Expression And Characterization Of The Bacillus Subtilis Aminopeptidase In Pichia Pastoris

Aminopeptidase is a kind of exonuclease which act on the N-amino-terminal ends of peptides and proteins.Aminopeptidases have significant applications,such as synthesizing pharmaceutical compounds,enhancing the flavor and nutritional value of foods,analyzing protein sequences,and acting as curing agents.Bacillus is one of the major sources of aminopeptidases and aminopeptidases from different species may vary considerably.Some studies had been showed on the expression of aminopeptidase from Bacillus,but the characterizations were quite different in aminopeptidases from various Bacillus isolate.In this study,we cloned and expressed the ywaD gene from this new Bacillus isolate in strain GS115,and characterized its enzymatic properties as well.The main content is described as follows:1.Cloning of the target gene and construction of recombinant vector pHBM905A-ywaDTwo primers:ywaD-F and ywaD-R,were designed according to the DNA sequence of the ywaD gene,without its native signal peptide,from B.subtilis subsp.subtilis str.BSP1 chromosomal DNA.The ywaD gene was amplified by PCR and cloned into vector pHBM905A.Then,the recombinant plasmid was identified by colony PCR and verified by DNA sequencing,and named as pHBM905A-ywaD.2.Transform of P.Pastoris and the screening of P.pastoris recombinantspHBM905A-ywaD was linearized with the restriction enzyme SalI and transformed into P.pastoris GS115 using polyethylene glycol(PEG)-1000.After a preliminary screening by yeast colony PCR,the supernatant of the cell culture in shake flask was obtained and SDS-PAGE was performed to detect whether the aminopeptidase YwaD was expressed.The recombinant Pichia pastoris was named as GS115-pHBM905A-ywaD.3.Expression and optimized induction conditions of YwaDThe P.pastoris GS115 strain harboring plasmid pHBM905A-ywaD was cultivated in buffered glycerol-complex medium(BMGY)at 28?(with shaking at 200 rpm)for approximately 48 h,and then the cells were transferred into buffered methanol-complex medium(BMMY).One percent(v/v)methanol was added every 24 h throughout the induction phase.After 288 h,the supernatant of the aminopeptidase YwaD was obtained.After a 288-h induction,the highest protein yield reached 734 mg/L.To explore the medium pH on recombinant YwaD expression,the recombinant P.pastoris GS115 cells were induced in BMMY at different pH(3.0,4.0,5.0,6.0,with 0.1 M phosphate buffered solution and without any phosphate buffered solution).The recombinant YwaD was stable in BMMY at pH 4.0,5.0 and with 0.1 M phosphate buffered solution,and achieved high protein yield 908 mg/L in BMMY at pH 5.0.To explore the medium cell density on recombinant YwaD expression,the recombinant P.pastoris GS115 cells at the same cell density were transformed into BMMY media with different volume(25 mL,50 mL,100 mL)and induced with 1%(v/v)methanol.The enzyme was stable in BMMY with three different volumes.However,the enzyme achieved the highest yield(1227 mg/L)in 25 mL of BMMY.4.Purification and glycoprotein staining of recombinant YwaDThe enzyme was purified by three sequential steps(ultrafiltration,Ni2+-affinity and gel filtration).The whole purification procedure resulted in a 5.19-fold purification and 59.82%recovery.It was worth mentioning that Ni2+-affinity chromatography was a very effective method for YwaD.As the recombined YwaD is a strong binder for immobilized metal ions and just a few protein was lost during ultrafiltration to exchange the buffer from BMMY to buffer A,the Ni2+-affinity chromatography resulted in a 3.61-fold purification and 79.61%recovery.The molecular size of the YwaD was approximately 55 kDa,which was higher than the calculated value of 47 kDa.Three potential N-glycosylation site and two potential O-glycosylation sites have been predicted.It could be inferred through glycosylation analysis that YwaD was glycosylated.5.Characteristics of recombinant YwaDYwaD obtained the maximal activity at pH 8.5,40?.The enzyme was relatively stable when incubated under the temperature 50? and was relatively active in pH 6.5-9.0.The aminopeptidase was completely lost its activity by EDTA,which indicate that YwaD is a metallo-aminopeptidase.Co2+ strongly increased the activity of YwaD.However,Cu2+(1 mM)and Ni2+(1 mM)intensively inhibited YwaD activity.Inhibition of YwaD activity was observed in the presence of dithiothreitol,and ?-mercaptoethanol,while YwaD was completely insensitive to PMSF.It was worth mentioning that YwaD has strong resistance to carbamide,which retained about 80%of its activity after treatment with up to 10 M carbamide.At last,we found that YwaD was most active toward Arg-pNA.