Enzymatic Synthesis Of Phenyl Ethyl Acetate In Aqueous Using The Immobilized Acyltransferase

Phenylethyl acetate is a high-value natural volatile ester with a smell similar to rose fragrance.It is widely used in medicines,cosmetics,soaps,food and beverages to enhance aroma or flavor.The development of a green synthesis process of phenethyl acetate is of great significance to enhance product value and expand product application fields.Traditional lipase or esterase catalyzes the synthesis of phenethyl acetate,and various organic solvents such as tert-butanol and n-hexane are often used as media.The organic solvent introduced into the reaction system reduces the operational stability of the enzyme protein;it adds a certain degree of difficulty to the separation and purification of subsequent synthetic products.At the same time,the treatment of the organic waste liquid generated will also increase the production cost of the enterprise.Mycobacterium smegmatis acyl transferase（MsAcT）is a multifunctional biocatalyst capable of catalyzing hydrolysis,transesterification and perhydrolysis.Because of its highly hydrophobic active center,it can catalyze transesterification reactions in aqueous solutions.This research aims to explore the development and use of MsAcT as a catalyst,water as a solvent,and immobilization technology to increase the ratio of[k_cat/K_m]_{Transesterification}/[k_cat/K_m]_Hydrolysiswhen enzyme protein catalyzes transesterification in an aqueous solution.,And use the immobilized MsAcT to catalyze the synthesis of phenethyl acetate.（1）MsAcT is immobilized by sol-gel.The kinetic parameters of free state recombinant MsAcT and fixed state recombinant MsAcT catalyzed hydrolysis reaction and transesterification reaction were measured and compared.The influence of microenvironment on enzyme molecules is also explored.The experimental results showed that the K_m of recombinant MsAcT to hydrolyze p NPA was 12.6μmol·L^-1,V_maxwas 386.9μmol·mg^-1·min^-1,and k_cat/K_m was 770.12 L/mmol·min;while the immobilized MsAcT enzyme hydrolyzed p NPA The scientific constant K_m is 136.7μmol·L^-1,V_max is284.3μmol·mg^-1·min^-1,and k_cat/K_m is 52.16 L/mmol·min.The K_m for the synthesis of phenethyl acetate catalyzed by recombinant MsAcT was 74.3 mmol·L^-1,V_max was 0.42mmol·mg^-1·min^-1,and k_cat/K_m was 0.14 L/mmol·min;while immobilized MsAcT catalyzed benzene acetate The K_m of the ethyl ester synthesis reaction is 64.4 mmol·L^-1,V_max is 0.16 mmol·mg^-1·min^-1,and k_cat/K_m is 0.06 L/mmol·min.Immobilized MsAcT[k_cat/K_m]_{Transesterification}/[k_cat/K_m]_Hydrolysis is 1.15×10^-3;recombinant MsAcT[k_cat/K_m]_{Transesterification}/[k_cat/K_m]_Hydrolysis is 1.82×10^-4;The immobilized MsAcT[k_cat/K_m]_{Transesterification}/[k_cat/K_m]_Hydrolysis is 6.33 times that of recombinant MsAcT.（2）The immobilization process of recombinant MsAcT is optimized.Also,the influence of different R values（water to silane ratio）and additives on the immobilization effect are explored.It reaches the maximum when R=14,the specific activity of immobilized MsAcT is 300.38±10.12 U/g,the activity recovery rate is 63.12±2.13%,the relative activity is 65.85±2.22%,and the conversion of immobilized MsAcT catalyzed synthesis of phenethyl acetate 89.86±0.01%.（3）The catalyzed synthesis of phenethyl acetate by immobilized MsAcT in the aqueous solution is optimized.The effects of different concentrations,different acyl donors,and different water content on the conversion rate were explored,and the time kinetic curve was determined.The optimal synthesis conditions are as follows:the molar ratio of vinyl acetate to phenethyl alcohol is 2:1,and the water content is 80%（w/w）,the maximum conversion rate of 95.37±0.51%is reached within 0.5 h at 40℃.