Abstract:[Objective] A novel bifunctional mannanase-acetyl esterase 44884 from lignocellulose-degraded consortium EMSD5 was investigated, including its interdomain synergism. The effect of carbohydrate-binding module (CBM) on the properties of catalytic domain was also studied, to provide references for improving and applying bifunctional enzymes. [Methods] Mannanase-acetyl esterase 44884 and its truncated mutants, as well as site-directed mutant were expressed in E. coli. The differences in the enzymatic properties of the wild type strain and mutants were evaluated by thin-layer chromatography (TLC) and dinitrosalicylic acid (DNS) assay. [Results] Mannanase-acetyl esterase 44884 and its mutants were successfully overexpressed in E. coli. The mannanase domain and acetyl esterase domain showed synergistic effect, and mannanase-acetyl esterase 44884 showed higher production of reducing sugars and acetic acid than the combination of mannanase domain and acetyl esterase domain. Two CBM65 from mannanase-acetyl esterase 44884 could bind mannan and Avicel, and not change the optimal conditions of catalytic domains. Although two CBM65 significantly decreased the thermostability of catalytic domain, they specifically improved the natural substrate hydrolysis of their adjacent catalytic domains.[Conclusion] The synergistic action between different domain of mannanase-acetyl esterase 44884 was illustrated, in which two CBM65 could improve the mannan hydrolysis of mannanase-acetyl esterase 44884 by binding to substrate.
