The Study On Biosynthesis Of Sucrose-6-acetate,a Key Intermediate Of Sucralose

Sucralose,which could be widely used instead of sucrose in many ways for its pure sweetness and good heat stability,is the most ideal sweetener developed recently.The preparation of sucrose-6-acetate(s-6-a)is a key step in the synthesis of sucralose.In this study,the strain GXU-011 was isolated,which could synthesizes glucose-6-acetate(g-6-a).The strain GXU-011 was Gram-positive,rod shaped cell.The physiological,phylogentic analysis and alignment analysis of 16S ribosomal DNA sequence showed that it belonged to the genus Bacillus sp.,and identified as Bacillus sp.GXU-011.The main composition of the fermentation medium was determined by the single factor experiments.Three statistically significant parameters(quantity of glucose,yeast extract and peptone)were determined by Placket-Burman design.Then Box-Behnken Design(BBD)response surface analysis was used for the optimization of g-6-a fermentation medium,the results showed that the optimal fermentation medium was as follows:glucose 48.28 g/L,yeast extract 8.67g/L,peptone 8.11 g/L,K2HPO4 0.5 g/L,KH2PO4 0.5 g/L,CaCO3 5g/L,MgSO4 0.5 g/L,KCL 0.2g/L.Single factor and orthogonal array design experiments were performed to obtain the optimum fermentation conditions for g-6-a production,the result showed the optimal culture conditions were cultivation temperature of 28?,5%inoculum concentration,liquid volume of 100ml/500ml,rotation speed of 190rpm.Under the optimal conditions,the highest yield of g-6-a reached 31.71 g/L,increased by 82.56%compared to the control.The separation of g-6-a was preliminary investigated,the results showed the purity and recovery ratio were increased by 95.84%and 88.37%by using organic solvent extracting,respectively.The synthesis reaction of s-6-a was performed using sucrose and g-6-a by immobilized fructosyltransferase(IFTS)from Aspergillus sp.GX-0010.The catalyzed performance of IFTS was evaluated and the optimal conditions were determined:temperature is 50?,pH is 6.5.Compared with free fructosyltransferase,temperature and pH stabilities of the IFTS were improved.The effects of reaction factors such as the time,g-6-a concentration,ratio of g-6-a to sucrose,and enzyme concentration were also investigated in this study.The optimal condition of IFTS in reaction was obtained as follows:50?,1h reaction,pH6.5,1:2 ratio of g-6-a to sucrose and 35g/L concentration of enzyme,and under this optimal condition,the g-6-a conversion ratio reached 25.18%.