Maltooligosyltrehalose trehalohydrolase (MTHase), which is derived from the Sulfolobus acidocaldarius ATCC 33909, is a key enzyme in the couple enzymatic production of trehalose. The mutant strain L202P/L218D/Y323G, which has a 1.9-fold increase in protein expression, was used for the determination of enzymatic properties and trehalose conversion. The optimum temperature，optimum pH，pH stability and 60 ℃ half-life of mutant strain L202P/L218D/Y323G SaMTHase were determined. The results showed that there was no significant change in the enzymatic properties of the mutant strain. The kinetic parameters of the mutant strain L202P/L218D/Y323G SaMTHase were determined using maltotetraosyl trehalose as substrate. Using Maltodextrin (DE 5-7) as substrate, maltooligosyl trehalose synthase (MTSase) was mixed with the mutant L202P/L218D/Y323G SaMTHase at different ratios. The trehalose conversion results showed that trehalose conversion ratio was up to 76.0%.