Enzymatic Reaction And Enzymatic Properties In Deep Eutectic Solvents

The non-aqueous phase enzyme catalyzed reaction has the advantages of mild reaction conditions,no environmental pollution,high rate,high selectivity,etc.,but the traditional organic solvent reduces the activity and stability of the enzyme,or hinders the dissolution of the substrate,thus limiting the enzyme in Industrial applications.In recent years,the use of eutectic solvents(Deep Eutectic Solvents,DESs)instead of traditional organic solvents as an enzymatic reaction medium has attracted more and more attention.In this paper,a variety of DESs were used as reaction medium to synthesize benzyl cinnamate by lipase-catalyzed reaction of cinnamic acid with benzyl alcohol,and the process conditions for the synthesis of benzyl cinnamate catalyzed by enzyme were optimized by ultrasound.First,urea,glycerol,ethylene glycol and xylitol are used as hydrogen bond donors,and choline chloride and betaine are used as hydrogen bond acceptors,and chlorination is carried out by magnetic stirring in a water bath according to a certain molar ratio.Various DESs such as choline-urea,choline chloride-glycerol,choline chloride-glycol,betaine-glycerol,betaine-urea and betaine-xylitol.Lipozyme TLIM and Novozym 435 were used as catalysts to synthesize cinnamic acid and benzyl alcohol to synthesize benzyl cinnamate in different DESs.The results were quantitatively analyzed by high performance liquid chromatography.The results showed that the yield of benzyl cinnamate was higher when Lipozyme TLIM enzyme was used as the catalyst and betaine/xylitol(molar ratio 1:2)was used as the reaction medium.Furthermore,the effects of temperature,water content,substrate molar ratio and enzyme loading on the reaction were investigated.The results show that the appropriate water content in the DESs reaction medium can promote the esterification reaction.This may be because cinnamic acid and benzyl alcohol form hydrogen bonds with the DES component,which affects the enzymatic esterification reaction,while the proper amount of water is both a hydrogen bond donor and a hydrogen bond acceptor.The cinnamic acid and benzyl alcohol are released from the hydrogen bond network to promote the esterification reaction.In addition,the effect of water on the reaction is also reflected in the fact that compared with traditional organic solvents,DESs can also ?lock? the water molecules formed during the esterification process,thereby affecting the water activity and enzyme activity of the system.The optimal reaction conditions were obtained by optimization.When the molar ratio of acid to alcohol was 1:3,the reaction temperature was 40 °C,the water content was 12.32%,the enzyme loading was 30 mg/mL,and the reaction was carried out for 22 h,lipase catalyzed the synthesis of benzyl cinnamate.The conversion rate can reach 30.9%.Secondly,the betaine/glycerol(molar ratio 1:2)was selected as the reaction medium,and the benzyl cinnamate was synthesized by ultrasonic-assisted lipase Lipozyme TLIM.The effect of ultrasonic intensity on the reaction was studied.Studies have shown that ultrasonic assist not only shortens the reaction time from 16 h to 5 h,but also increases the yield of benzyl cinnamate by 2.5 times compared with the shaker reaction mode,which promotes the esterification reaction.Going on.Furthermore,the effects of water content,temperature,substrate molar ratio,and enzyme loading on the yield of benzyl cinnamate under ultrasound-assisted conditions were investigated.Finally,the experiment also studied the reusability of lipase Lipozyme TLIM under ultrasonic conditions.The results showed that the catalytic activity of the enzyme decreased only 16% after repeated use at 45 °C for 4 times,indicating lipase Lipozyme in DESs.TLIM is reproducible under ultrasound-assisted conditions.