Overexpression Of Carboxypeptidase Y From Actinomucor Elegans In Pichia Pastoris

Carboxypeptidase is a protease that can hydrolyze amino acid residues from the carboxyl-terminus of proteins and it has a wide range of substrate specificities.Compared with other carboxypeptidases,carboxypeptidase Y does not contain metal ions,and it also exhibits activities for proline residues that are hard to be degraded by other carboxypeptidases.Carboxypeptidase Y is widely used in amino acid sequencing,mass spectrometry analysis,and debittering of protein hydrolysates for its broad substrate specificity and the ability to decompose proline residues.The currently well-studied carboxypeptidase Y is derived from Saccharomyces cerevisiae,while less studies have been conducted on other species,especially derived from filamentous fungi.In our laboratory,a novel carboxypeptidase Y gene was cloned from the Actinomucor elegans genome,and expressed in Pichia pastoris,but the expression level was not high.In this paper,the efficient expression of carboxypeptidase Y from Actinomucor elegans was achieved by selecting appropriate promoters,constructing double-plasmid integration recombinant bacteria,and co-expressing secretion-promoting factors.The carboxypeptidase Y zymogen was activated by protease K in vitro.The main research results include:?1?Construction of recombinant Actinomucor elegans carboxypeptidase Y-expressing strains containing the AOX1 promoter and the GAP promoter,respectively.Through screening methods for increasing the concentration of antibiotics,a highly efficient recombinant strain was screened and the total extracellular protein content reached 275±20 mg·L^-1 after methanol induction.?2?On the basis of the obtained highly-recombinant recombinant strain two-plasmid integration strains Z?-600-1+9K and Z?-600-1+GAP were constructed.The recombinant strains were screened by increasing the concentration of antibiotics,and the expression of the highly expressed recombinant strains increased the secretory expression of Actinomucor elegans carboxypeptidase Y to 1.51 and 1.47 times the recombinant strain Z?-600-1,respectively.?3?Co-expresses the secretion promoting factor Binding protein?BIP?,Protein disulfide isomerase?PDI?,and Endoplasmic oxidoreductin 1?ERO1?in recombinant strains with high copy number,and secretory expression of Actinomucor elegans carboxypeptidase Y was 1.87fold,1.76 fold,and 1.26 fold higher than that of recombinant strain Z?-600-1,respectively.The recombinant strain with the best secretory effect was induced by methanol,and the total extracellular protein secretion reached 525±15 mg·L^-1.?4?The protease K was successfully used in vitro to activate the Actinomucor elegans carboxypeptidase Y zymogen,and a new activation method for the Actinomucor elegans carboxypeptidase Y zymogen was found.