Cross-linked laccase aggregates from Coriolopsis polyzona - Optimization of production and partial characterization

Production of cross-linked enzyme aggregates (CLEAs), consisting of aggregation/precipitation (step 1) and covalently cross-linking (step 2) enzyme molecules, has been shown to be a valuable technique for insolubilization of enzymes facilitating their retention in reactor systems. However, to fully exploit the potential of this technique, high conversion yields of free enzymes to CLEAs need to be achieved and implemented in simplified protocols. Although of high interest for biotechnological applications, reported conversion efficiencies for oxidative enzymes, especially laccases, are far away from fulfilling these needs. Thus, relevant factors for the production of CLEAs of laccases from the white-rot fungus Coriolopsis polyzona were identified and optimized by applying Taguchi orthogonal experimental design and response surface methodology. Among other tested factors, concentration of the precipitant polyethyleneglycol (PEG), concentration of the cross-linking agent glutaraldehyde (GLU) and reaction time were identified to be most important. Optimal values for these parameters were 70% (w/w) PEG, 18 mM GLU and 21.5 h of incubation, resulting in a complete conversion of the applied laccase to CLEAs, i.e. without loss of activity. In parallel, methodology for the production of CLEAs was simplified leading to a one-step protocol combining steps 1 and 2 without necessity of a prior purification of the enzyme. The resulting CLEAs were characterized regarding their stability against temperature and pH, proving an increased robustness over free laccase. A uniform size distribution of the CLEAs around 1-2 µm in diameter was shown using light microscopy and environmental scanning electron microscopy. The results will be exploited in future studies aiming at an application of the CLEAs in continuously operated membrane reactors for the biotransformation of micropollutants such as bisphenol A or nonylphenol