Enzymatic Synthesis Of Turanose

Turanose?3-O-?-D-glucosyl-D-fructose?is a reducing disaccharide naturally present in honey.As an isomer of sucrose,its sweetness is half of sucrose.Turanose is easy to crystallize and dissolve,and it is not easily hydrolyzed and used by caries-causing microorganisms,so it is suitable for people with obesity,hyperlipemia,hypertension,diabetes,etc.In the food industry,it has the potential to replace sucrose as a new functional sweetener and has broad application prospects.Currently,there are only two methods for enzymatic preparation of turanose.Cyclodextrin glucosyltransferase from Bacillus stearothermophilus can efficiently synthesize turanose with using?-cyclodextrin as the best glucosyl donors,and its yield is 45%.However,?-cyclodextrin is expensive and not suitable for industrial production.The other one,amylosucrase?ASase?derived from Neisseria polysaccharea could directly isomerize sucrose to turanose and its yield is 56%.In particular,the yield of turanose can be increased to 73%if fructose is added as the receptor.However,by-products such as malto-oligosaccharides are still formed during the reaction,which affect the final conversion rate.In addition,in order to further increase the possibility of application of turanose in the food field,we need to select a food-grade host to achieve efficient expression.Because CGTase has the potential to produce high value-added products by using inexpensive starch derivatives as raw materials,and the conversion rate of amylosucrase is high,the two enzymatic preparation processes were studied.On the one hand,we used corn starch-derived maltodextrin instead of?-cyclodextrin as the donor.Then the enzymatic conversion conditions for the synthesis of turanose catalyzed by B.stearothermophilus CGTase were optimized.On the other hand,after synthesizing the amylosucrase gene from N.polysaccharea,we mutated the amino acid residue at a single point to reduce the formation of by-products and transformed them into Bacillus subtilis WS11,then carried out protein purification and determined enzymatic properties.On this basis,the natural enzyme was used as the control to investigate the performance of the mutant to catalyze the preparation of turanose.Finally,the shake-flask fermentation conditions of the recombinant strain were optimized and the high-density culture in the 3-L fermentor was investigated.The main results were as follows:?1?Preparation of CGTase by E.coli/pET-20b?+?-cgt^opt constructed in the laboratory.In order to save the cost,with fructose as the receptor,using corn starch-derived maltodextrin instead of?-cyclodextrin as the donor,the enzymatic conversion conditions for the synthesis of turanose were optimized.The results showed that when the maltodextrin dextrose equivalent[DE]was 16-20,the donor and acceptor concentration were both 300 g·L^-1,and the amount of enzyme was 10 U·mL^-1,at 50°C and pH 6.0 for 24 h,then after treatment with glucoamylase the yield of turanose could reach 150 g·L^-1.?2?Optimized the synthesis of amylosucrase gene npas derived from N.polysaccharea and performed site-directed mutagenesis,the glycine?Gly?at position 396 at the+2 subsite was mutated to be serine?Ser?,which increased steric hindrance.The way could interfere with the extension of?-glucan to reduce the production of by-product malto-oligosaccharides.Then the recombinant plasmids pHY300PLK-npas and pHY300PLK-npas G396S were constructed using the food-grade strain B.subtilis as the host cell.The enzyme activities after culturing in shake flask for 20 h were 0.8 U·mL^-1 and 0.6 U·mL^-1,respectively.Protein electrophoresis showed that the target band appeared at about 66.2 kDa.Then the natural and mutant enzymes were purified and the specific activities were determined to be 1.11 U·mg^-1and 0.94 U·mg^-1.Moreover,the enzymatic properties of the two were similar:the optimum temperature was 50°C and the optimum pH was 7.0.The half-life of 30°C was 40 h,and the relative enzyme activity could be maintained above 90%in the range of pH 6.0-8.0.?3?The performance of mutant enzyme NpAS G396S for the preparation of turanose was studied and its enzymatic conversions were optimized.The results showed that with 684 g·L^-1sucrose as substrate,the yield of turanose was 410 g·L^-1 when the amount of enzyme was 0.8U·mL^-1,at 30°C and pH 7.0 for 48 h,which increased 50 g·L^-1 than that of NpAS,and the yield could reach 60%.On this basis,fructose was additionally added as the receptor,and the optimal concentration was reduced from 135 g·L^-1 to 90 g·L^-1 compared with NpAS,not only did the cost of the substrate be saved,but the yield of turanose was increased from 73.3%to76.75%,whose yield was 525 g·L^-1.?4?The shake flask fermentation conditions of the B.subtilis/pHY300PLK-npas G396S were optimized,and the optimal composition of the medium included 10 g·L^-1 XiWang bean powder,15 g·L^-1 XiWang soy peptone,5 g·L^-1 glycerol or glucose,1 mM Mg²⁺,12.54 g·L^-1K₂HPO₄ and 2.31 g·L^-1 KH₂PO₄.The activity of recombinant enzyme reached 1.3 U·mL^-1.Then we explored the expansion of the culture in the 3-L fermentor and found that when using glucose as the carbon source,the ASase produced by the recombinant B.subtilis was mainly concentrated in intracellular part,however extracellular secretion was rare,and significantly degraded in the late fermentation stage.But if using glycerol as the carbon source,the decrease of enzyme activity in the late fermentation period could be effectively inhibited,and the proportion of extracellular secretion of the enzyme increased.Under the conditions of33°C,pH 7.0 and dissolved oxygen 30%,when the fermentation continued for 80 h,the OD₆₀₀ could reach 120,and the total enzyme activity was about 12.5 U·mL^-1 which was about10 times that of the shake flask fermentation.