Maltooligosyltrehalose synthase (MTSase) is a key enzyme for the production of trehalose from starch. Thermophilic MTSases offer advantages for trehalose production but suffer from low yield. In this study, directed evolution was used to increase the production of Sulfolobus acidocaldarius MTSase (SaMTSase) in Escherichia coli. Mutant libraries constructed using error-prone polymerase chain reaction were assessed using high-throughput activity assays. Three mutants with enhanced activities were obtained, the best of which (mutant D-4) exhibited 2.4 times greater activity than wild-type SaMTSase. The specific activity and catalytic efficiency of D-4 were also greater than those of wild-type SaMTSase. The D-4 activity (624.7 U·mL-1) produced in a 3 L fermenter was 2.0 times greater than that of wild-type SaMTSase. Because the same trehalose yield was obtained using an equal amount of either D-4 or wild-type SaMTSase activity, using D-4 will significantly lower the cost of trehalose production. The activities of the individual mutations present in the three SaMTSase mutants obtained using directed evolution were analyzed. Mutants F284V and T439A exhibited the greatest increases in enzyme activity. Homology models suggested that the decreased side-chain size, weakened hydrophobicity, and decreased interaction might enhance the flexibility of the loop containing catalytic residue Asp443, which was conducive to catalysis.
Keywords: directed evolution; high-throughput screening; maltooligosyltrehalose synthase; trehalose.