Synergistic Mechanism Of Microfluidic Biocatalysis Technology Used In The Enzymatic Synthesis Of Natural Products

Nowadays, microreactors have shown promise as a novel method in biocatalysis. Thesehave some advantages such as rapid heat and mass transfer rates, higher yield, safelyoperation and easily enlarge, which can realizes highly efficient and rapid enzymaticsynthesis. However, no report has been published concerning microfluidic biocatalysistechnology used in enzymatic synthesis of natural product ingredients. Thus, based onenzymatic synthesis of isoquercitrin and cafffeic acid phenethyl ester (CAPE) which areboth rare natural product ingredients, microfluidic biocatalysis technology and processmechanism of continuous flow and segmented flow patterns in microreactors wereinvestigated, respectively. It has important significance for high-efficient production ofhigh-value natural product ingredient. The concrete results were as following:A novel continuous flow biosynthesis of isoquercitrin using thehesperidinase-catalyzed selective hydrolysis of rutin in a microchannel reactor wasestablishsed. The effects of channel length, hesperidinase concentration, rutin concentrationand temperature on isoquercitrin yield were studied. The maximum isoquercitrin yield(98.6%) was achieved under the optimum conditions as follows: rutin concentration of1g/L,hesperidinase concentration of0.1g/mL, reaction temperature of40°C, and flow rate of2μL/min. The activation energy value (Ea) of the enzymatic reaction was4.61kJ/mol.Compared with a batch reactor, the reaction time was shorten from10h to40min, and thereaction rate was increased by16.1-fold.Based on a new system of reaction coupling with separation, a novel segmented flowcontaining “buffer-ionic liquid/solvent” in a multi-channel microreactor was applied tosynthesize isoquercitrin by the hesperidinase-catalyzed selective hydrolysis of rutin. Usingthe developed segmented flow microreactor, the process conditions were optimized by thesingle-factor experiments. The optimal conditions were obtained as followed: flow rate of4μL/min, rutin concentration of2g/L, hesperidinase concentration of1g/L and reactiontemperature of30°C, and the maximum yield of isoquercitrin reached100%. Comparedwith a batch reactor, the reaction time was shorten from1h to20min, and the reaction ratewas increased by3-fold. This is due to the two phase system of“buffer-[Bmim][BF4]/triacetin” forming a slug flow in microchannel, which significantlyincreased the mass transfer. The volume production in a multi-channel microreactor was0.439g/min, which was almost5.7-fold better as compared to a single channel microreactor (0.076g/min).Based on transesterification strategy, a novel continuous flow biosynthesis of CAPEfrom alkyl caffeate and phenethanol using a packed bed microreactor was successfullyestablishsed. Among the tested alkyl caffeates and lipases, methyl caffeate and Novozym435, respectively, were selected as the suitable substrate and biocatalyst in conventionalreactor. Process conditions of synthesis of CAPE in a continuous flow packed bedmicroreactor were investigated. Response surface methodology (RSM) was successfullyused to determine the optimal conditions for CAPE synthesis. A maximum CAPE yield of93.21%was achieved in2.5h under the optimized conditions as follows: molar ratio ofmethyl caffeate to phenethanol of1:20, reaction temperature of60°C, flow rate of2μL/minand methyl caffeate concentration3g/mL. Compared with a batch reactor, the CAPE yieldwas increased by26.8%, while the reaction time was reduced by0.1-fold.Density functional theory (DFT) and reaction kinetic model were used to explore theprocess mechanism of enzymatic synthesis of CAPE. The results shown that methyl caffeatehad relatively longer bond length and higher energy than other alkyl caffeate, so it was theoptimal substrate for transesterification. The kinetic of enzymatic transesterificationsynthesis of CAPE in a continuous flow packed bed microreactor was described byLilly–Hornby model. At2μL/min, the kinetic constant km’ was14.04mM. The kinetic ofenzymatic esterification and transesterification synthesis of CAPE in a batch reactor wasdescribed by Ping-Pong Bi-Bi model with inhibition by phenethanol.Therefore, microfluidic biocatalysis technology used in the enzymatic synthesis ofnatural product ingredients has distinct advantages over the batch reactor. Syntheticefficiency and volumetric productivity have risen greatly and it is easy improve productivityby numbering up approach with parallel microreactors. In the furture, the application ofmicrofluidic biocatalysis technology offers broad prospects for high-efficient productinghigh-value natural product ingredients.