Flow microreactors are emergent engineering tools for the development of continuous biocatalytic transformations. Exploiting enzymes in continuous mode requires their retention for multiple rounds of conversions. To achieve this goal, immobilizing the enzymes on microchannel walls is a promising approach. However, protein immobilization within closed structures is difficult. Here, we describe a methodology based on the confluent design of enzyme and microreactor; fusion to the silica-binding module Zbasic2 is used to engineer enzymes for high-affinity-oriented attachment to the plain wall surface of glass microchannels. As a practical case, the methodology is described using a sucrose phosphorylase; the assayed reaction is synthesis of α-D-glucose 1-phosphate (αGlc 1-P) from sucrose and phosphate using the immobilized enzyme microreactor. Procedures of enzyme immobilization, reactor characterization, and operation are described. The methodology is applicable for any other enzymes fused to Zbasic2 and silica (glass)-based microfluidic reactors.
Keywords: Flow biocatalysis; Immobilization; Microreactor; Sucrose phosphorylase; Zbasic2.