Bacterial chitinases recruited multiple accessory domains for the conversion of recalcitrant polysaccharides to simple soluble sugars/amino sugars. Here, we report detailed properties of a multi-domain GH18 chitinase from Enterobacter cloacae subsp. cloacae (EcChi2) that preferred β-chitin as substrate. EcChi2 exhibited transglycosylation (TG) activity on oligomeric substrates from DP4-DP6. The high amount of DP2 is indicative of exo mode activity of EcChi2. We generated EcChi2 variants (truncated and fusion chimeras) and elucidated the role of catalytic and accessory domains. The catalytic efficiency of truncated GH18 and fusion chimera of GH18+ChBD1-ChBD2 decreased to 22 and 17-fold, respectively, than EcChi2, and lost the hydrolytic activity on polymeric substrates, except colloidal chitin. On the other hand, the catalytic activity of truncated PKD1-GH18-PKD2 on polymeric and oligomeric substrates was similar to EcChi2, suggesting that PKD domains are essential for increasing the rate of hydrolysis. Moreover, the truncated ChBD1-ChBD2 and fusion PKD1 + PKD2 participated in chitin-binding.

中文翻译：

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来自阴沟肠杆菌亚种的多结构域转糖基化几丁质酶的催化效率。泄殖腔 (EcChi2) 受多囊肾病域的影响

细菌几丁质酶招募了多个辅助结构域，用于将顽固的多糖转化为简单的可溶性糖/氨基糖。在这里，我们报告了来自阴沟肠杆菌亚种的多域 GH18 几丁质酶的详细特性。首选 β-几丁质作为底物的阴沟（EcChi2）。EcChi2 对来自 DP4-DP6 的寡聚底物表现出转糖基化 (TG) 活性。大量的 DP2 表明 EcChi2 的外模式活动。我们生成了 EcChi2 变体（截断和融合嵌合体）并阐明了催化域和辅助域的作用。截短的 GH18 和 GH18+ChBD1-ChBD2 的融合嵌合体的催化效率分别比 EcChi2 降低了 22 倍和 17 倍，并且失去了对除胶体几丁质外的聚合物底物的水解活性。另一方面，截短的 PKD1-GH18-PKD2 对聚合和寡聚底物的催化活性与 EcChi2 相似，表明 PKD 结构域对于提高水解速率至关重要。此外，截短的 ChBD1-ChBD2 和融合 PKD1 + PKD2 参与了几丁质结合。