Process Optimization Forenzymatic Synthesis Of Salidroside Based On Kinetic-controlstrategy

Based on the strategy of kinetic control,we optimized the biosynthesis of salidroside through enzymatic catalysis.We started with the basic researches towards the reaction mechanism and kinetic parameters,then followed by the optimization of the semi-continuous process,as well as the continuous process with a reaction coupling separation,and the design of the reaction route and reactor,finally we optimize the various operating conditions and parameters.Accordingly,the best methods to synthesis of salidroside have been achieved for such a particular reaction system.Under the basic researches of kinetiCs,we found Km of M-CLGAs is 55.469 mmol/L,and Vmax is 3.977 μ mol/(min-mL).Reaction order of tyrosol is 0.47.When the concentration of glucose was 0.5 mol/L,the concentration of tyrosol was more than 0.9 mol/L,the substrate inhibition could be observed markedly.The enzyme dose is proportional to the reaction rate.When tyrosol concentration was 1.5 mol/L,glucose concentration was more than 0.6 mol/L,the substrate inhibition also could be seen obviously.When the cumulative amount of salidroside was less than 30 mmol/L,there was no negative effect found on the reaction rate.But with the increasing concentration of salidroside,for example at more than 45 mmol/L,the product feedback inhibition occurred.To determine whether the by-product disaccharides generated or not,we used the methods of thin layer chromatography and evaporative light-scattering detector for the investigation..As for the results of the optimization of semi-continuous process,we found the approach that tyrosol pre-adding and glucose constantly-adding could effectively reduce the by-product,which may be the most important thing for this method in effectively using of all substrates,and maximally improving the Conversion ratio and yield.When the rate of adding glucose was 0.01 mol/L,the maximum yield could reach 12.515%at 72 h.According to this way,the concentration of glucose was fixed at 1 mol/L for the optimization of the concentration of tyrosol.When the concentration of tyrosol reached 0.5 mol/L,the yield of salidroside could be the highest at 14.46%.We established the dynamic equation which described as h=vR/vs=K1/K2=nAα12nBβ12nRγ1V-α12-β12nAα12nBβ12nγ1V-α12-β12 and then inferred the formula of reaction selectivity to reason about the effects of the feeding ways,amounts and rates on the reaction rate.Through the screening experiments,DA-201 was found to be the best macroporous resin for the separation of product from the reaction medium.It not only has a good specific salidroside adsorption capacity and higher desorption,but also has a good selectivity in the mixed system.Based on it,we designed and constructed the route of the reaction system which was in line with the continuous operation of coupling the synthesis and separation,as well as building the reactor system,following by the optimization of the various operation parameters.We defined K value as the amount of resin was added in the system.K value was optimized at 0.091 g/mL,it means 1 mL of medium needs 0.091 g resin.Moreover,the resin needs to be replaced every 24 h.The flow rate of medium in the separator is 0.30 mL/min,the adding rate is 0.25 mL/min.The flow rate of r2 is 0.05 mL/min,and the flow rate of r1 is 0.25 mL/min.The reactor needs to add 150 mmol/L tyrosol,50 mmol/L glucose first,and then the feeding substrate concentration should be 450 mmol/L tyrosol,150 mmol/L.Under this strategy the optimized yield of salidroside reached up to 40.67%.