Optimization Of Pilot Preparation Process Of ?-cyclodextrin Glucosyltransferase And Evaluation Of Its Application

Cyclodextrin(CD)is a series of cyclic oligosaccharides,which are produced by the action of cyclodextrin glucosyltransferase(CGTase)on amylose.Cyclodextrina are usually divided into ?-,?-and ?-cyclodextrin,composed of 6,7 or 8 glucose residues,formed by the connection of ?-1,4-glycosidic bond.Cyclodextrin has a special nature of hydrophobic,internal cavity hydrophobic.It can be embedded in a large number of hydrophobic object molecules,Changing stability,solubility,volatility and chemical reaction properties of them.Due to the properties,Cyclodextrins are so widely used in pharmaceutical,food cosmetics,environmental protection,industry as well as agriculture.The cyclodextrin was mainly prepared by enzyme rather than chemical synthesis.The main principle of biological enzyme conversion method is using CGTase acting on starch or other related compounds to produce cyclodextrin.CGTase is a multifunctional enzyme,it can catalyze the glycosylation reaction,including cyclization,coupling,disproportionation and hydrolysis.In the previous study,a strain Y112 from Bacillus subtilis was screened for a production of ?-CGTase,and its strain was identified.The conditions of shake flask culture were optimized,as well as its purification and enzymatic characteristics.On the basis of the experiment,the fermentation of 20 L fermentor of Bacillus subtilis Y112 and the preparation process of ?-CGTase enzyme are optimized.And preparation of cyclodextrin and modification conditions of stevioside by CGTase are optimized too.The results are as follows:Optimization of fermentation conditions for CGTase pilot fermentation:Based on the single factor experiment,the fermentation conditions were as follows: the volume of liquid 10 L,inoculation amount 6%,27 ?,rotation speed 300 r/min,pH9.5,dissolved oxygen 40%.The average enzyme activity was 2140 U/mL,32.1% higher than the original fermentation activity.And the experiment of small-scale fermentor wastested by pilot-scale experiment.Solid enzyme powder was produced after flocculation,high-speed centrifugation,ultrafiltration concentration and freeze-drying.The total enzyme recovery was 72.8%.Study on the production of cyclodextrin by CGTase:According to single factor and response surface method,the optimum conditions were as follows: concentration of potato starch 5%,amount of enzyme 200 U/g(starch),pH8.4,temperature 30 ?,200r/min for 6 h.The yield of ?-CD was 28.67%,which was 2.48 times higher than that before optimization.Enzymatic modification of Stevioside by CGTase:Through single factor and orthogonal experiments,the optimum conditions for the transformation of stevioside were as follows:Sugar donor as corn starch,temperature 40 ?,pH 8.0,substrate ratio 1:1,enzyme dosage 10 U/g(stevioside),reaction 2.5 h.The final conversion rate reached79.98% by the determination of HPLC.