Department of Bioscience and Biotechnology, University of the Ryukyus, Okinawa, 903-0213, Japan.
Okinawa Institute of Science and Technology Graduate University, Okinawa, 904-0412, Japan.
Planta. 2021 May 13;253(6):120. doi: 10.1007/s00425-021-03639-8.
A chitin-binding domain could contribute to the antifungal ability of chitinase through its affinity to the fungal lateral wall by hydrophobic interactions. Complementary DNA encoding the antifungal chitinase of gazyumaru (Ficus microcarpa), designated GlxChiB, was cloned and expressed in Escherichia coli cells. The results of cDNA cloning showed that the precursor of GlxChiB has an N-terminal endoplasmic reticulum targeting signal and C-terminal vacuolar targeting signal, whereas mature GlxChiB is composed of an N-terminal carbohydrate-binding module family-18 domain (CBM18) and a C-terminal glycoside hydrolase family-19 domain (GH19) with a short linker. To clarify the role of the CBM18 domain in the antifungal activity of chitinase, the recombinant GlxChiB (wild type) and its catalytic domain (CatD) were used in quantitative antifungal assays under different ionic strengths and microscopic observations against the fungus Trichoderma viride. The antifungal activity of the wild type was stronger than that of CatD under all ionic strength conditions used in this assay; however, the antifungal activity of CatD became weaker with increasing ionic strength, whereas that of the wild type was maintained. The results at high ionic strength further verified the contribution of the CBM18 domain to the antifungal ability of GlxChiB. The microscopic observations clearly showed that the wild type acted on both the tips and the lateral wall of fungal hyphae, while CatD acted only on the tips. These results suggest that the CBM18 domain could contribute to the antifungal ability of chitinase through its affinity to the fungal lateral wall by hydrophobic interactions.
一个几丁质结合域可能通过疏水相互作用与真菌的侧墙的亲和力有助于几丁质酶的抗真菌能力。编码榕属（榕属）的抗真菌几丁质酶的 cDNA 被克隆并在大肠杆菌细胞中表达，该几丁质酶被命名为 GlxChiB。cDNA 克隆的结果表明，GlxChiB 的前体具有内质网靶向信号的 N 端和液泡靶向信号的 C 端，而成熟的 GlxChiB 由 N 端碳水化合物结合模块家族 18 结构域（CBM18）和 C 端糖苷水解酶家族 19 结构域（GH19）组成，带有短的连接子。为了阐明 CBM18 结构域在几丁质酶抗真菌活性中的作用，使用重组 GlxChiB（野生型）及其催化结构域（CatD）在不同离子强度下进行定量抗真菌测定，并对绿色木霉进行了微观观察。在本试验中使用的所有离子强度条件下，野生型的抗真菌活性均强于 CatD；然而，CatD 的抗真菌活性随着离子强度的增加而减弱，而野生型的抗真菌活性则保持不变。高离子强度下的结果进一步验证了 CBM18 结构域对 GlxChiB 抗真菌能力的贡献。微观观察清楚地表明，野生型作用于真菌菌丝的尖端和侧墙，而 CatD 仅作用于尖端。这些结果表明，CBM18 结构域可能通过与真菌侧墙的疏水相互作用，有助于几丁质酶的抗真菌能力。
