Cross-linked enzyme lyophilisates (CLELs) of urease: A new method to immobilize ureases

Abstract

In this study, we presented a new approach for immobilizing JBU (Jack bean urease), by producing urease cross-linked enzyme lyophilisates (CLELs). Through the use of bovine serum albumin (BSA), lyophilisation, cross-linking with dextran polyaldehyde (DPA), and optimizing cross-linker pH, the urease-CLELs produced show an increase in relative catalytic activity that is 1.47 times higher than that of free urease, while remaining stable up to temperatures of 85 °C. Urease-CLEL activity increases in direct proportion with the increasing BSA content due to the offered additional lysine (Lys) groups which are potential cross-linking points providing better immobilization and retention of JBU, while lyophilisation also enables stabilization by eliminating solvating water molecules and intra-molecular reactions that may block the cross-linking residues. Two most commonly used cross-linkers that are reacting with the available Lys groups, i.e.glutaraldehyde (GA) and bulkier alternative DPA, have been selected for the immobilization of urease. The catalytic activity increase with DPA suggests an improved access to the active site through hindering blockage, while the increase with alkaline pH of the cross-linkers indicates decreased buffer inhibition. The long lifetime (113% residual activity after 4 weeks), recyclability (132% residual activity after 10 cycles) and thermal stability (276% relative activity at 85 °C) of these urease-CLELs demonstrate that they are technologically attractive as green biocatalysts, while our immobilization approach offers an alternative to conventional methods for proteins that are difficult to immobilise.

Keywords: Cross-linking; Enzyme aggregates; Enzyme immobilization; Lyophilisates; Recyclable catalyst; Urease.