| It is a rapid method for screening the cyclodextrin glycosyltransferase producer according tothe size of the colorless ring by using phenolphthalein as a indicator in the agar solid mediumcontaining soluble starch. The mutant 7-12 which produces as much as 5600u/ml ofcyclodextrin glycosyltransferase was obtained by using the rapid screen method after Bacillusalkalophilus1177 was mutagenized by ultraviolet (UV) and γirradiation with 60Co. And theenzyme activity was raised by 1.76 times. The continuous 5 generations test shows that themutant 7-12 is stable in the cyclodextrin glycosyltransferase production.The conditions of cyclodextrin glycosyltransferase(CGTase) production by Bacillus alk-alophilus12-7 was studied. The optimal fermentation conditions for cyclodextrin glycos-yltransferase production were determined by a single-factorial experiment and orthogon-al experiment. The results showed that the optimal conditions as follows: amount ofinoculum 3%;incubation temperature 30℃;pH 10.5;the compositions of fermentationmedium are maize powder 2%, yeast extract 1.5%, corn steep liquor 5%;load of 250ml-flask is 30ml;shaking incubation is 3 days at 270rpm and the enzyme activityrise up to 5400 U/ml. In 10L-fermentor enzyme activity is for 5820U/ml. This enzym-e activity is higher than that of the literature reported.Cyclodextrin glucantransferase (EC 2.4.1.19) of Alkalophilic Bacillus sp.7-12 was purified byammonium sulfate fractionation, DEAE-cellulose column chromatography and sepharoseCL-6B column chromatography. The final preparation thus obtained only a single band bySDS-PAGE, The enzyme showed a Km of 1.24 mg/ml and Vmax 114.9 μg/min when starchwas used substrate. The Km value indicates that the enzyme has very high affinity for starchby comparison with that of the previously reported. Molecular weight of the purified enzymewas 69,000 by SDS polyacrylamide gel electrophoresis.The purified enzyme protein denatured either by β-mercaptoethanol or boiling water bath hadthe same relative mobility by SDS-PAGE, which indicated that the enzyme had no subunits,or monomeric in nature. Therefore, it demonstrated that our novel enzyme was not completelyidentical with those reported previously.The optimum conditions for activity were temperature 60℃,pH 8.5, stable in the range ofpH6.0 -10.0 and below 70℃. However, the enzyme decreased its activity drastically beyondthat range, and almost lost its activity below pH 4.0 and above pH 11.0. When kept in bufferat pH 8.5, the enzyme showed a wide thermal stability from 40°C to 70°C, though the enzymerapidly lost its activity above 70oC, and only 14% activity left at 80oC.The enzyme activity was strongly inhibited by Ag+,Cu2+,Mg2+,Al3+,Co2+,Zn2+,Fe2+ slightlyinhibited by Sn2+,Mn2+, and not affected by K+, Ca2+.The effects of some protein modification reagents on the activity of the cyclodextringlucantransferase (EC 2.4.1.19) of Alkalophilic Bacillus sp.7-12 have been studied. Theenzyme was not affected by DIC, PMSF, DTT and Ch-T modification, suggesting thatguanido, hydroxyl, disulfide bond and methionine residues were not necessary to enzymeactivity. The enzyme activity was remarkably decreased after DEPC, NBS, EDC modification.The results indicated that carboxyl groups, imidazolyl group and indolyl group seemed to beessential to the catalytic activity of cyclodextrin glucantransferase. The study on enzyme ofBender shows tryptophan residues participates in the combination with the substrate mainly,has a little effect on catalysis;histidine residues not only relates to the fact that thing iscombined at the substrate, but also may be very important to the catalysis. The result of studyof Hofmann indicates the structure of amino acid carboxyl groups participates in the activecenter.The substrate would protect the enzyme. This was because substrate and enzyme would formmedium after the CGTase mixed with starch, when it was modified with NBS. The resultshowed substrate-complex after they contacted, and oxidation of tryptophan residues by NBScould be prevented. The protection of enzyme through substrate would be put up.We find emission spectrum that produces at 295 nm excited all by the endogenousfluorescence of the enzyme molecule, the biggest wavelength locate in 345nm. There isprotective action on the combination of CGTase and NBS in the glycerines.Dithiothreitol (DTT) has no effect on enzyme activity. This has indicated after DTT hasreduced disulfide bond of the enzyme. It have not destroyed the advanced structure of theenzyme, has not influenced the stability of the enzyme either. There are no subunits in thisenzyme;its electrophoresis explained this too at the same time.PH, temperature, E/S, kinds of starch and reactive time have effect on production of γ-cyclodextrin by single factor experiments. The influence of pH and temperature isremarkable, the influence of enzyme quantity is not remarkable;the influence of mutualfunction of every factor is not remarkable by Response Surface Methodology experiment andanalysing. That is to say pH the most influential, temperature takes second place, theinfluence of the concentration of enzyme is minimum in the range of this experiment. Theoptimum conditions of production of γ-cyclodextrin were pH8.12, temperature 60℃, E/S1200U/g starch. Under the conditions, amount of γ-cyclodextrin was 14.31mg/ml.The experiment showed: glycyrrhizin can choice improvement of output of γ-CD the enzymefrom alkalophilic Bacillus.DE value has a little effect on production of γ-CD in the range of 2-15. When greater than 15,the output of γ-CD drops fast;When DE value is in the range of 2-12, influence on theproduction of β-CD not very great. When greater than 12, the output of β-CD drops fast.Pullulanase doesn't increase much the output of γ-CD (5.3%), do not increase so obviously asoutput of β-CD (10.5%). When the enzyme exceeds 40U, the output of γ-CD and β-CD bothdrop to some extent. The output of the enzyme to CD increases limitedly. CGTase can workamylopectin and amylose.Gamma cyclodextrin was synthesized from beta cyclodextrin containing seven carbonsatoms in the presence of glycyrrhizin, using Alkalophilic Bacillus cycloddextrin gluca-notransferase (EC 2.4.1.19). More than 30% of beta cyclodextrin substrate was conver-ted to the corresponding gamma cyclodextrin under the conditions given below, 400 u-nits enzyme/g ofβ-CD, β-CD concentration of 20mmol/L, glycyrrhizin concentrationof 3%,a reaction pH and temperature of 7 and 65℃, respectively, reaction time 27 h.Glucose, maltose and maltotriose can increase coupling reaction of cyclodextrin glucanotransferase, but the effect of glucose is maximal.Identification of the gamma cyclodextrin was performed by means of paper chromatography(PC), high performance liquid chromatography (HPLC), infrared spectrum (IR), nuclearmagnetic resonance (13C-NMR) spectrum, scanning electron microscope (SEM) anddifferential scanning calorimetry (DSC).Decomposition temperature of γ-cyclodextrin is 286℃. By the Kissinger's equation andOzawa's equation, the activation energy of thermal degradation of γ-cyclodextrin was 191.3kJ/mol and 190.8kJ/mol, respectively. The results were in good agreement. Thepreexponential factor (A) was 3.559×1017 min-1 according to method of Ozawa. The enthalpychange was 21.2kJ/mol and entropy change 0.0376kJ/mol﹒K, speed constant 0.722 min-1,half life 57.88 second.Grind method was used in preparing the VD2-γ-CD complex. Formation of the complex wasidentified by UV, IR, DSC and phase solubility diagram method, and the molecular ratio ofVD2 to γ-CD in the complex was 1:1, the complex constant was 2562 L/mol. The aqueoussolubility of was significantly increased after formation of complex, and more than β-CD.
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