt and amino acids, thereby causing liver cells to de novo synthesize bound bile salt from cholesterol and reduce the serum cholesterol level in the body. Bile salt hydrolase has become one of the research hotspots at home and abroad because it can reduce the cholesterol level in serum and prevent cardiovascular diseases. [0003] There are mainly 6 kinds of bile acids in the human body, including glycochenodeoxycholic acid (26%), glycodeoxycholic acid (26%), glycodeoxycholic acid (22%), taurocholic acid (9%), Taurodeoxycholic acid (9%) and taurochenodeoxycholic acid (9%). Bile salt hydrolyzing enzymes from different sources have dif...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/80C12N15/55C12N15/70A23L1/29C12R1/19
Inventor 张娟陈坚周晓玲堵国成
Owner JIANGNAN UNIV
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