[Objective] Three β-glucan degrading glycoside hydrolases from an extreme thermophilic anaerobic bacterium Caldicellulosiruptor sp. F32 were investigated, including the synergistic effect. The effect of glycosylation on β-glucanase F32EG5 thermostability was also studied.[Methods] Two β-glucanases (F32EG5, Lam16A-GH) and β-glucosidase (BlgA) were heterologously expressed in E. coli. The synergistic effect of all these enzymes on β-glucan degradation was evaluated by 3,5-Dinitrosalicylic acid (DNS) and Thin-layer chromatography (TLC) assays including substrate tolerant abilities. Furthermore, the glycosylated p-F32EG5 was expressed in Pichia pastoris, and compared with F32EG5 from E. coli.[Results] F32EG5 and Lam16A-GH released oligosaccharides with different degrees of polymerization (DP) after hydrolyzing β-glucan. The proportion of low-DP oligosaccharides was increased, when two enzymes used together. BlgA showed excellent synergistic effect with F32EG5 and Lam16A-GH, respectively. Although the glycosylated p-F32EG5 from Pichia pastoris did not obviously change its optimum pH and temperature when compared with E. coli-expressed F32EG5, both thermal stability and catalytic efficiency were found two-folds higher than those of E. coli-expressed F32EG5 at the extreme-high temperature (80-90℃).[Conclusion] F32EG5 and Lam16A-GH showed excellent synergistic effect and substrate tolerant abilities with BlgA. The heterologous glycosylation by Pichia pastoris could improve the thermal stability of F32EG5 under extreme thermophilic environment, which was a benefit during the granulation process of enzyme.