Secretory Expression And Modification Of Substrate-binding Properties Of Bile Salt Hydrolase From Lactobacillus Plantarum

Bile salt hydrolase （EC3.5.1.24, BSH） catalyzes the hydrolysis of conjugated bilesalts into free bile salts and glycine/turine. This will increase the de novo synthesis ofconjugated bile salts from cholesterol, thus reducing the serum cholesterol level in vivo.Therefore, oral administration of BSH-producing cells，pure enzyme and related productsfor curing hypercholesterolemia has good prospect for applications. Furthermore, BSHbecomes one of the focused researches at home and abroad now. However, there are stillsome controversies about the mechanism of hypocholesterolemic effect by BSH, thesubstrate-binding mechanism and safety of BSH. In this study, a lactic acid bacteriumwith high BSH activity and good cholesterol-lowering effect in vitro was screened.Besides, its bsh gene was extracellularly expressed by twin-arginine signal peptides in E.coli, and the recombinant BSH was then characterized and modified to change thesubstrate specificity. Finally, we achieved the food-grade expression of BSH. The mainresults were as follows:1) A lactic acid bacterium, with high BSH activity and excellent hypocholesterolemic effectin vitro, was screened by qualitative and quantitative methods. This strain was furtheridentified and named as Lactobacillus plantarum BBE7. The bsh gene of this bacteriumwas then cloned and expressed inside the cells of E. coli. As analyzed, BSH, a proteinencoded by324amino acids, showed high similarities to other BSHs and belonged toN-terminal nucleophilic （Ntn） hydrolase family. According to the results of BSH assay, theintracellular BSH activity of recombinant bacterium was1.85U mg-1, which was13timeshigher than that of wide-type strain.2) BSH was successfully secreted outside the cells of E. coli by twin-arginine signalpeptides. As analyzed by SDS-PAGE and BSH assay, these three signal peptides oftwin-arginine translocation pathway allowed both the intracellular and extracellularexpression of BSH in recombinant bacteria compared to signal peptide PelB from Secpathway, although most of the BSH expressed was inclusion bodies inside the cells andonly a few was liberated into the medium. Because signal peptide DMSA had higherGRAVY score, stability and aliphatic index than other two signal peptides, its secretionefficiency was the highest. In addition, the effects of the four hosts on the expression ofBSH differed, which could be attributed to the fact that the utilization of twin-argininesignal peptides varies greatly among different strains of the same species. Thus the bestcombination of signal peptide and host that allows effective secretion of BSH could befound. The extracellular BSH activity of recombinant bacterium was optimized byorthogonal experimental design and increased by68.1%.3) BSH was purified from the supernatant of recombinant bacterium by ammonium sulfate precipitation, dialysis, Ni²⁺colomn and gel colomn. The purification fold and yiled were39.34and12.55%, respectively. N terminal sequencing demonstrated that the extracellularBSH was secreted by Tat pathway but not caused by cell leakage. The optimal pH andtemperature of the purified BSH were5.6-6.4and37°C, respectively. Purified BSH wasstable in pH7.0-8.0, with a more than96%residual activity. After this enzyme wasincubated at40°C for60min, it remained about83.5%residual activity; but a nearlycomplete loss of activity was observed after incubation for10min at60°C. The acitivty ofBSH was increased by45.1%and86.8%by10mM EDTA and DTT, respectively. Butagents that oxidize thiol (Cu²⁺and sodium periodate) strongly inhibited the activity ofBSH. PMSF and other metal ions inhibited BSH activity at different levels. Besides, BSHwas more efficient at hydrolyzing glyco-conjugated bile salts than tauro-conjugated bilesalts. The Vmax, Km, kcatand kcat/Kmof the puribvfied BSH were142.8μmol （min mg）-1,2.07mM,88.127s-1and42.57L （s mmol）-1, respectively.4) As indicated by amino acid sequence alignment, when the amino acid at position65ofBSH was a nonpolar residue, BSH showed preference for tauro-conjugated bile salts, butwhen it was a polar residue, BSH displayed a preferential hydrolysis of glyco-conjugatedbile salts. Homology modeling and superimposition showed that Tyr65located near theisovaleric side chain of deoxycholic acid （DCA） which was near the hydroxyl substituents,and more hydrophilic amino acids added here can alter the affinities toward thecorresponding substrates. The importance of the polarity of residue at position65washighlighted by site-directed mutagenesis and when it was C or N, BSH lost activity; butwhen it was A, F or I, BSH was an active enzyme. Tauro-conjugated bile salts hydrolysiswas all increased by mutants Y65A, Y65F and Y65I （up to4fold）. Besides, amino acidmoieties and hydroxyl substituents at C7and C12of steroid moiety had effects on thehydrolysis of corresponding substrates by BSH.5) BSH was successfully expressed both intracellularly and extracellularly in L. lactisNZ3900, using food-grade selection marker lacF of vector pNZ8149and food-gradesignal peptide Usp45of plasmid pNZ8112. The bsh gene was optimized according to thecodon bias of L. lactis, which increased the intracellular and extracellular BSH activity ofrecombinant bacteria by12fold and9.5%, respectively. Propeptide LEISSTCDA with twonegative charges was used to optimize the charge balance between the c-region of signalpeptide and mature moiety, thus the extracellular activity of BSH encoded by bsh2genewas increased by8.8%. And the extracellular BSH activity was increased by11.3%by thecombination of codon optimization and the acidic propeptide LEISSTCDA. Besides, thetwo negatively charged residues were proved to be necessary for the secretion-enhancingeffect of LEISSTCDA.