Expression And Directed Evolution Of Neutral Protease I From Aspergillus Oryzae In Pichia Pastoris

Neutral protease from Aspergillus oryzae is one of the enzymes produced inmetabolism of Aspergillus oryzae, and is mainly used in soy sauce brewing industry,which can degrade the protein in raw materials into sorts of amino acids. As aeukaryote, Pichia pastoris is a very useful industrial protein expression system whichhas many of the advantages of higher eukaryotic expression systems such as proteinprocessing, protein folding, and posttranslational modification. Glycosylation is oneof the most common modifications occurring in the synthesis of proteins in Pichiapastoris. Directed evolution has been proven to be an effective method for proteinsengineering. As for NPI, it is an efficient way to construct a mutation library andscreen the ideal mutated variants which have improved properties. The researchresults are mainly described as follows:1Expression, purification and enzymatic characteristics of neutral protease IPlasmid pPIC9K-NPI-6His was constructed and eletrotransformated into P.pastoris to express. After72hours’ expression inducted by methanol in BMMY, thesupernatant was collected and the neutral protease’s activity reached46U/mL. Thecrude enzyme was treated with solid (NH4)2SO4at70%saturation and purified bynickel-affinity chromatography column, and the molecular weight of rNPI was about55kDa. The purified rNPI’s optimum pH was7.5and it had the best pH stability atpH7. The optimum temperature of rNPI was55oC and there’s little influence to theenzyme’s activity under50oC. Recombinant NPI was inhibited by Cu2+, Zn2+,TritonX-100, and totally inactive after addition of EDTA and Phen.2Glycosylation analysis of recombinant neutral proteaseThe recombinant neutral protease (rNPI) was confirmed to be N-glycosylated byPAS staining and Endo H digestion. Moreover, the deglycosylated protein’smolecular weight decreased to43.3kDa from54.5kDa analyzed by SDS-PAGE andMALDI-TOF-MS, and the hyperglycosylation extent was20.5%. TheN-glycosylation site of rNPI was analyzed by nano LC-MS/MS after digesting bytrypsin and Glu-C, and the unique potential site Asn41of mature peptide was found tobe glycosylated. Homology modeling of the3D structure of rNPI indicated that there was a great distance away from the active site of the enzyme to the glycosylation site.Mutation of N41A was carried out to eliminate glycosylation in order to determine itseffects on enzyme’s activity. After mutation, the mass of expressed protein wasdecreased to43kDa analyzed by SDS-PAGE, and it had little neutral protease’sactivity compared to the wild-type protein, which demonstrated glycosylation wascritical important in the expression of recombinant NPI.3Improve thermostability of NPI by directed evolutionA random mutated library with2×104variants was constructed by error-pronePCR, and50%of the variants still had neutral protease’s activity. A thermostablemutated strain whose93rdamino acid was mutated to Ala from Ser was screened out.The S93A mutant’s optimum temperature increased from55oC to60oC, and its half–life of inactivation at56oC increased from7.9min to16.7min, which was2.1-foldof that of the widl-type.3D structures of wild-type and mutated NPI were analyzed.