Jiangsu Key Laboratory of Phylogenomics & Comparative Genomics, School of Life Science, Jiangsu Normal University, Xuzhou 221116, Jiangsu Province, China.
Enzyme Microb Technol. 2023 Jan;162:110118. doi: 10.1016/j.enzmictec.2022.110118. Epub 2022 Aug 31.
Carbohydrate-binding modules (CBMs) are commonly found within chitinases, but their contributions to chitinolytic systems are poorly understood. To address this knowledge gap, full-length chitin-acting enzymes (ChiA, ChiB, ChiC, and CBP21) of Serratia marcescens BWL1001 and CBM-truncated versions (ChiB-dCBM and ChiC-dCBM) were heterologously expressed for enzymological analysis. The CBM5 of ChiB and the CBM12 of ChiC both exhibited an affinity for α-chitin, while only CBM12 could bind colloidal chitin based on adsorption assays and affinity electrophoresis. Consistent with their ligand specificity, both CBMs were essential to α-chitin hydrolysis, while only CBM12 enhanced the hydrolytic efficiency of colloidal chitin by individual chitinases. Analysis of synergistic hydrolysis with separate full-length and CBM-truncated chitinases revealed that the two CBMs promoted the synergistic activity of chitinases on crystalline and amorphous chitin. The two CBMs also promoted the hydrolysis when chitin was mixed with non-substrate polysaccharides. This study reveals not only the effects of CBMs on enzymatic characteristics of individual chitinases, but also their contributions to the overall efficiency of chitinolytic systems during synergistic hydrolysis.
碳水化合物结合模块 (CBMs) 通常存在于几丁质酶中，但它们对几丁质降解系统的贡献尚不清楚。为了解决这一知识空白，本研究异源表达了粘细菌 BWL1001 的全长几丁质作用酶（ChiA、ChiB、ChiC 和 CBP21）及其 CBM 截断变体（ChiB-dCBM 和 ChiC-dCBM），并进行了酶学分析。ChiB 的 CBM5 和 ChiC 的 CBM12 均对 α-几丁质表现出亲和力，而仅 CBM12 可根据吸附测定和亲和电泳结合胶体几丁质。与它们的配体特异性一致，这两个 CBM 对 α-几丁质水解都是必需的，而仅 CBM12 提高了单个几丁质酶对胶体几丁质的水解效率。用单独的全长和 CBM 截断几丁质酶进行协同水解分析表明，这两个 CBM 促进了几丁质酶对结晶和无定形几丁质的协同活性。当几丁质与非底物多糖混合时，这两个 CBM 也促进了水解。本研究不仅揭示了 CBMs 对单个几丁质酶酶学特性的影响，还揭示了它们在协同水解过程中对几丁质降解系统整体效率的贡献。
