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Structural analysis and construction of thermostable antifungal chitinase
bioRxiv - Biochemistry Pub Date : 2021-09-18 , DOI: 10.1101/2021.09.17.460877
Dan Kozome , Keiko Uechi , Toki Taira , harumi fukada , Tomomi Kubota , Kazuhiko Ishikawa

Chitin is a biopolymer of N-acetyl-D-glucosamine with β-1,4-bond and is the main component of arthropod exoskeletons and the cell walls of many fungi. Chitinase (EC 3.2.1.14) is an enzyme that hydrolyzes the β-1,4-bond in chitin and degrades chitin into oligomers. It has been found in a wide range of organisms. Chitinase from Gazyumaru (Ficus microcarpa) latex exhibits antifungal activity by degrading chitin in the cell wall of fungi and is expected to be used in medical and agricultural fields. However, the enzyme’s thermostability is an important factor; chitinase is not thermostable enough to maintain its activity under the actual applicable conditions. We solved the crystal structure of chitinase to explore the target sites to improve its thermostability. Based on the crystal structure and sequence alignment among other chitinases, we rationally introduced proline residues, a disulfide bond, and salt bridge in the chitinase using protein engineering methods. As a result, we successfully constructed the thermostable mutant chitinases rationally with high antifungal and specific activities. The results provide a useful strategy to enhance the thermostability of this enzyme family.

中文翻译:

耐高温抗真菌几丁质酶的结构分析与构建

几丁质是具有β-1,4-键的N-乙酰-D-氨基葡萄糖的生物聚合物,是节肢动物外骨骼和许多真菌细胞壁的主要成分。几丁质酶 (EC 3.2.1.14) 是一种水解几丁质中的 β-1,4-键并将几丁质降解为低聚物的酶。它已在广泛的生物体中发现。来自 Gazyumaru ( Ficus microcarpa ) 的几丁质酶) 乳胶通过降解真菌细胞壁中的几丁质表现出抗真菌活性,有望用于医疗和农业领域。然而,酶的热稳定性是一个重要因素;几丁质酶的热稳定性不足以在实际适用条件下保持其活性。我们解析了几丁质酶的晶体结构以探索目标位点以提高其热稳定性。基于其他几丁质酶的晶体结构和序列比对,我们利用蛋白质工程方法在几丁质酶中合理引入脯氨酸残基、二硫键和盐桥。结果,我们成功地构建了具有高抗真菌活性和比活性的热稳定突变几丁质酶。结果提供了一种有用的策略来增强该酶家族的热稳定性。
更新日期:2021-09-21
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