Improving the catalytic performance of laccase using a novel continuous-flow microreactor

Abstract

A novel, inexpensive and efficient method for the preparation of laccase-immobilized microreactors has been proposed, based on the formation of an enzyme-polymeric membrane on the inner wall of microtubes (500 μm inner diameter) as a result of the cross-linking polymerization reaction between the laccase and bifunctional cross-linkers agents (paraformaldehyde and glutaraldehyde). Under the optimum conditions, an immobilization yield of 72% and an activity of 45 μM/min, determined using 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as substrate in a continuous-flow assay, were detected. The biochemical characterization of the laccase-immobilized microreactors demonstrated their enhanced characteristics: they exhibited a broad range of optimum pH and temperature and excellent stability under different conditions of pH, temperature, chemical inactivating agent, storage and long-term operation. The laccase-immobilized microreactors were applied for the biotransformation of model compounds to demonstrate their efficiency and performance. Important reaction yields were obtained, even at lower residence times compared with conventional bioreactors. Furthermore, it was designed a two-stage bioreactor for the application of laccase-mediated reactions, preventing the biocatalyst from inactivation. The great performance of the microreactor system, possibly due to the more rapid mass transfer and the larger area to volume ratio, makes the presented technologies excellent platforms for increasing the laccase uses and improving its catalytic action in several fields such as biotransformations or bioanalyses.