Activity, stability and structure of laccase in betaine based natural deep eutectic solvents

Natural deep eutectic solvents (NADES) play a role as alternative media to water in living organisms. They are formed by mixing two or more natural compounds in certain ratios producing a liquid having a lower melting point than those of the individual NADES components. Although, the eutectics medium bring several advantages as enhanced solubility of non-polar substrates and/or products, however, these advantages would often be limited by a lower stability of biocatalysts in these systems. To examine this matter, biochemical characterization, thermal stability and tertiary structure of laccase from Bacillus HR03 was investigated as a model in betaine and choline based NADES. In eutectics containing choline, a sudden drop in enzyme activity and stability was observed. Betaine based eutectics exhibited a better media for the laccase stability in comparison with the aqueous buffer and choline chloride eutectics. The enzyme highest activity was observed in 20% (v/v) glycerol:betaine (2:1). Among betaine based eutectics, the enzyme exhibited its highest stability in sorbitol:betaine:water (1:1:1) and glycerol:betaine (2:1) compared to the aqueous buffer at 80 and 90°C. Associated conformational changes caused by solvents were monitored using fluorescence technique. Finally, the effects of NADES on the enzyme activity and stability were discussed.