PURIFICATION AND CHARACTERIZATION OF A THERMOSTABLE CARBOXYPEPTIDASE (CARBOXYPEPTIDASE TAQ) FROM THERMUS-AQUATICUS YT-1

A thermostable carboxypeptidase, which we named carboxypeptidase Taq, was purified from Thermus aquaticus YT-1 and characterized. The molecular weight of the enzyme was estimated to be about 56,000 and 58,000 on SDS-polyacrylamide gel electrophoresis and gel filtration, respectively, indicating that the enzyme has a monomeric structure. The optimum pH of the enzyme was 8.0, and the optimum temperature for the reaction was 80-degrees-C. The enzyme activity was dependent on cobalt ion and was inhibited by metal-chelating reagents, indicating that the enzyme is a metalloenzyme. The enzyme had high thermostability independent of cobalt ion; about 90% of its activity remained even after treatment at 80-degrees-C for 5 h. The enzyme showed broad substrate specificity, although proline at the C-terminus of peptides was not cleaved. The enzyme released amino acids sequentially from the C-terminus.