The Expression And Characterization Of Carboxypeptidase Y From Saccharomyces Cerevisiae In Pichia Pastoris GS115

Carboxypeptidase Y (CPY; EC 3.4.16.5) is one of vacuolar proteases derived from Saccharomyces cerevisiae, first isolated by Doi et al. Carboxypeptidase Y is encoded by PRC1 gene, expressed as a precursor. The amino acid sequence of carboxypeptidase Y precursor (preproCPY) contains a 20-residue signal peptide, a 91-residue propeptide and a 421-residue mature region. This protein precursor is synthesized in endoplasmic reticulum, then is transferred into Golgi apparatus. There are four potential N-glycosylation sites in this protein precursor. Mature CPY can hydrolyze the C-terminal amino acids of small peptides with broad substrate specificity and also release the proline residue which is difficult to hydrolyze by most carboxypeptidases.Based on the potential hydrolysis of small peptides, this characterization of carboxypeptidase was worth to investigate. We attempted to apply CPY to the hydrolysis of small peptides derived from the feather hydrolysate. The procedure of this study was described as follows.1. The construction of recombinant plasmid pHBM905A-proCPYThe gene of proCPY(proCPY) was cloned from S. cerevisiae INVScl by PCR with two primers CPY-F and CPY-R, and was cloned into the P. pastoris expression vector pHBM905A. The recombinant plasmid was sequenced and named as pHBM905A-proCPY.2. The screening and culture of P. pastoris recombinantsThe recombinant plasmid pHBM905A-proCPY was transformed into P. pastoris GS115. Recombinants were screened and cultured with 1%(v/v) methanol induction.3. Expression and deglycosylation analysis of recombinant proCPYThe main band (68 kDa) was larger than the calculated one (58 kDa) through SDS-PAGE detection. Western blot analysis showed the proCPY was expressed efficiently. the expression level of the proCPY reached 605 mg/L after induction for 168 h. Deglycosylation analysis with Endo-H implied the N-glycosylations happened to the synthesis of recombinant proCPY.4. Purification and Enzyme activity measurement of CPY with different activation waysThrough treatment with protease K, the proCPY was activated, and the properties of CPY was consistent with reported results. However, our study also demonstrated that proCPY could be transformed into a small form(CPY) in the culture supernatant after treatment at 10℃ for two weeks. By a series of purification steps, the peptidase activity of CPY reached 305 U/mg, compared with initial 39 U/mg of crude enzyme with 7.9 fold purification. In addition, the esterase activity of CPY was also detected by TLC (Thin-Layer Chromatography) with hydrolyzing the ester.5. The characterization of CPYThe optimal reaction temperature and pH of recombinant CPY were 30℃ and pH 6.0, respectively. This recombinant enzyme was stable below 40℃, as well as at pH4.5 to pH7.0.6. Hydrolysis of Feather peptide by CPYAbout 1 L peptide sample (100 mg/ml) was performed to investigate that recombinant CPY hydrolyze the peptides into free amino acids. Through the analysis of amino acid analyzer S433D (SYKAM, Germany), several kinds of amino acids Serine, Leucine and Glutamic acid etc. were detected and accounted for the 23.12%(w/w) of peptide sample.