Facile preparation of an enzyme-immobilized microreactor using a cross-linking enzyme membrane on a microchannel surface

The enzyme microreactor has considerable potential for use in biotechnological syntheses and analytical studies. Simplifying the procedure of enzyme immobilization in a microreactor is attractive, and it is achievable by utilizing enzyme immobilization techniques and taking advantage of the characteristics of microfluidics. We previously developed a facile and inexpensive preparation method for an enzyme-immobilized microreactor. The immobilization of enzymes can be achieved by the formation of an enzyme-polymeric membrane on the inner wall of the microchannel through cross-linking polymerization in a laminar flow. However, this method is unsuitable for use in conjunction with electronegative enzymes. Therefore, a novel preparation method using poly-L-lysine [poly(Lys)] as a booster and an adjunct for the effective polymerization of electronegative enzymes was developed in this study. Using aminoacylase as a model for an electronegative enzyme, the reaction conditions for the enzyme-cross-linked aggregation were optimized. On the basis of the determined conditions, an acylase-immobilized tubing microreactor was successfully prepared by cross-linking polymerization in a concentric laminar flow. The resulting microreactor showed a higher stability against heat and organic solvents compared to those of the free enzyme. ne developed method using poly(Lys) was applicable to various enzymes with low isoelectric points, suggesting that this microreactor preparation utilizing a cross-linked enzyme in a laminar flow could be expanded to microreactors in which a broad range of functional proteins are employed.