Expression Of Pseudomonas Aeruginosa Elastase Gene In Pichia Pastoris And Effects Of Mutagenesis Of N-glycosylation Sites On The Recombinant Enzymes

Elastase may be a promising biocatalysis in several aspects, especially in processing ofsubsidiary agricultural products and poultry feathers and peptide synthesis in organic solvents.A bacterium strain C11tolerant to organic solvents was identified as Pseudomonasaeruginosa based on morphological and biochemical characterization and analysis of16SrRNA sequences. The33-kDa protease secreted by the strain showing high stability andactivity in organic solvents was identified as P. aeruginosa elastase (PAE) by peptide massfingerprint analysis. The characterization of PAE was also studied in the paper.The lasB gene from P. aeruginosa C11(the portion of coding the propeptide and matureprotein) shared highest similarity to that of P. aeruginosa PST-01(Accession No. AB029328),showing a slight diversity in one nucleobase, leading to the alteration of the54th amino acidresidue in the propeptide (the residue at this site of the propeptide of elastase from P.aeruginosa C11is aspartic acid, while that from P. aeruginosa PST-01is alanine). Thesequence of the lasB gene was inserted into pPIC9K and then successfully expressed inPichia pastoris. The recombinant elastase (rPAE), containing three types of proteins withvaried molecular weights (33、35and37kDa), exhibited a expression level of0.13g·L-1. RPAEshowed greater stabilities than PAE in aqueous or organic solvents, while its catalyticactivities of hydrolyzing casein were highly similar with those of the native enzyme.The characterization of organic solvents exhibited great effects on the rates and yields ofpeptide synthesis catalyzed by rPAE and PAE; DMSO was the best solvent for peptidesynthesis, wherein the rates and yields of synthesis reaction were highest. When the reactioncondition was in50%(V/V) DMSO, at pH8.0and under temperature from20to30℃, theyield of the peptide synthesis catalyzed by rPAE or PAE attained its maximum. Theconcentrations of rPAE or PAE above0.06mg·mL-1gave rise to the best results in the aspectof synthesis yield. Equilibrium of peptide synthesis catalyzed by rPAE or PAE appeared after48-hour reaction.The three potential N-glycosylation sites (N43, N212, and N280) of rPAE were mutatedusing site-directed mutagenesis; the asparagine (Asn) residues were converted to glutamine(Gln). The deglycosylation analysis revealed the glycosylation level of rPAE: the33,35and37-kDa recombinant proteins were non, mono, and bi-glycosylated, respectively.N-glycosylation improved rPAE stability both in aqueous medium and in organic solvents.The results indicated that the influence of N-glycosylation on its activity was insignificant.The non-and glycosylated isoforms of rPAE displayed similar kinetic parameters forhydrolyzing casein in aqueous medium, and when catalyzing bipeptide synthesis in organicsolvents, they exhibited identical substrate specificity and activity, and produced similaryields. Any potential N-glycosylation site of rPAE was involved in its high-level expression inP. pastoris; N-glycosylation at N212or N280was more critical. Additional N-glycosylation sites were introduced into rPAE by site-directed mutagenesis(I38T, A69T and N266T); as a result, the glycosylation levels of I38T and N266T mutantproteins were considerably enhanced comparative to the wild-type rPAE. I38T mutationdecrease rPAE thermostability, while N266T mutation affected that slightly. Neither I38T norN266T mutation exerted significant effects on the rPAE solvent stability. I38T rather thanN266T mutation increased the caseinolytic activity of rPAE. Accordingly, the peptidesynthesis catalyzed by I38T mutant rPAE gave higher rates and yields than the wild-typerPAE, while its substrate specificity did not alter. On the other hand, N266T mutant exertednon-significant effects on the rPAE-catalyzing synthesis reactions. The mutagenesis at thesethree sites inhibited rPAE expression in P. pastoris to varied degrees; the expression level ofI38T or N266T mutant were72.5or74.8%of the wild-type rPAE, respectively, while A69Tmutant was not detected in broth of the transformant P. pastoris.