Droplet-Based Biomimetic Cell Microreactor With Aqueous Two-Phase Systems For Continuous-Flow Catalysis

Whole cell catalysis has drawbacks including low reaction rate,generating by-products and low reuse ability of cells.To address these drawbacks,droplet is proposed as an artificial microreactor to simulate the compartmentalized cells for processing catalytic reactions.These artificial droplet microreactors avoid the generation of by-products and purification process of products.In addition,using these droplet microreactors,product can be separated while enzymes can be remained inside the droplets for the next catalytic cycle.However,the current artificial droplet microreactors lead to the rapid loss of enzymes,resulting in poor reusability and low reaction rate of the droplet microreactors.To tackle the issue,we proposed a PEGylated enzyme-based W/W/W droplet microreactors,which is with a high reaction rate and a high enzyme containing ability,benefiting from the inherit two-layer interfaces of the proposed microreactor.The PEGylated enzyme-based W/W/W droplet microreactor can potentially be applied to the microfluidic biocatalysis and synthesis of high value pharmaceutical intermediates.To construct the proposed microreactor,PEGylated enzymes mixed with Dextran(Dex)was used as the inner phase,while PEG was used as outer phase to generate droplets in the glass capillary microfluidic device.Then the droplets were collected and transferred to a home-made circulation device for cycling the droplets for biocatalysis.Using the proposed microreactor and the circulation device,the droplet microreactors can be reused without the traditional emulsion formation,demulsification and re-emulsion formation process,enabling the droplet biocatalysis to be simple,green and efficient.To demonstrate the continuous-flow catalysis ability of the proposed droplet microreactor,urease was PEGylated can used for constructing the microreactor.As a result,90% of urease activity can be retained after 60 cycles,indicating the high reusability of the microreactor due to the two-layer w/w interfaces which can retain the enzymes inside the droplet and avoid the enzyme leakage.In addition,reaction rate of the droplet microreactors was 5.8 times that of the batch reaction,indicating the high reaction rates of the proposed microreactor benefit from local enrichment of enzymes in droplet.Consequently,the proposed droplet microreactor provides a general solution for enhancing the reusability and reaction rate of the continuous flow catalysis.To demonstrate the cascade biocatalytic ability of the proposed droplet microreactor,the glucose oxidase(GOx)and the horseradish peroxidase(HRP)were PEGylated and self-assembly into a conjugate for constructing the droplet microreactors.As a result,the reaction rate of the proposed droplet microreactor was 5.7 times higher than that of the unconjugated enzymes due to the enzyme conjugates.As a result,benefit from this metabolic channel,the proposed droplet microreactors also provide a general solution to the construction of cascadic biocatalysis with a high enzymic activity.