Isolation And Identification Of Actinomucor Elegans PEP001 And Cloning, Expression Of Its Carboxypeptidase Y Gene

The white flake, mostly consists of free L-tyr, was composed by overstaturated free amino acids which were released by sufu mucor proteases. Free L-tyr was producted from soybean protein by endopeptidases and carboxypeptidases while carboxypeptidases made a great contribution in this process. Although carboxypeptidases played an important role in forming white flake, there is no report about carboxypeptidases gene sequences from mucor which was used to product sufu. In this study, A. elegans PEP001 was screened, identified and named from sufu fermentation and full-length CPY gene was cloned form A. elegans PEP001. The proCPY was expressed in E. coli Rosetta and P. pastoris GS115, and the enzymatic properties of mature enzyme were analyzed after purification and in vitro activation of proCPY. Main results are listed as follows:(1) White flake was made up of 92.6% free amino acids with more than 90% free L-tyr. The differences of amino acids concentration between the first stage and the end stage of sufu maturation period showed that L-tyr content had a ninefold increase during sufu maturation period. It indicated that most of free amino acids was released by mucor proteases during sufu maturation period, especially exopeptidases.(2) A. elegans PEP001 was screened, identified and named from sufu fermentation while the activity of carboxypeptidase, releasing L-tyr form carbon-terminal of peptides, was detected and presumed to be CPY. The part length of CPY gene was cloned by degenerate primers, which were designed based on conserved sequence of CPY from its homologous fungi, and the end sequence of 5’ and 3’ were obtained using rapid amplification of c DNA ends(RACE) technology. The 1557 bp sequences of full-length CPY gene, which coding 518 amino acids, was cloned sucessfully. Sequence alignment and software analysis of CPY showed that it consists 23 amino acids signal, 67 amino acids propeptide and 428 amino acids mature peptidase. Its catalytic central sites are S228, D434 and H495.(3) The recombinant proCPY was expressed in E. coli Rosetta(DE3) as inclusion body while it was successfully setcretory expressed in P. pastoris GS115. The recombinant proCPY, expressed in P. pastoris GS115, was purified and activated in vitro by trypsin.The specific activity of purified mature CPY was 157.2 U·mg-1. It was indicatied that the optimal pH and temperature of purified mature CPY was 6.0 and 45 ℃. Mature CPY was stable below 40 ℃,but it will be inactivated immediately at 60 ℃. Also, it was stable between pH 4.0-7.0.