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Bile salt hydrolase (BSH) mutant and use thereof

A technology of bile salt hydrolyzing enzyme and mutants, which is applied in the field of genetic engineering to achieve the effect of improving the hydrolysis ability

Active Publication Date: 2014-08-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, there is still some controversy about the recognition site of BSH to the substrate, and bile salt hydrolase may recognize both the core part of the steroid and the amino acid group

Method used

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  • Bile salt hydrolase (BSH) mutant and use thereof
  • Bile salt hydrolase (BSH) mutant and use thereof
  • Bile salt hydrolase (BSH) mutant and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Construction method of pET-20b(+)-dmsA-bsh

[0055] The specific operation is as follows:

[0056] Plasmid pUC57-Tat (Tat-dependent protein targeting in prokaryotes and chloroplasts) was used as a template to amplify the dmsA gene. The PCR conditions were: pre-denaturation at 95°C for 10 minutes; 30 cycles at 98°C for 10s, 55°C for 30s, and 72°C for 20s; and extension at 72°C for 10 minutes. At the same time, using the genomic DNA of L. plantarumBBE7 as a template, PCR amplified (with an extension time of 1 min) the bsh gene without a stop codon, the two fragments were gel-recovered, and the two fragments were used as templates in equimolar amounts to obtain Fusion fragment dmsA-bsh.

[0057] 2) Carry out double digestion with NdeI and XhoI, and connect with the correspondingly digested plasmid pET-20b(+), to obtain the recombinant plasmid pET-20b(+)-dmsA-bsh. The recombinant plasmid was transformed into E.coliJM109 competent cells, and positive recombinant...

Embodiment 2

[0058] Example 2: Alignment Analysis of Amino Acid Sequences

[0059] The amino acid sequences of BSH with known substrate specificities were aligned and analyzed using the online tool ClustalW2 (http: / / www.ebi.ac.uk / Tools / msa / clustalw2 / ). The bile salt hydrolase genes for comparative analysis include the following 23 species, the specific amino acid sequences are shown in figure 1 :

[0060] LSB-30514_bsh1, L. salivariusB-30514BSH1 (AFP87505.1);

[0061] LSUCC118_bsh1, L. salivariusUCC118BSH1 (ACL98201.1); LSLGM14476_bsh1, L.

[0062] salivariusLGM14476BSH1(ACL98197.1);LSLGM14476_bsh2,L.salivariusLGM14476(ACL98205.1);

[0063] LSJCM1046_bsh1.L.salivariusJCM1046BSH1(ACL98194.1);

[0064] LPST-III-bsh1, L.plantarum subsp.plantarumST-IIIBSH1 (ADO00098.1); LPBBE7_bsh, L.

[0065] plantarum BBE7BSH;

[0066] LPWCFS1_bsh1,L.plantarumWCFS1(CCC80500.1);

[0067] LP80_bsh,L.plantarum80(AAB24746.1);

[0068] LRCRL1098_bsh,L.reuteriCRL1098(ACH81023.1);

[0069] LJ100_chshalpha,...

Embodiment 3

[0084] Example 3: Homologous structure modeling and superposition

[0085] The homology simulation of the 3-D structure of L. plantarumBBE7BSH was performed using the online software SwissModel, and the template used for homology modeling was the mutant C2a (PDBID: 2rf8B) of C. perfringens13CBAH. The homology of the two sequences is 37.69%. The estimated absolute model quality (QMEANZ-Score) and the root mean square deviation (Root-Mean-SquareDeviation, RMSD) of the α-carbon atoms of the BSH model after simulation were -3.61 and Superimpose this model with the complex structure of CBAH and substrate (PDBcode: 2BJF, 2BJG) (RMSD is ), obtained the structure of the complex of L.plantarumBBE7BSH and deoxycholic acid (DCA), taurine ( figure 2 a). figure 2 Superposition of L.plantarumBBE7BSH and C.perfringens13CBAH structures in a, dark gray is BSH, light gray is CBAH; figure 2 b depicts the conserved amino acids (Cys2, Arg16, Asp19, Asn79, Asn170, and Arg223) in L. plantar...

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Abstract

The invention discloses a bile salt hydrolase (BSH) mutant and a use thereof, and belongs to the technical field of gene engineering. Through comparison analysis of a BSH amino acid sequence and homology modeling and overlay analysis of a 3-D structure, a BSH substrate specific amino acid mutation site is determined, through a site-specific mutagenesis technology, the substrate specific amino acid mutates into a nonpolar amino acid, and the mutant is transferred into a host bacterium so that a mutant strain is obtained. The BSH produced by the mutant is active enzyme. Compared with a wild-type bacterium, the mutant has a reduced glycine-combined bile salt hydrolysis capability and an improved taurine-combined bile salt hydrolysis capability. The BSH mutant lays a foundation for improving a taurine-combined bile salt hydrolysis capability of BSH and uncovering a substrate combination mechanism of BSH.

Description

technical field [0001] The invention relates to a bile salt hydrolyzing enzyme mutant and its application, belonging to the technical field of genetic engineering. Background technique [0002] Through the study of substrate specificity, it was found that bile salt hydrolase has strong substrate selectivity, and its ability to hydrolyze glyco-conjugated bile salts is much higher than that of tauro-conjugated bile salts. ), which is consistent with the reported substrate specificity of most BSHs. However, the ratio of glycine-bound bile salts to taurine-bound bile salts in normal humans is 3:1, which will cause taurine-bound bile salts to not be hydrolyzed well by BSH. [0003] The substrate of bile salt hydrolase is composed of a steroid nucleus and an amino acid group (glycine / taurine). Huijghebaert and Hofmann found that modifying the amide bond of the substrate or reducing the negative charge of the terminal group of the amino acid part of the substrate can significantl...

Claims

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Application Information

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IPC IPC(8): C12N9/14C12R1/25
CPCC12N9/80C12Y305/01024
Inventor 陈坚张娟董自星周晓玲堵国成李华钟
Owner JIANGNAN UNIV
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