Efficient Immobilization of Enzymes on Microchannel Surface Through His-Tag and Application for Microreactor

Abstract

We developed a simple immobilisation method for His-tagged enzymes on a microchannel surface. It facilitates immobilisation of protein molecule on microchannel surface through Ni-complex, using crude or purified protein solutions. By this method, we could immobilize proteins on microcapillary constantly. This method might be useful for further development of microreactor with reversibly immobilized enzymes.

Silica-immobilized enzyme reactors; application to cholinesterase-inhibition studies

“…A recent report describing immobilization of enzymes onto a microreactor surface using his-tag attachment was limited to commercially available or highly purified enzymes and resulted in very low enzyme loading [24]. The location of the his-tag on the silica-nucleating peptide rather than on the protein eliminates the need for recombinant modification of the protein of interest in order to use this method.…”

Self‐renaturing enzymes: Design of an enzyme‐chaperone chimera as a new approach to enzyme stabilization

“…Water-miscible organic co-solvents can increase the solubility of nonpolar substrates, suppress hydrolysis reactions, and shift reaction selectivity towards an otherwise unstable product (Castro and Knubovets, 2003;Schmid et al, 2001). One strategy for improving enzyme stability in general is to immobilize the enzyme (Grazu et al, 2005;Miyazaki et al, 2005), which offers the additional advantages of enabling continuous operation of the enzyme reactor and reuse of the enzyme, recycling of substrates, and steady removal of products to alleviate product degradation and inhibition (Bornscheuer, 2003;Polizzi et al, 2007). Immobilization has also been employed to increase enzyme stability in organic co-solvents (Fernandez-Lafuente et al, 2001); however, a common drawback of most immobilization methods is a concomitant reduction in the enzyme's intrinsic activity (Fernandez-Lafuente et al, 2001;Kranz et al, 2007;Wehtje et al, 1993).…”

Tailored adsorption of His6-tagged protein onto nickel(ii)–cyclam grafted mesoporous silica

“…However, the application of biocatalsyts in many industrial applications is restricted as the process of removing the enzyme from the reaction mixture can cause denaturation of the biocatalyst. Immobilized enzymes can display activities superior to that of the free enzyme [1][2][3][4] while remaining stable and exhibiting significant catalytic activity in harsh media such as non-aqueous solvents 5,6 . However, more often than not immobilized enzymes display activities significantly less than free enzyme 7 .…”