Abstract

Mannanases catalyze the cleavage of β-1,4-mannosidic linkages in mannans and have various applications in different biotechnological industries. In this study, a new β-mannanase from Verrucomicrobiae DG1235 (ManDG1235) was biochemically characterized and its enzymatic properties were revealed. Amino acid alignment indicated that ManDG1235 belonged to glycoside hydrolase family 26 and shared a low amino acid sequence identity to reported β-mannanases (up to 50% for CjMan26C from Cellvibrio japonicus). ManDG1235 was expressed in Escherichia coli. Purified ManDG1235 (rManDG1235) exhibited the typical properties of cold-active enzymes, including high activity at low temperature (optimal at 20 °C) and thermal instability. The maximum activity of rManDG1235 was achieved at pH 8, suggesting that it is a mildly alkaline β-mannanase. rManDG1235 was able to hydrolyze a variety of mannan substrates and was active toward certain types of glucans. A structural model that was built by homology modeling suggested that ManDG1235 had four mannose-binding subsites which were symmetrically arranged in the active-site cleft. A long loop linking β2 and α2 as in CjMan26C creates a steric border in the glycone region of active-site cleft which probably leads to the exo-acting feature of ManDG1235, for specifically cleaving mannobiose from the non-reducing end of the substrate.

摘要

甘露聚糖酶催化甘露聚糖中β-1，4-甘露糖苷键的裂解，并且在不同的生物技术产业中具有多种应用。在这项研究中，对Verrucomicrobiae DG1235（ManDG1235）的一种新的β-甘露聚糖酶进行了生物化学表征，并揭示了其酶学性质。氨基酸比对表明ManDG1235属于糖苷水解酶家族26，与报道的β-甘露聚糖酶具有较低的氨基酸序列同一性（来自日本Cellvibrio japonicus的Cj Man26C高达50％）。ManDG1235在大肠杆菌中表达。纯化的ManDG1235（rManDG1235）具有冷活性酶的典型特性，包括低温下的高活性（在20°C下最佳）和热不稳定性。rManDG1235的最大活性在pH 8时达到，表明它是一种弱碱性的β-甘露聚糖酶。rManDG1235能够水解多种甘露聚糖底物，并且对某些类型的葡聚糖具有活性。通过同源性建模建立的结构模型表明，ManDG1235具有四个甘露糖结合亚位点，对称分布在活动位点裂口中。像Cj一样连接β2和α2的长环Man26C在活动部位裂口的糖基区域中创建一个空间边界，这可能导致ManDG1235的外显作用特征，用于从底物的非还原末端特异性切割甘露二糖。