Studies On Enzymatic Production Of α-cyclodextrin And γ-cyclodextrin

Cyclodextrins are cyclic a-1,4-glucans having the shape of a hollow truncated cone. They can form inclusion complexes with various hydrophobic guest molecules, changing their physical and chemical properties. These properties make cyclodextrin attractive for various applications in many different fields:food, chemical, pharmaceutical and textile industries as well as in biotechnology, agriculture and environmental protection. They are commonly produced from starch or starch derivates via an intramolecular transglycosylation reaction catalyzed by cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase).The industrial production of cyclodextrins is mainly through the enzymatic production, but no domestic reports aboutα-,γ-cyclodextrin production was found. In this paper, the enzymatic production process ofα-,γ-cyclodextrin by a-CGTase and y-CGTase has been systemically studied.The main research contents are as follows:1. The enzymatic preparation parameter of a-cyclodextrin by mutation Y89D from Peanibacillus macerans JFB05-01 was optimized, including reaction time, starch source (potato, corn, cassava, soluble starch), the enzyme dosage, tempreture, pH, polar organic solvents (ethanol, isopropanol, butan-1-ol, decan-1-ol), starch concentration and different adding way of enzyme. The optimized condition was found to be:15% potato starch,5 U of CGTase per gram of starch, pH 5.0,30℃,5% decan-1-ol,6 h. In this condition,55%(w/w) conversion to cyclodextrins was obtained, the ratio ofα:β-cyclodextrin formed was 87:13, with negligible formation ofγ-cyclodextrin.2. The separation and extraction process of a-cyclodextrin was studied. a-Cyclodextrin was obtained through steam distillation, concentration, crystallization, drying method. The a-cyclodextrin product was analyzed by HPLC, the yield of a-cyclodextrin could reach 90%-95% of the theoretical value and the purity of a-cyclodextrin was 98%. The product was also analyzed by gas chromatography (GC), the residual concentration of decanol was only 3.Oppm, meet the safe requirements of production.3. The enzymatic preparation parameter of y-cyclodextrin by y-CGTase from Alkalophilic Bacillus clarkii 7364 was optimized, including reaction time, starch source (potato, corn, cassava, soluble starch), the enzyme dosage, tempreture, pH, polar organic solvents (ethanol, cyclohexane, butan-1-ol, glycyrrhizic acid), starch concentration and different adding way of enzyme. The optimized condition was found to be:15% soluble starch,5 U of CGTase per gram of starch, pH 12.0,55℃,2% glycyrrhizic acid, after 6 h reaction,66.7%(w/w) conversion to cyclodextrins was obtained, the ratio ofγ:β-cyclodextrin formed was 95:5, with negligible formation of a-cyclodextrin.4. The separation and extraction process of y-cyclodextrin was preliminary studied. Compared with macroporous adsorption resin and acid precipitation method, we found the acid precipitation method was better. There is total 56% of the glycyrrhizic acid separated in reaction solution. This provides a theoretical basis for separation and purification of refined y-cyclodextrin.