Study On Production, Gene Cloning And Expression Of An Acid Protease From Marine Yeast Metschnikowia Reukaufii W6b

After screening of more than 400 yeast strains from different sources in marine environments,it was found that strain W6b could secrete an acid protease on the plates with 2%skim milk.After protease activity of the yeast cells suspension was estimated,it was found that the acid protease secreted by W6b was cell-bound enzyme.The acid protease was named SAP6.The strain W6b was identified to be Metschnikowia reukaufii according to the results of routine yeast identification and molecular methods.The optimal conditions for acid protease production by strain W6b were examined. The optimal production medium was made with sea water at pH 5.0 containing 0.5% glucose,1.5%casein,0.5%yeast extraction.The inoculation size was 7.5×10⁷ cells per 50 mL of the production medium.The cultivation temperature was 28℃and the agitation speed was 140rpm.The optimal conditions for action of the acid protease were pH3.4 and temperature 40℃.The full-length cDNA of the acid protease(SAP6) from strain W6b was cloned using SMART RACE PCR.It was 1755 bp long and contained an open reading frame of 1527 bp encoding 508 amino acids.The protein sequence deduced from the cDNASAP6 gene exhibited 18.11%,20.52%and 28.6%identity with that of acid proteases from Aspergillus oryzae,Saccharomyces cerevisiae and Candida parapsilosis.The deduced amino acid sequence included a signal peptide of 16 amino acids.The consensus motif contained a VLLDTGSSDLRM active site and a ALLDSGTTITQF active site which were highly conserved in the eukaryotic and viral aspartyl proteases family(EC 3.4.23).The Kyte and Doolittle hydropathy profiles of SAP6 and Cwp1 from Saccharomyces cerevisiae were both characteristic of glycosylphosphatidylinositol cell-wall proteins GPI-CWPs. The cDNA of SAP6 without 48 bp signal peptides sequences was subcloned into an expression plasmid pET-24a(+) and fused with a 6-His Tag and transformed into E. coli BL21(DE3) for recombinant expression of the protease.Following induction by IPTG,active enzyme was found within the E.coli cells.The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 54 kDa was found.The crude acid protease of the culture of strain W6b and the crude recombinant acid protease had milk clotting activity.All these evidences showed that SAP6 gene cloned in this study indeed encoded the acid protease.The present work is the first report of the cloning and expression of cDNA encoding an aspartic protease from Metschnikowia spp.The recombinant acid protease rSAP6 was purified with affinity chromatography using a Ni Sepharose^TM 6 Fast Flow column.SDS-PAGE analysis and Western Blotting reveal that the purified rSAP6 was predicted to be 54 kDa.The optimal temperature and pH for the purified rSAP6 were 40℃and 3.4,respectively.The enzyme was stable below 40℃and between pH 2.6 and 5.0,respectively.Mn²⁺ had a weak activating effect on the enzyme while Cu²⁺ acted as a strong inhibitors of the enzyme.The purified rSAP6 was characterized as an aspartic protease as it was inhibited by pepstatin,an aspartic protease-specific inhibitors.