Isolation of alkaline protease-deficient mutants. Alkaline
protease mutants were identified by their failure to produce a
zone of clearing on skim milk plates when incubated at 37°C
for 48 h. After three independent EMS mutageneses, a total
of 21,500 colonies were examined. Colonies with no zones of
clearing were subcultured for heavy growth on skim milk
agar and incubated at 370C for 48 h. A total of 19 presump-tive alkaline protease mutants, which produced no zones of
clearing under either condition, were subcloned. These
presumptive mutants were grown in liqu