Effects of Linker Length and Flexibility on the Activity of GH18 Chitinases in Bacteria

IF 6.2 1区农林科学 Q1 AGRICULTURE, MULTIDISCIPLINARY

Journal of Agricultural and Food Chemistry Pub Date : 2025-10-07 DOI:10.1021/acs.jafc.5c05839

Pengfei Li, Lin Kong, Shihan Yang, Haonan Chen, Xiumei Wang, Lixia Pan, Mingbo Qu

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Abstract

Chitinases that play pivotal roles in chitin biodegradation for high-value bioconversion often adopt multimodular architectures with catalytic and binding domains connected by linkers. The linkers have been proven to possess functional roles, yet their mechanistic contributions remain poorly defined. In this study, a systematic analysis of 1242 bacterial glycoside hydrolase family 18 chitinases revealed a preferred linker length of 30–45 amino acids and an enrichment in threonine–proline (TP) motifs. Engineered Serratia marcescensSmChiC variants with controlled lengths (15–60 residues) demonstrated that a 45-residue linker (GS9) optimally enhances hydrolytic efficiency (22.4–39.8% activity increase) across α-/β-/colloidal chitins, with QCM-D confirming enhanced substrate binding/hydrolysis dynamics. Strikingly, engineered SmChiC variants with different rigidities demonstrated that the TP-linker variant outperformed the wild-type, flexible (GS9), and rigid (EK) variants, exhibiting rapid substrate association as revealed by HS-AFM. These findings highlight the critical role of linker design in optimizing chitinase function and provide a foundation for engineering enhanced glycoside hydrolases.

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连接体长度和柔韧性对细菌GH18几丁质酶活性的影响

几丁质酶在几丁质生物降解和高价值生物转化中发挥关键作用，通常采用多模块结构，催化和结合结构域由连接物连接。连接体已被证明具有功能作用，但它们的机制贡献仍然不明确。在本研究中，对1242个细菌糖苷水解酶家族18几丁质酶进行了系统分析，发现优选的连接体长度为30-45个氨基酸，并富集了苏氨酸-脯氨酸（TP）基序。长度控制（15-60个残基）的工程粘质沙雷氏菌smchic变异表明，45个残基的连接体（GS9）最优地提高了α-/β-/胶体几丁质的水解效率（活性提高22.4-39.8%），QCM-D证实了底物结合/水解动力学的增强。引人注目的是，具有不同刚性的工程SmChiC变体表明，tp连接子变体优于野生型，柔性（GS9）和刚性（EK）变体，HS-AFM显示出快速的底物关联。这些发现强调了连接体设计在优化几丁质酶功能中的关键作用，并为工程增强糖苷水解酶提供了基础。

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来源期刊

Journal of Agricultural and Food Chemistry 农林科学-农业综合

CiteScore

9.90

自引率

8.20%

发文量

1375

审稿时长

2.3 months

期刊介绍： The Journal of Agricultural and Food Chemistry publishes high-quality, cutting edge original research representing complete studies and research advances dealing with the chemistry and biochemistry of agriculture and food. The Journal also encourages papers with chemistry and/or biochemistry as a major component combined with biological/sensory/nutritional/toxicological evaluation related to agriculture and/or food.