Study On The Synthesis Of Geranyl Propionate In Non-aqueous Phase Using Yarrowia Lipolytica Lipase

As fatty acid esters of the terpene alcohol,geraniol esters are widely used in food,cosmetic and pharmaceutical industries in recent years.Compared with the traditional chemical synthesis,the biosynthesis of geranyl propionate in organic solvents has the advantages of high selectivity,high catalytic efficiency and mild reaction conditions.Up to now,most of the researches working on the synthesis of geranyl propionate are focused on the batch experiments to study the factors influencing the esterification ratio,but working on the continuous synthesis are very few.In this paper,the feasibility of geranyl propionate synthesis using Yarrowia lipolytica Lipase Lip2(Lip2)in non-aqueous phase was first studied,on this basis,the free Lip2 was immobilized in organic solvent and then was used to catalyze the continuous synthesis of geranyl propionate in a Continuous Stirred Tank Reactor(CSTR).By comparing the effects on the catalysis of geranyl propionate synthesis of several lipases in organic solvents,Lip2 from Yarrowia lipolytica was proved to be of good catalytic ability and could be applied in the esterification of short-chain fatty acids.Factors influencing the esterification ratio of geranyl propionate in organic solvents were investigated and the optimal conditions were found to be: enzyme loading 7 mg/mL,isooctane as solvent,acid/ alcohol molar ratio 1:1.5,temperature 35 oC,molecular sieve loading 20 mg/mL,shaking speed 200 r/min.Under these conditions the esterification ratio of 88.6% was obtained after 3.5 h of reaction.The reaction product was separated and identified as geranyl propionate by means of FT-IR,GC-MS and NMR.At the same time,the kinetics of the synthesis of geranyl propionate in organic solvent catalyzed by Lip2 was investigated,the results showed that the reaction followed the Ping-Pong Bi-Bi mechanism with competitive inhibition by both propionic acid and geraniol.To further investigate the value of Lip2 in industrial application,the free Lip2 was immobilized on the macroporous adsorptive resin DA201-C in n-heptane.The optimal conditions were: lipase loading 0.01 g/g resin,25 oC and adsorption for 3 h.Under these conditions the immobilization efficiency could be up to 98.6% and the specific activity of lipase immobilized was 318.5×103 U/g with the activity recovery 128.5%.At the same time,investigation on the properities of immobilized Lip2 indicated that the optimal temperature was 35 oC and its optimal pH was 8.0,indicating that they were kept the same as that of the free enzyme.When the immobilized and free Lip2 were incubated at high temperature for 2 h,the residual activity of immobilized Lip2 was much higher than that of free enzyme,indicating an obvious improvement of the thermal stability.The immobilized Lip2 was used to catalyze the synthesis of geranyl propionate for 12 times and the esterification ratio could be kept over 80%,showing good stability.Finally,the immobilized Lip2 was used to generate the geranyl propionate in CSTR.The optimal conditions were: enzyme loading 3.0 g,feed flow rate 6.0 mL/h(average residence time more than 4 h),molar ratio of acid/alcohol 1:1.5,temperature 35 oC.Under these conditions the esterification ratio could be stabilized at about 72.8% for 12 h.