| Optically pure secondary alcohols are very essential chiral units.Enzymatic resolution of chiral secondary alcohols can overcome the harsh conditions,high cost,and environmental pollution problems of traditional chemical methods,which has received widespread attention.However,it is restricted by the lack of a matching system and leads to difficulties in separating substrates and products,reusing enzymes,and poor selectivity,which severely restrict its development,and there is an urgent need to develop a new enzymatic catalytic system.In recent years,the three-liquid-phase system(TLPS)has its unique advantages in terms of product separation and improvement of enzyme catalytic efficiency,and it is expected to solve many problems in some current enzyme resolution systems.Subsequently,a three-liquid-phase system suits for enzymatic resolution was constructed in this paper,and the preparation of(R)-1-(4-methoxyphenyl)ethanol by enzymatic resolution was taken as an example to discuss the probability of the system.The feasibility of secondary alcohols,and the micro-mechanism and continuous application of the catalytic reaction were explained and verified.The research content is as follows:(1)We constructed different three-liquid-phase systems and explored the effects of different enzymes,solvents and reaction systems on the efficiency of hydrolysis and resolution of 1-(4-methoxyphenyl)ethanol acetate.The isooctane/sodium sulfate/PEG600system with CALB lipase is the optimal system,Under this system,not only can the product and the substrate be effectively separated,but also 1-(4-methoxyphenyl)ethanol acetate can be resolved by hydrolysis.Compared with the traditional system,its conversion rate can reach49.87%(theoretical conversion rate is only 50%)and ee_p can reach 98.72%.In addition,in this system,the enzyme was mainly concentrated in the middle phase and can be effectively reused.After 8 times of recycling,the catalytic efficiency of the enzyme can still reach more than 95%of the initial reaction efficiency.The enzymatic separation of carbon chain fatty secondary alcohols and aromatic secondary alcohols verified that the system has good versatility.(2)Aiming at the problem of a large amount of hydrophobic solvent in the three-liquid-phase system constructed in the early stage,the direct use of its liquid substrate to construct a new solvent-free three-liquid-phase enzymatic resolution system was discussed.It was found that the substrate/PEG2000/phosphate system was the optimal system.The solvent-free system had better catalytic efficiency and the conversion rate as well as selectivity of its chiral resolution were improved to a certain extent compared with the solvent system.The p H,speed,temperature and other factors of the system were explored to optimize the efficiency of hydrolysis resolution.The optimal conditions were established:200rpm,45℃,16%phosphate,19%PEG2000,p H=8.At the same time,as a new type of liquid immobilized enzyme,the conversion efficiency of the middle phase was still 91.67%of the first time after being reused8 times.(3)Aiming at the phenomenon that the catalytic efficiency of enzymes in the three-liquid-phase system has been significantly improved,the microstructure and particle size of the three-liquid-phase system composed of solvent-free and solvent systems were analyzed by Laser Confocal Microscope and Malvern particle size analyzer.It was found that the solvent-free dispersed emulsion droplets had a smaller particle size.Furthermore,the reason for the improvement of the reaction was explained through the phase transfer catalysis mechanism,and the continuous process application of the multi-liquid-phase system was finally realized.A relatively long-lasting bio-transformation of the catalytic reaction was obtained,that is,the reaction could be maintained for more than 48 hours. |
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