Improving the expression of Cyclodextrin Glucanotransferase gene of Bacillus macerans in Bacillus subtilis

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The cgt gene had been constructed into a shuttle vector and transformed to B. subtilis in our laboratory. However,there was no identified promoter sequence in the upstream of cloned cgt gene. The host could not express CGTase without addition of antibiotics. In order to improve the expression of cgt gene of B. macerans in B. subtilis. The G3b promoter of Bacillus phage φ29,amyE promoter of B. subtilis and the cgt gene of B. macerans were synthesized by polymerase chain reaction （PCR） and inserted into a vector -pWP18（a promoter cloning vector）, pWGG and pWαG was constructed and transformed to the protease negative B. subtilis strain DB430. B. subtilis harboring pWGG and pWαG could express CGTase on log phase and early stationary phase,respectively. And high amount of CGTase was produced as B. subtilis was grown in medium B（a medium for growing B. macerans ）. The inserted promoter region was sequenced and the amount of CGTase produced in cell lysate or medium by B. subtilis harboring pWαG and pWGG was determined. The results indicated that a base of G3b promoter -10 region was not correct, and secretion rate is too slow to secret CGTase completely. Therefore, CGTase in B. subtilis harboring pWGG was not overexpressed. The plasmid stability of B. subtilis harboring pW αG reached 90% after 60 hours when cultures were renewed without selection every 12 hours. But B. subtilis harboring pWGG lost almost all plasmids after 36 hours at the same condition. Besides，when B. subtilis CGTase（pWαG、pWGG）was treated with N-acetyl-β-D-glucosamidase,the activity of CGTase decreased gradually,and through the results of SDS-PAGE and Ochterlong double diffusion,we predict that CGTase may be a glycoprotein.