Abstract: The gene of maltooligosyl trehalose synthase（MTSase）from Sulfolobus solfataricus ATCC 35092 was amplified by using polymerase chain reaction (PCR).The expression plasmids，pTrc99a-MTSase and pET-28a（+）-MTSase，were constructed by inserting the DNA fragments into E. coli expression vectors，pTrc99a and pET-28a（+）.These two plasmids were separately transformed into E.coli BL21（DE3），JM109（DE3）and BL21-Codonplus（DE3）-RIL，and the highest specific activity of MTSase was obtained in BL21（DE3）/pTrc99a system，which was 31.3 U·（g wet cell）^-1.To increase the expression level，the rare codons in the 5′-and 3′-end of the gene were substituted with E.coli preferred ones，and the present termination codon，UAG，was substituted with efficient translational termination sequence UAAU.By these modifications in the DNA sequence of MTSase gene，the specific activity of MTSase in E.coli cell was doubled.