Cloning And Expression Of Maltooligosyltrehalose Trehalohydrolase In E.coli From Micrococcus Roseus QS412

Trehalose is a non-reducing disaccharide consisting of twoα-1,1 linked glucose molecules and has about half the sweetness of sucrose. It is an unusual sugar as compared with other disaccharides, and is widely distributed in various organisms.A gene encoding the maltooligosyltrehalose trehalohydrolase (MTHase) from Micrococcus roses QS412 was cloned and inserted into pET expression system. Then the gene was expressed in E. coli strain BL21(DE3). IPTG was used to induce the recombinant plant E. coli BL21(DE3)( pET28-MTH). The molecular mass of the recombinant enzyme was estimated to be 69 kDa at 8% SDS-PAGE. And also, we studied that the recombinant enzyme can specifically hydrolyzed the maltooligosyltrehalose to release free trehalose, and the enzymatic activity was determined to be 44U/L.The His-MTHase expressed from pET-MTH was mainly existed in the form of insoluble inclusion bodies. Temperature might be the most important factor which influences the expression level of His-MTH, So induction was carried out at various temperatures between 4℃to 37℃. And also, IPTG concentration was varied from 0.04mM to 1mM to find a good induction condition. After E. coli cells were lysed by sonication, the crude inclusion bodies were washed twice and solubilized in 8mol/L urea containing 50mmol/L Tris-HCl(pH 8.0). Then the solution was desalted by the dialysis bag and the enzyme activity of it was tested. However, it shows no specific bioactivity.In an attempt to obtain more soluble His-MTH protein, a new expression system was used to construct a new recombinant plasmid, pGEMKT-HPf—MTH. But no specific bioactivity was found in the host cell E. coli. It may due to the complicated structure of MTHase.