Improvement of subtilisin-like serine alkaline protease by directed evolution for cold-adaptation

The activities in low temperatures of a mesophilic alkaline protease TC4 was improved by directed evolution. The process involved random mutagenesis by error-prone PCR and DNA shuffling followed by first screening on skim-milk plate and second screening by 96-well plate with AAPF as the substrate. It yielded a mutant T31, with a kcat 5 times and a catalytic efficiency k(cat)/K-m 6.33 times greater than that of wild type TC4 at 20 C-circle. The relative activity of T31 at 20 C-circle. was improved as much as 25% compared to that of wild type enzyme TC4 with casein as the substrate. Four amino acid substitutions were found in T31 and the results showed that these amino acid substitutions were sufficient to generate a mutant whose low temperature activity was greater than that of TC4.