2006 Volume 53 Issue 2 Pages 91-97
The thermal stability of sucrose phosphorylase (EC 22.214.171.124) from Streptococcus mutans was enhanced using random and site-directed mutageneses. Random mutagenesis studies revealed that eight single amino acid substitutions, T47S, S62P, Y77H, V128L, K140M, Q144R, N155S and D249G, contributed to the enhancement of thermal stability. These mutated enzymes retained their activity even after heat treatment at 55°C for 20 min, while the wild-type enzyme was drastically inactivated. The combinations of these eight mutations showed that the increase in the number of combined amino acid substitutions resulted in the higher thermal stability of the enzyme. The thermostable sucrose phosphorylase with all eight mutations retained more than 60% of initial activity after heating at 60°C for 20 min and exhibited the highest thermal stability among these mutated enzymes. The optimum temperature and pH of the thermostable sucrose phosphorylase were confirmed to be substantially similar to those of the wild-type enzyme. The thermostable sucrose phosphorylase was easily purified by heating at 65°C for 20 min with 20% sucrose, and the purified enzyme can be directly employed for the production of amylose without further purification.