Structural basis for the cold activation and adaptation of an α-agarase from marine bacterium Catenovulum agarivorans STB13

Abstract

Enzymes have high activity at low temperatures are defined as cold-adapted enzymes, but few enzymes show an activation effect after low-temperature incubation. In this study, we cloned an agarase from the marine bacterium Catenovulum agarivorans STB13 (Ca-AGA) and expressed the protein in Bacillus subtilis. The enzyme exhibited a peculiar cold activation, which was enhanced by 5-fold after incubation at −20 °C or 4 °C for 6 h. Fluorescence and CD spectra studies confirmed the secondary and tertiary structural changes in Ca-AGA at low temperature, which formed compact protein structures after low-temperature treatment. The enzyme also showed outstanding cold adaptation, retaining approximately 60% of its maximum activity at 10 °C. Based on a bioinformatics analysis, the carbohydrate-binding modules (CBMs) and long linker region were presumed to play a crucial role in cold adaptation due to a relatively high percentage of acidic residues and a negative charge density compared to the catalytic domain. A mutation that truncated the linker-CBM region resulted in a 59.97% reduction of the activity at 10 °C. This study describes an α-agarase of cold activation and adaptation in detail, which may have great potential for producing functional agaro-oligosaccharides in food and pharmaceutical industrial applications at ambient temperature.