ISOLATION AND CHARACTERIZATION OF UREA-RESISTANT STAPHYLOCOCCUS-AUREUS V8 PROTEASE DERIVATIVES

Three different urea-resistant Staphylococcus aureus Y8 protease deriv atives (V8 Delta 53-U1, V8 Delta 53-U5 and V8 Delta 53-U8) were obtain ed by random mutagenesis using a polymerase chain reaction (PCR) metho d. From DNA sequence analysis of the mutants, three different amino ac id substitutions, D44E, N71S and K147R, were identified. The half-live s of these mutant enzymes were 3 to 5 times longer than that of the wi ld type in the presence of 5 M urea, and they also showed increased st ability to sodium dodecyl sulfate. Measurements of the kinetic paramet er values of each mutant enzyme indicate that these mutations substant ially conferred the stability to these denaturants without markedly ch anging their kinetic parameter values. In addition, we demonstrate tha t these mutant enzymes were applicable to digestion of a human calcito nin fusion protein produced in Escherichia coli.