Fermentation And Application Of CGTase From Bacillus Stearothermophilus Expressed In Bacillus Brevis

Cyclodextrin glycosyltransferase(Cyclodextrin Glycosyltransferase, CGTase in abbreviation, EC 2.4.1.19) is a member of α- amylase family(family 13). Among its four different reactions. Cyclization reaction is responsible for converting the starch into cyclodextrins(CDs), and β-cyclodextrin is the most common and most widely used cyclodextrins.In the previous study, the CGTase from Bacillus stearothermophilus was cloned and expressed in E. coli. But its endotoxin has security risk. So the protein product in E. coli is generally not used in food production. Thus, choosing a safer expression host is necessary for CGTase and its efficient production.The present study focused on expression, fermentation optimization and application of CGTase. The main contents were listed as follows:(1) The cgt gene was sub-cloned from p ET-20b(+)-cgt and the plasmid p NCMO2-cgt was constructed and transformed into B. brevis. SDS-page showed CGTase had been successfully expressed in B. brevis. Then different culture conditions were tested in shake flask conditions. The optimal results were as follows: The B. brebis was cultured in 30°C with industrial peptone as the nitrogen source, glucose as the carbon source, added 1 mmol×L-1 Mg SO4 and the highest yield CGTase reached 93.6 U·m L-1. Then different temperature, different media formulations, and the effects of different initial glucose concentration were investigated in 3.6 L fermenter to further enhance the CGTase yield and the results were as follows: The combinant B. brevis was cultured under 30% DO concentration, 30°C and p H 7.0. The medium contained 40 g·L-1 industrial peptone and 10 g·L-1 beef extract. The highest CGTase yield reached 607.1 U·m L-1.(2) Xylose promoter from B. megaterium was substituted for P2 promoter in the plasmid p NCMO2. Different induction temperature and concentration of xylose were investigated in flask and 3.6 L fermenter. The optimal conditions were as follows: When OD600 reached 5, the induction was performed with 0.5 g×L-1 xylose in 37°C and the CGTase activity reached 98.7 U·m L-1.(3) The optimal conditions for β-CD production were studied as follows: The potato starch of 15%(w·v-1) was liquefied and cooled to room temperature. The CGTase of 13.0 U per gram starch and cyclohexane of 5%(w·v-1) were added into the reaction system at p H 6.0. After 18 h of incubation at 50°C, the yield of CD reached 73.9%(w·w-1) with 98.7% ratio of β-CD.Pullulanase was added into the reaction system to further increase the conversion rate, the optimal conditions for β-CD were studied as follows: The potato starch of 15%(w·v-1) was liquefied and cooled to room temperature. The CGTase of 10.0 U per gram starch, pullulanase of 50.0 U per gram starch and cyclohexane of 5%(w·v-1) were added into the reaction system at p H 6.5. After 18 h of incubation at 40°C, the yield of CD reached 81.4%(w·w-1) with 97.5% ratio of β-CD.