Kuba Yumani, Takashima Tomoya, Uechi Keiko, Taira Toki
a Graduate School of Agricultural Science , Kagoshima University , Kagoshima , Japan.
b Department of Bioscience and Biotechnology , University of the Ryukyus , Okinawa , Japan.
Biosci Biotechnol Biochem. 2018 Oct;82(10):1742-1752. doi: 10.1080/09168451.2018.1491285. Epub 2018 Jul 2.
Chitinase-A from a lycophyte Selaginella doederleinii (SdChiA), having molecular mass of 53 kDa, was purified to homogeneity by column chromatography. The cDNA encoding SdChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1477 nucleotides and its open reading frame encoded a polypeptide of 467 amino acid residues. The deduced amino acid sequence indicated that SdChiA consisted of two N-terminal chitin-binding domains and a C-terminal plant class V chitinase catalytic domain, belonging to the carbohydrate-binding module family 18 (CBM18) and glycoside hydrolase family 18 (GH18), respectively. SdChiA had chitin-binding ability. The time-dependent cleavage pattern of (GlcNAc) by SdChiA showed that SdChiA specifically recognizes the β-anomer in the + 2 subsite of the substrate (GlcNAc) and cleaves the glycoside bond at the center of the substrate. This is the first report of the occurrence of a family 18 chitinase containing CBM18 chitin-binding domains.
AtChiC: Arabidopsis thaliana class V chitinase; CBB: Coomassie brilliant blue R250; CBM: carbohydrate binding module family; CrChi-A: Cycas revolute chitinase-A; EaChiA: Equisetum arvense chitinase-A; GH: glycoside hydrolase family, GlxChi-B: gazyumaru latex chitinase-B; GlcNAc: N-acetylglucosamine; HPLC: high performance liquid chromatography; LysM; lysin motif; MtNFH1: Medicago truncatula ecotypes R108-1 chitinase; NCBI: national center for biotechnology information; NF: nodulation factor; NtChiV: Nicotiana tabacum class V chitinase; PCR: polymerase chain reaction; PrChi-A: Pteris ryukyuensis chitinase-A; RACE: rapid amplification of cDNA ends; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SdChiA: Selaginella doederleinii chitinase-A.
来自石松类植物江南卷柏（Selaginella doederleinii）的几丁质酶A（SdChiA），分子量为53 kDa，通过柱色谱法纯化至同质。通过cDNA末端快速扩增和聚合酶链反应克隆了编码SdChiA的cDNA。它由1477个核苷酸组成，其开放阅读框编码一个由467个氨基酸残基组成的多肽。推导的氨基酸序列表明，SdChiA由两个N端几丁质结合结构域和一个C端植物V类几丁质酶催化结构域组成，分别属于碳水化合物结合模块家族18（CBM18）和糖苷水解酶家族18（GH18）。SdChiA具有几丁质结合能力。SdChiA对（GlcNAc）的时间依赖性切割模式表明，SdChiA特异性识别底物（GlcNAc）+2亚位点中的β-异头物，并在底物中心切割糖苷键。这是关于含有CBM18几丁质结合结构域的18家族几丁质酶存在的首次报道。
AtChiC：拟南芥V类几丁质酶；CBB：考马斯亮蓝R250；CBM：碳水化合物结合模块家族；CrChi-A：苏铁几丁质酶A；EaChiA：木贼几丁质酶A；GH：糖苷水解酶家族；GlxChi-B：gazyumaru乳胶几丁质酶B；GlcNAc：N-乙酰葡糖胺；HPLC：高效液相色谱；LysM：溶菌基序；MtNFH1：蒺藜苜蓿生态型R108-1几丁质酶；NCBI：美国国立生物技术信息中心；NF：结瘤因子；NtChiV：烟草V类几丁质酶；PCR：聚合酶链反应；PrChi-A：琉球凤尾蕨几丁质酶A；RACE：cDNA末端快速扩增；SDS-PAGE：十二烷基硫酸钠-聚丙烯酰胺凝胶电泳；SdChiA：江南卷柏几丁质酶A
