β-CGTase From Bacillus Cereus: Fermentation, Cloning And Enzymatic Properties

β-CGTase because of its extensive application value, especially theproduction of β-cyclodextrin and drug molecules modification has greatprospects. Firstly, the strains produced β-CGTase was screened from soil filterto get a high yield of β-CGTase strains. The strain was determined by16SrDNA analysis as Bacillus cereus BC-0801. Bacillus cereus BC-0801wassubjected to mutagenesis by means of low energy ion implantation of40×2.5×10¹³ions/cm²at10keV. After culture in selective medium, a mutantstrain producing a high yield of β-cyclodextrin glucanotransferase wasselected and designated as BC-0802. The optimum medium of the strains is3%of corn flour,1%of corn steep liquor,1%of peptone,1.5%of yeastextract,0.05%of K2HPO4,0.01%of MgSO47H2O. The optimumfermentation of the pH is9.0, the inoculum size is4%, the fermentation timeis36h, the fermentation temperature is30℃.The purified β-CGTase enzymes exhibited a single band with molecularweight of75kDa on SDS-PAGE. Biochemical characterization of the enzymeshows an optimum temperature of60℃, and optimum pH of7.0. The enzymewas stable between pH6and9.5and temperature up to65℃. The enzymeactivity was enhanced by Ca²⁺, while was strongly inhibited by Mg²⁺, Al3+,Zn²⁺, Fe²⁺, Fe3+, Mn²⁺and Co²⁺.By means of molecular biology, β-CGTase gene was cloning fromBacillus cereus, and constructed in expression vector pBV220plasmid weretransformed into E. coli DH5α. Engineering bacteria induced conditions asfollows: bacteria concentration of OD600is1.0, the induction time for4h, thetemperature of medium is30℃, pH is8.0. The distribution of the inductiontemperature was39℃,2h,41℃,1h,42℃,1h. By the steps of ammoniumsulfate precipitation, dialysis, Sephadex G-150column chromatography andSDS-PAGE, the purified β-CGTase enzymes exhibited a single band withmolecular weight of85kDa on SDS-PAGE. Km of β-CGTase was calculatedto be2.4508g/L-1. The optimum temperature of the enzyme is65℃. Theenzyme activity have50%decreased significantly after4h at90℃, and have70%decreased significantly after2h at100℃. The optimum pH of the enzymeis9.0. The enzyme activity was enhanced by Ca²⁺and Fe²⁺, while was strongly inhibited by Mg²⁺, Zn²⁺, Co²⁺and Tris Co²⁺.Then β-CGTase was constructed in pET28plasmid at the host of E·coliDH5α and the pAX01plasmid at the host of Bacillus subtilis. β-CGTase couldbe expressionin the two bacterias.The research from strain Screening, mutagenesis, the optimization offermentation conditions and the molecular biology research, we got a highyield of β-CGTase of Bacillus cereus mutant strains and a high yield addictedto hot β-CGTase engineering strain. The research built a systematic of theresearch in enzyme engineering, and made a foundation for industrialproduction in future.