Expression And Characterization Of Acid Protease From Aspergillus Usamil In Pichia Pastoris

Supplementation of animal feed with acid protease has proven to be an effective strategy to enhance amino acids and peptide absorption in gastric animals. Currently, all industrial acid protease are produced by Aspergillus strains, but the yields were too low. The aim of the present research was to obtain high-level expressions of acid protease in the methylotrophic yeast Pichia pastoris.The main results of the study were as follows:1. The synthesis of acid protease (PepA) gene from Aspergillus usamilThe acid protease (PepA) gene of Aspergillus usamil is1182bp and encodes a protein of394amino acids, including a signal peptide of20amino acids at the N-terminal, a propeptide of50amino acids and mature enzyme of324amino acids. The coding region, apart from the signal peptide, was synthesized according to the codon usage bias of P. pastoris, without changing the amino acid sequence.2. Construction of the recombinant vector and expression in P. pastoris GS115The synthesized pepA was cloned into the pHBM905A vector and successfully expressed in the yeast Pichia pastoris.The recombinant strain was named as PEPASA.However, the yield of PepA in strain PEP ASA were modest (0.578mg/ml). Therefore, we have used two approaches to increase the productivity of PepA expression in Pichia pastoris.One is that we have constructed a new expression vector pHBM905BDM containing mutant AOX1promoter and the synthetic signal peptide (designated MF4I).The yield of PepA with the pHBM905BDM vector is only1%higher than that of PepA with the pHBM905A vector.Another is that a panel of Pichia clones carrying increasing copies of the pepA expression cassette was created using an in vitro multimerization approach. P. pastoris clones carrying one, two, three, and four copies of the PepA gene were generated were named as PEPAS1, PEPAS2, PEPAS3, PEPAS4, respectively.The gradual increase of the transgene dosage from one to three copies under the control of mutant AOX promoter and MF4I signal had an additive effect on PepA yield, but PEPAS4with four copies brought about a decrease in expression levels. The highest PepA yield produced by strains PEPAS1, PEPAS2, PEPAS3, PEPAS4was0.593mg/ml,0.669mg/ml,1.058mg/ml,0.975mg/ml, respectively.A similar trend was observed in mRNA levels by qRT-PCR. Therefore, these results indicate that increasing gene copy numbers can increase transcription and the yield, but not necessarily with a linear relationship.High cell density fermentation of PEPAS3with three copies in30-L bioreactor led to a production level of4.3mg/ml after72h of induction. The strategy described in this work can also be adapted to express other important industrial enzymes.3. Purification of Recombinant PepAThe PepA was purified by the step of ammonium sulfate precipitation. After the purification, the PepA was purified1.3-fold with total yield and a specific activity of4127.3U/mg.The purified PepA was analyzed by SDS-PAGE.Native PepA with molecular mass of44kDa was glycosylated in P. pastoris to have a molecular mass of76kDa.One potential N-glycosylation site and three potential O-glycosylation sites have been predicted.4. Enzymatic properties of the purified PepAThe optimum assay temperature and pH for the PepA were found to be45℃and2.5, respectively.The activity of the PepA was not affected in the presence of phenylmethylsulfonyl fluoride, which suggests that the enzyme is an aspartic protease. It was activated by Ca2+,Zn2+,Fe2+,NH4+.but was inhibited by Cu2+,Co2+,Mg2+,Fe3+,Sodium Dodecyl Sulfate.