| Summary: | 碩士 === 國立中興大學 === 食品科學系 === 86 === AbstractThe 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.
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