| Cyclodextrins are the cyclized products of starch and could form inclusion complexes with a variety of molecules for the tubular structure, a hydrophobic inner and a hydrophilic outer, to change the characteristics of the guest molecules. Thus cyclodextrins are widely used in the food, pharmaceutical, chemical, light industry and other fields. However, only α-, β-and y-cyclodextrin and their derivatives are widely used on the market. The application of the cyclodextrins, the degree of polymerization more than9, named cycloamylose, is almost no, which is mainly limited by their yield. And the known information about cycloamylose is limited also affects their application. This dissertation was focused on the nature of the cycloamylose synthase, the optimization of the synthesis conditions, the separation of the single degree of polymerization (degree of polymerization of11-15) and inclusion performance of cycloamylose. The main results are as follows:1. The impact of substrate concentration, the dosage of enzyme, temperature, pH, maltose, ethanol and dimethyl sulfoxide on conversion rate were studied with the substrate amylose. The results showed that the concentration of amylase, too high or too low, will all lessen the conversion rate. The dosage of enzyme has little effect on the final conversion rate. The optimum pH and temperature of the cyclization were6.5and40℃. And maltose inhibits the conversion rate. Ethanol and dimethyl sulfoxide improve conversion rates.2. With design-expert software and the substrate high amylase content of corn starch, first, three most significant components affecting cycloamylose production were selected out of seven components using Plackett-Burman design, namely the enzyme dosage, reaction temperature and time. Based on this reaction system, the substrate concentration of starch10mg/mL, pH6.5, ethanol8%, DMSO15%, the maximum corresponding area of the three factors were approached with the steepest ascent experiment, and the results show that the optimal center is the point, enzyme dosage20U/g starch, reaction temperature38℃, reaction time10h. Box-Behnken design and response surface analysis were adopted to further investigate the mutual interaction between the variables and obtain optimal values that bring maximum cycloamylose yield. By solving the regression equation we got the optimized reaction conditions:enzyme dosage20.8U/g starch, reaction temperature37.5℃, reaction time10.3h. Under this condition, the conversion rate was29.62%close to the predictive value of29.20%, and than that of before it increased by81.05%.3. Inactivating enzyme by boiling water bath, removing protein by centrifugation, precipitation by mixing5-fold volume ethanol and vacuum drying were successively conducted for crude cycloamylose from the reaction system. The recovery rate for the whole separation process was about46%. The cycloamylose monomer (degree of polymerization,11-15) can be obtained with the purity of66%-71%by a Bio-Gel P-4column (160cm×1.2cm i.d.). The cycloamylose monomer (degree of polymerization,11-15) can be obtained with the purity of84%-92%by a silica gel column (18cm×1.2cm i.d.).4. The inclusion ability of the P-cyclodextrin and the cycloamylose (degree of polymerization,11-15) with benzoic acid and DNS were studied. The results showed that the inclusion reaction can be completed with the maximum absorption wavelength of benzoic acid and DNS respectively transform from228nm to293nm and from335nm to455nm. The phase solubility curves for P-cyclodextrin (0-3.5mM) and cycloamylose (degree of polymerization,11-15,0-0.5mM) with benzoic acid and DNS are AL type. The inclusion constants of β-cyclodextrin and cycloamylose (degree of polymerization,11-15) with benzoic acid and DNS are215M-1,43M-1,41M-1,48M-1,53M-1、52M-1and296M-1,156M-1,173M-1,200M-1,220M-1,207M-1, respectively. About the inclusion constants β-cyclodextrin is the largest. Between degree of polymerization11-14the inclusion constants shows the same change with the degree of polymerization. And the inclusion constant turns smaller when the degree of polymerization is15. |
PDF Full Text Request