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Understanding the structural basis for thermostable lichenin-degrading enzyme is important for β-1,3-1,4-mixed linkage glucan degradation and industrial application.A novel lichenase F32EG5 belonging to the glycoside hydrolase (GH) family 5 was identified from an extremely thermophilic bacterium Caldicellulosiruptor sp.F32.Biochemical analysis indicated that F32EG5 is a non-metalloprotein lichenase with the optimal temperature and pH at 80℃ and 5.5, respectively, and a half-life at 80℃ of 8 h.It exhibited a specific and high activity on β-1,3-1,4-glucan.Thin-layer chromatography and NMR analyses further indicated that F32EG5 hydrolysed the β-1,3 linkage after a β-1,4 linkage, as well as the β-1,4 linkage before a β-1,3 linkage, which is different from the extensively studied GH family 16 lichenase with a cleavage site on the β-1,4 linkage after a β-1,3 linkage.The crystal structure of F32EG5 was determined to 2.8 (A) resolution, which showed a typical (β/α)8-barrel fold of the GH family 5 proteins.Analysis of the structure revealed that the exit subsites of substrate binding sites contribute to thermostability and substrate specificity of F32EG5.The catalytic mechanism and substrate selectivity of F32EG5 was further verified by site-directed mutagenesis and molecular docking of different glucotetraoses.F32EG5 is a novel type of lichenase with a distinct cleavage pattern for β-1,3-1,4-mixed linkage glucan and shows potential in industrial processes, such as the brewing and feed industries.