The Cold-Active Endo-β-1,3(4)-Glucanase from a Marine Psychrophilic Yeast, Glaciozyma antarctica PI12: Heterologous Expression, Biochemical Characterisation, and Molecular Modeling

Glaciozyma antarctica is a psychrophilic yeast that was isolated from the surface of Antarctic sea ice. A key adaptation of psychrophilic microorganisms is to synthesize cold-active enzymes for survival at low temperatures. A full-length cDNA encoding β-glucanase (GaEgl) from G. antarctica PI12 was amplified by reverse-transcription polymerase chain reaction (RT-PCR). The cDNA encoded a 394-residue polypeptide with a putative signal peptide of 22 residues. Subsequently, the novel GaEgl was expressed in E. coli and purified with nickel affinity chromatography as an approximately 44 kDa protein. The biochemical characterisation of purified recombinant GaEgl (rGaEgl) revealed typical cold-active enzyme characteristics, such as maximal activity at 20 °C and pH 7.0. However, the enzyme was still active at 5-15 °C and alkaline pH values of 8-10. The activity of recombinant GaEgl was enhanced in the presence of Co2+ and Mn2+ metal ions. The Km and Vmax values of the enzyme using lichenan as the substrate were 8.87 mg mL-1 and 37.45 U mg-1, respectively. The enzymatic hydrolysis analysis of laminarin using HPLC showed that the main hydrolysis products were monosaccharides, disaccharides and trisaccharides. An analysis of the three-dimensional structure of the enzyme was carried out and compared with homologous mesophilic endo-β-1,3(4)-glucanase. The results of the comparative structural study revealed that the psychrophilic GaEgl contains longer loops, fewer hydrogen bonds and salt bridges, and a higher total solvent-accessible surface area which enhanced the protein flexibility for high catalytic efficiency at low temperatures.