新型甘露聚糖酶-乙酰酯酶双功能酶的功能和结构域协同研究

目的] 分析鉴定高效木质纤维素降解菌群EMSD5来源的新型甘露聚糖酶-乙酰酯酶双功能酶44884，解析催化域间的协同关系，以及碳水化合物结合模块（CBM）对催化域特性的影响，拓展对该类双功能酶的认识，为甘露聚糖酶的升级改造和应用提供依据。方法] 通过大肠杆菌异源表达甘露聚糖酶-乙酰酯酶双功能酶44884，并构建截短和定点突变的突变体，利用TLC和DNS法比较野生型和突变体的酶学性质。结果] 成功对44884全长和突变蛋白进行克隆表达，并发现44884中2个催化域能够彼此促进各自产物的释放，而且以双功能酶形式存在时，这种促进效果更为明显。44884中的2个CBM65均有甘露聚糖和结晶纤维素结合活性，且CBM65的存在并不改变甘露聚糖酶和乙酰酯酶的最适反应条件和水解模式。虽然CBM65显著降低了2个催化域的热稳定性，但水解天然底物时，2个CBM65对各自临近催化域的水解具有明显的促进效果。结论] 本研究首次发现并探究了新型甘露聚糖酶和乙酰酯酶形成的双功能酶44884的功能，解析了催化域之间高效的协同效应，以及新型甘露聚糖结合模块CBM65对双功能酶水解的促进作用。

Functional characterization of a novel bifunctional mannanase-acetyl esterase focusing on the synergistic mechanism between different domain

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.