Expression And Characterization Of Acid Protease In Pichia Pastoris

Acidic protease is an aspartic protease that can hydrolyze proteins in acidic conditions(pH 1.0-5.0).It exists in a number of microbial sources and animal innards,and has wide applications in the fields of food,animal husbandry,pharmaceutical,tanning and fisheries.Industrial microbial acidic proteases are usually produced from solid state fermentation with Aspergillus strains,including A.niger,Monacus anka,A.flavescens,A.oryzae and so on,but the production has met some problems,such as the long fermentation period,great labor intensity,and difficult purification procedure.Pepsin from porcine gastric mucosa is the main industrial acidic proteases from animal innards,but the industrial extraction of porcine pepsin is limited by the low yield,high production-cost and the scale of live-pig slaughtering.In recent years,with the development of genetic engineering,more and more attentions have been paid to Pichia pastoris for the production of heterologous protein.P.pastoris has the benefits of low secretion of non-target protein,low cost of culture and mature fermentation process,and many heterologous proteins have been cloned and expressed in this expression system.However,there have been few reports and low enzyme activity on the expression of acidic protease from A.oryzae and porcine pepsin.In this study,an acid protease gene(pepA)from A.oryzae was obtained from fermented soy based on metagenome sequencing.The sequencing results showed that the gene pepA consisted of 1 215 bp,encoding 287 amino acids with a signal peptide from the initial 20 amino acids.Then the gene pepA was linked to pPIC9K to construct pPIC9K-pepA expression vector and transformed into P.pastoris GS115 for heterogenous expression.The shaking flash fermentation culture conditions of the recombinant strain yzl was also optimized.The results showed that under the optimal starting pH at 6,methanol concentration of 1%and fermentation time of 4 days,the activity of acid protease in the culture supernatant was the highest and reached up to 50.62 U/mL.The characteristic of recombinant PepA was further investigated.As shown by SDS-PAGE,its molecular mass was about 50 kDa,and almost no other proteins except PepA in the supernatant were observed.The optimum pH and temperature of PepA were pH 4.5 and 50?.Mn2+ and Cu2+ enhanced its activity,whereas Fe3+,Fe2+ and Ca2+ had inhibitory effects on its activity.In this study,meanwhile,a full porcine pepsinogen(pA)gene was synthesized,according to the published nucleotide sequence in GenBank(No.EF108301).The gene pA consisted of 1 227 bp,encoding 385 amino acids with a signal peptide from the initial 15 amino acids.To investigate the influence of ?-factor signal peptide on the heterogenous expression of pA,pPIC9K-pA expression vectors with and without the?-factor signal peptide were constructed,transformed into P.pastoris GS115 for heterogenous expression,and the recombinant strains were named as yz2 and yz3,respectively.The results showed that the enzymatic activity of PA was 20 and 113 U/mL in the culture supernatant of yz2 and yz3,respectively,indicating that the removal of?-factor signal peptide could improve the expression of PA.The shaking flash fermentation culture conditions of the recombinant strain yz3 were further optimized.The results showed that under the optimal starting pH at 6.5,methanol concentration of 0.5%and fermentation time of 9 days the activity enzymatic activity of PA in the culture supernatant was the highest and reached up to 252.63 U/mL,with a 1.3-fold increase than that before optimization.The characteristic of recombinant PA was also investigated.The optimum pH and temperature of PA were pH 2.5 and 50?,and this recombinant enzyme had a good stability at pH 2.0-4.0 and under 45?.Mn2+and Cu2+could enhance the enzymatic activity of PA,whereas Co2+and Ca2+ had weak inhibitory effects on its activity.The above findings on the expression,fermentation optimization and characterization of acidic proteases in P.pastoris,might lay a good foundation for industrial application of acid protease from microbial sources and animal innards.