Facile Preparation of an Enzyme‐Immobilized Microreactor using a Cross‐Linking Enzyme Membrane on a Microchannel Surface

Abstract

Abstract: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 [polyA C H T U N G T R E N N U N G (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 enzymecross-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. The developed method using polyA C H T U N G T R E N N U N G (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.

Simple and Rapid Proteomic Analysis by Protease-Immobilized Microreactors

“…The solutions were introduced to the PTFE microtube from gas tight syringes by syringe pumps. The combination of GA (0.25%, v/v) and paraformaldehyde (PA) (4%, v/v) provided better cross-linking yields between the proteases maintaining their activities (Honda et al, 2005;Honda et al, 2006). Because the pI value of CT (8.6) is close to the pH value of the reaction buffer (8.0), this probably leads to low polymerization yield.…”

“…These results indicate that the stability of proteases was improved after formation of enzyme-polymerization. This enhancement in the efficiency of activity of the immobilized protease compared to free protease can be ascribed to minimization or elimination of the auto-digestion of proteases (Dulay et al, 2005;Shui et al, 2006) and possible stabilization of the structure of proteases by cross-linking, thus resulting www.intechopen.com in higher accessibility of the substrate to the catalytic-site of the enzyme (Honda et al, 2005;Honda et al, 2006;Dulay et al, 2005;Shui et al, 2006). The operational stability of the protease-immobilized microreactors was also tested based on the digestions of cytochrome c (Cyt-C) for TY-microreactor and β-casein for CT-microreactor (Yamaguchi et al, 2010a).…”

“…www.intechopen.com (Honda et al, 2006;Yamaguchi et al, 2009). The cross-linker solution was supplied to the substrate PTFE microtube through a silica capillary, corresponding to a central stream in the concentric laminar flow.…”

“…A microfluidics-based enzyme-polymerization technique (Honda et al, 2005;Honda et al, 2006) was used for the preparation of protease-immobilized microreactors. The procedure for immobilizing protease on the internal surface of the PTFE microtube by forming an enzyme polymeric membrane through a cross-linking reaction between Lys residues on the protein surface are presented in Fig.…”

“…In addition, reusability is also an important feature required for laboratory use. We developed the procedure for immobilizing enzymes on the internal surface of the polytetrafluoroethylene (PTFE) microtube by forming an enzyme polymeric membrane through a cross-linking reaction in a laminar flow between lysine (Lys) residues on the protein surfaces (Honda et al, 2005) or between the mixture of proteins with isoelectric point pI < 7.0 and poly-Lys (Honda et al, 2006). A typical sample preparation for proteolysis involves multi-steps (denaturation, reduction and alkylation) that are expected to produce enhancement of digestion efficiency.…”