Maltooligosyltrehalose synthase (MTSase) is a key enzyme in trehalose production. MTSase from Arthrobacter ramosus has poor thermostability, limiting its industrial use. In this study, mutant G415P was obtained by directed evolution and S361R/S444E was subsequently generated based on a structure analysis of the region around G415. The t 1/2 of G415P and S361R/S444E at 60 °C increased by 3.0-and 3.2-fold, respectively, compared with the wild-type enzyme. A triple mutant (G415P/S361R/S444E) was obtained through a combination of the above mutants, and its t 1/2 significantly increased by 19.7fold. Kinetic and thermodynamic stability results showed that the T 50 and T m values of the triple mutant increased by 7.1 and 7.3 °C, respectively, compared with those of the wild-type enzyme. When the triple mutant was used in trehalose production, the yield reached 71.6%, higher than the 70.3% achieved with the wild-type. Thus, the mutant has a potential application for industrial trehalose production.

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