Expression Of Aspergillus Pseudoglaucus Acidic Protease Aspergillopepsin ? For Protein Hydrolysis

Acidic protease is a kind of enzyme with hydrolysis activity in acidic environment.Since there are two aspartic acid residues in its active centre,it was also named aspartic protease.Acidic proteases efficiently hydrolyze the protein under acid conditions.They are widely used in food,feed,fur and leather processing and pharmaceutical industry and they have a good industrial application prospect.In this study,the genes of mature peptide?App?and mature peptide with propeptide?proApp?of Aspergillopepsin?were cloned using PCR amplification technique,the recombinant plasmid was constructed and the recombinant proteins were expressed in Escherichia coli BL21.The results showed that propeptide had an important influence on the expression of acidic protease.The effects of the different propeptides and the different vectors on the expression and function of acidic protease and the enzymatic properties were determined.The conditions of hydrolysis of soy protein and milk protein were optimized.This work provides the basis of microbial acid protease for the theoretical research and industrial application.The main results are as follows:?1?The genes of App and proApp were amplified from synthetic Aspergillopepsin?gene from A.pseudoglaucus using PCR method,the four different propeptide genes?CP,PP,RP,SP?were obtained from the genes of Candidapepsin from Candida tropicalis,proteinase A from Saccharomyces cerevisiae,proteinase from Rhizomucor miehei and aspartic proteinase 2 from Candida albicans.The propeptides were cloned at 5'-terminal of App gene by overlap extension PCR,respectively.Eight recombinant strains:E.coli BL21/pET-App,E.coli BL21/pET-proApp,E.coli BL21/pGEX-App,E.coli BL21/pGEX-proApp,E.coli BL21/pET-CP-App,E.coli BL21/pET-PP-App,E.coli BL21/pET-RP-App and E.coli BL21/pET-SP-App were constructed.By the optimization of the induction temperature,the recombinant proteases were efficiently expressed as soluble form at 20°C,except that E.coli/pET-App was not expressed in E.coli.?2?After the proteases were purified by HisTrap HP affinity and GSTrap HP affinity chromatography,SDS-PAGE analysis showed that the purified protease with a single band were obtained,with a molecular weight of App and proApp,CP-App,PP-App,RP-App,SP-App were about 50 kDa and 55 kDa,which were insistant with the therotical molecular weight.The results of enzyme assay showed that only proApp has enzymatic activities towards the substrate casein.Circular dichroism spectra analysis showed that App and proApp exhibited the similar secondary structures,while CP-App,PP-App,RP-App and SP-App have obvious difference with App and proApp in their secondary structures.These results indicated that propeptide has an important effect on the protein folding and function of acidic protease.The thermal denaturation experiment showed that the denaturation temperature?T_m?of proApp was4.1°C higher than App.?3?The enzymatic properties of proApp from E.coli BL21/pET-proApp and E.coli BL21/pGEX-proApp were determined.Their optimal temperatures were 55°C,while the optimal pH of proApp from E.coli BL21/pET-proApp was 2.8 and proApp from E.coli BL21/pGEX-proApp was 3.0.Under the optimal conditions,the proApp from E.coli BL21/pET-proApp showed the higher activity than that of E.coli BL21/pGEX-proApp.The relative enzyme activities of proApp from E.coli BL21/pET-proApp and E.coli BL21/pGEX-proApp were 87%and 72%at 35°C,respectively.When the temperature was over 45°C,the relative enzyme activities of both proApp decreased to blow 40%.The result of pH stability showed that the relative enzyme activity of proApp from E.coli BL21/pET-proApp was above 85%in the range of pH 2.5–7.0,whereas the relative enzyme activity of proApp from E.coli BL21/pGEX-proApp was below 80%at pH>6.0.?4?The optimal temperature and pH for the hydrolytic activities of proApp towards soy protein and milk protein were determined.The results showed that the maximum hydrolytic activity of proApp towards soy protein was 104 U·mg^-1 at 55°C and pH 3.0,and the maximum hydrolytic activity of proApp towards milk protein was 252 U·mg^-1 at pH 55°C and 2.2,SDS-PAGE analysis showed that the soy protein and milk protein were effectively hydrolyzed into small peptides.The MALDI-TOF/MS results showed the small peptides of hydrolysates with a molecular weight lower than 2 kDa.Most of the proApp cleavage sites were upstream or downstream of hydrophobic amino acid.