Study On Microfluidic Microreactors With Immobilized Enzyme And Biofilm

Microreactor technology has becoming an important means for the enhancement of biocatalysis and conversion processe,which can significantly increase the enzyme reaction rate and catalytic selectivity.However,low solubility of the substrate,severe product inhibition,and high cost limit their application.Fast inactivation of free enzyme due to the lack of cell micro-environment reduces the catalytic efficiency in microchannel reactors.Therefore,an immobilized enzyme microchannel reactor modified by temperature stimuli-responsive materials was constructed and a catalytic recombinant Escherichia coli biofilm expressing a glycosidase gene thrived in a mircoreactor by segmented flow.The mechanism of ionic liquids/deep eutectic solvents on enhancing the cell membrane permeability was analyzed.Based on synthesis of isoquercitrin from rutin biotransformation,the two constructed bioreactors were studied to strengthen the rule of biocatalytic processes.The main research contents are as follows:?1?Design a temperature-controlled immobilized enzyme reactor,which the surface of the microchannel was modified with photopatterned temperature stimuli-responsive materials.The enzyme amount per unit area reached 0.31 g/cm²and the reaction yield was91.35%with the reaction time of 5 min.The enzyme of reversible release and immobilization on microchannel surface showed at temperature range of 20-25°C and40-45°C.Release efficiency of enzyme immobilized on microchannel surface could reached 93.63%,which indicated that above 90%of the enzyme could be replaced with fresh enzyme.Compared with the conventional reactor,the reaction time was reduced to1/12,and the product of unit time per unit enzyme?g/g/h?increased by 2.1 times.Six cycles of enzymatic hydrolysis reaction were successively completed and relative enzyme activity above 56%was maintained.These results indicated that the affinity between substrate and enzyme immobilized on the microchannel surface by temperature-stimuli-responsive material was enhanced.The stability of enzyme activity was also increased.?2?To explore the mechanism of ionic liquids/deep eutectic solvents on enhancing cells permeability of recombinant Escherichia coli BL21-pET21a-rhaB1.The cells were respectively treated with five types of ionic liquids and deep eutectic solvents in different ways.Among them,6%?v/v?choline chloride/urea?ChCl/U?showed the best biocompatibility and improved cell membrane permeability with isoquercitrin yield 93.0%,increasing by 32.8%.Compared with the catalytic results of crude enzyme solution obtained from cell disruption,the reaction time was reduced by 4/5 and the substrate concentration was increased by 4 times.The catalytic activity was relatively stable at pH 6.5 and reaction temperature of 40°C.Meanwhile,the whole-cell catalyst could be reused five times and the yield remained above 52%.?3?Study the static colonization of recombinant Escherichia coli BL21-pET21a-rhaB1on the microchannels surface modified with silane reagents.The“air/culture medium containing nano-materials”modulated the growth of recombinant strains biofilm by segmental flow in the microchannels.Under this regulation method,rapidly and stably growth of biofilm in the microchannel was achieved.The mature biofilm of stability foramation time was 20 hours,which was 33.3%shorter than the conventional method,although the minimum inhibition rate of graphene nanomaterials on biofilm growth was3.2%.The air/water solution flow rate was 45?L/min,the growth temperature was 32°C,and the ambient pH was 7.0.?4?The glycosyl-directed modification of the natural products with microfluidic recombinant strains biofilm was first proposed.The recombinant Escherichia coli BL21-pET21a-rhaB1 biofilm catalyst was prepared in a microchannel reactor by segmented-flow.The correlation discipline between formation process and catalytic specificity of biofilm was studied.When the flow rate of air?oxygen?increased by 3 times,the biomass of catalyst biofilm increased by 50%,and the yield of isoquercitrin catalyzed by the biofilm catalyst increased by 1.92 times.When the substrate concentration was 0.01 g/L and the reaction temperature was 35°C,the yield of the product was 1.12?g/L_tube/d,indicated that the biofilm had a good morphological stability and catalytic performance as a"living catalyst".Therefore,the microchannel surface-immobilized enzyme reactor modified by temperature stimuli-responsive material and the microfluidic recombinant Escherichia coli BL21-pET21a-rhaB1 biofilm reactor are effective ways to prepare natural products with high activity,low content and complex structures,which showed distinct advantages over the batch reactor.The application greatly improves the reuse of biocatalysts,and provides a new approach for microfluidic biosynthesis of rare natural products.