CLONING AND EXPRESSION OF THE CARBOXYPEPTIDASE GENE FROM ASPERGILLUS-SAITOI AND DETERMINATION OF THE CATALYTIC RESIDUES BY SITE-DIRECTED MUTAGENESIS

Carboxypeptidase from Aspergillus saitoi removes acidic, neutral and b asic amino acids as well as proline from the C-terminal position at pH 2-5, cpdS, a cDNA encoding A. saitoi carboxy peptidase, was cloned an d expressed. Analysis of the 1816-nucleotide sequence revealed a singl e open reading frame coding for 523 amino acids, When A. saitoi carbox ypeptidase cDNA was expressed in yeast cells, carboxypeptidase activit y was detected in the cell extract and was immunostained with a 72 kDa protein with polyclonal anti-(A. saitoi carboxypeptidase) serum. The recombinant enzyme treated with glycopeptidase F migrated with an appa rent molecular mass of 60 kDa on SDS/PAGE, which was the same as that of the de-N-glycosylated carboxypeptidase from A. saitoi. Site-directe d mutagenesis of the cpdS indicated that Ser-153, Asp-357 and His-436 residues were essential for the enzymic catalysis. It can be concluded that A. saitoi carboxypeptidase has a catalytic triad comprising Asp- His-Ser and is a member of serine carboxypeptidase family (EC 3.4.16.1 ).