Enhancement of thermostability of kojibiose phosphorylase from Thermoanaerobacter brockii ATCC35047 by random mutagenesis

Random mutation by error-prone PCR was introduced into kojibiose phosphorylase from Thermoanaerobacter brockii ATCC35047. One thermostable mutant enzyme, D513N, was isolated. The D513N mutant enzyme showed an optimum temperature of 67.5-70°C (the wild type, 65°C), and thermostability up to 67.5°C (the wild type, up to 60°C). The half-lives of D513N were estimated to be 135 h at 60°C, 110 min at 70°C and 6 min at 75°C, respectively. They were about 1.6-fold, 7-fold and 6-fold longer than those of the wild-type enzyme, respectively.