| Summary: | 博士 === 國立中興大學 === 食品科學系 === 88 === The study was aimed to investigate the structure and functional relationships the mutant Bacillus macerans cyclodextrin glucanotransferases (CGTase) generated by mutagenesis. And the investigate production of cyclodextrin (CD) with B. subtilis (pHG) CGTase.
The plasmid - pUG containing the cloned B. macerans cgt gene was mutated with random mutagenesis. Mutated plasmids were transformed into E. coli. Seven mutant strains were selected on solid screening plates from 20,000 clones, and DNA sequencing identified mutations of the cgt gene. Among the seven strains, five showed one-amino-acid substitution, and two showed two-amino-acid alteration. All mutant CGTases still had the activities for cyclization and coupling reactions. The relative specific activities for either reaction differed with the mutants（p<0.05）. The relative specific activity γ-CD formation by J-8 (L304F) CGTase was 41 % of than the wild-type. In cyclization and coupling assay, the J-11 (A125R) CGTase showed higher specific activity than wild-type, but it was found that the activity was decreased dramatically after storage at 4℃ for 40 days. In order to stabilize J-11 CGTase, the mutant was stored in 10 mM Tris-HCl buffer (pH 7.2) containing either 4 mM CaCl2 or NaCl. Ninety-five percent of the enzymatic activity was maintained after storage at 4℃ for 40 days. It revealed that we could ameliorate the stability of the unstable mutant enzyme to improve their industrial application. Furthermore, Trp101 and Leu304 are located near the active site pocket of CGTase from B. macerans. The roles of Trp101 and Leu304 of CGTase in the structure and function were investigated. The pUG, was used for the construction of mutants via site-directed mutagenesis. Acting on soluble starch, the wild-type CGTase mainly produced α-CD in the early stage. However, W101L, L304Y, L304W, W101F/L304W and W101L/L304W CGTases produced mainly β-CD. A clear change in CD product ratio was observed in W101F/L304W and W101L/L304W CGTases. They produced less 1 in α-CD/β-CD product ratio than wild-type CGTase for 8 hours. And W101L/L304W CGTase almost lost the ability to produced γ-CD at all incubation periods (48 hours). These results indicated that the amino acid residues, Trp101 and Leu304 in CGTase, were related to the product specificity.
Besides, as Bacillus subtilis (pHG) was cultivated in medium B containing 4 % wheat bran, 0.5 % (NH4)2SO4, 0.5 % CaCO3, 0.075 % K2HPO4, 0.05 % NaHPO4·12H2O and 0.03 % MgSO4·7H2O for CGTase production. The optimum condition for B. subtilis (pHG) CGTase production in 500 mL hinton flask was found as following : cultural volume, 100 mL; incubation temperature, 37℃; shaking speed, 120 rpm and incubation time, 72 hours. The CGTase was purified by a procedure including β-CD coupling affinity and DEAE Sepharose CL-6B chromatographs. The cgt gene from B. macerans was expressed in B. subtilis (pHG) and 100 ~ 110 mg/L of the enzyme was produced, which is about 10 times high as that of the parental strain. And the host could produce CGTase without adding any antibiotics (eg. tetracycline). The biochemical properties of B. subtilis (pHG) CGTase were similar with parental strain CGTase. Furthermore, effect of the substrate and enzyme concentration on the production of CD using response surface methodology. Results was applied to each variable to determine the productivity of total CD, α-CD and β-CD, the substrate and enzyme concentration were found to be significantly related (p<0.05). When a low concentration starch solution, was employed, it was found that the productivity of total CD and β-CD increased as the enzyme concentration increased. However, the productivity of α-CD was increased with decreasing CGTase concentration. The conditions for high productivity of total CD and β-CD were as follows : starch, 2.8 % ; B. subtilis (pHG) CGTase, 0.014 % ; agitation, 100 rpm ; incubation time, 2 hour and incubation temperature, 37℃. The productivity of total CD, α-CD and β-CD was 50 %, 19 % and 26 %, respectively. After the same amount of CGTase, was applied, a higher CD yield in same reaction volume could be obtained at a starch concentration of 5.2 %. Under this condition, the productivity of total CD was 45 % ; that of α-CD was 16 %; and that of β-CD was 22 %. No significant differences were observed between the experimental data obtained under the various conditions and the predicted values, which indicated that the regression equation established in this study was acceptable.
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