Correlation Between Signal Peptide Structure And Protease Secretion

The suitable signal peptide and its optimized structure is essential for high efficiency secretory expression of a specific enzyme molecule.In this thesis protein-glutamine amidohydrolase(PGase,EC 3.5.1.44)was selected as a model enzyme protein to screen the suitable Tat signal peptides from Bacillus licheniformis genome and then to optimize the signal peptide structure by cloning and site-directed mutagenesis for mediating its secretory expression.The following beneficial results were obtained:(1)The genes coding pro-PGase and PGase were cloned and heterologously expressed in Escherichia coli and verified by SDS-PAGE analysis,Recombinant E.coli expressed 35 kDa and 25 kDa bands,respectively,with the same size predicted.However,No PGase activities were detectable in E.coli.(2)Three TAT signal peptides,namely,AG,CG,and HG,derived from B.licheniformis genome were selected and the secretory expression vectors containing the signal peptide sequences were constructed.The gene encoding pro-PGase was cloned into the secretory expression vectors and transformed into Bacillus subtilis WB600,yielded the recombinant strains WB600(pHY-PAG),WB600(pHY-PCG),and WB600(pHY-PHG).The PGase activity expressed by WB600(pHY-PAG)was obviously expressed by the shake flask fermentation based on the PGase activity assay.(3)For N-terminal positive charge number in the structure of the Bacillus sp.signal peptide highly effects on the processing efficiency of the signal peptide and consequently on the secretion efficiency,the N-terminal site-directed mutagenesis of the AG sequence was made.Mutant PAG ? could mediated the highest secretory expression of PGase in WB600(pHY-PAG ?)with the enzyme activity of 0.23 U/mL.(4)The PGase fermentation condition by WB600(pHY-PAG ?)were further optimize.The optimized condition was inoculum age of 8 h,inoculation capacity of 5%,working volume of 75 mL in 250 mL flask,shaking speed of 200 r/min.After 24 h fermentation in media with lactose as carbon source and peptone and NH4NO3 as nitrogen source,the highest yield of PGase reached 0.55 U/mL.(5)The recombinant PGase was examined and its potential application was valuated.It performed the highest activity at pH 7.0 and 60? and kept stability amonst pH 6-8 and at 20?-40?.Ca2+ion promoted PGase obviously while Fe3+,Cu2+,Ag+,SDS had obvious inhibitory effect on PGase.The PGase was able to deamidate high-molecular-weight proteins(caseins)and soybean protein isolates.