Cloning and characterization of a small family 19 chitinase from moss (Bryum coronatum).
Cloning and characterization of a small family 19 chitinase from moss (Bryum coronatum).
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DOI：
10.1093/glycob/cwq212
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发表时间：
2011-05
期刊：
影响因子：
4.3
通讯作者：
T. Taira;Yoko Mahoe;N. Kawamoto;Shoko Onaga;H. Iwasaki;T. Ohnuma;T. Fukamizo
中科院分区：
文献类型：
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作者：
T. Taira;Yoko Mahoe;N. Kawamoto;Shoko Onaga;H. Iwasaki;T. Ohnuma;T. Fukamizo
通过几个步骤的柱色谱从苔藓 Bryum coronatum 中纯化几丁质酶-A (BcChi-A)。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测纯化的BcChi-A分子量为25 kDa，等电点为3.5。通过cDNA末端的快速扩增和聚合酶链式反应克隆了编码BcChi-A的cDNA。它由1012个核苷酸组成，编码228个氨基酸残基的开放阅读框。预测的成熟BcChi-A由205个氨基酸残基组成，分子量为22,654。序列分析表明，BcChi-A 是糖苷水解酶家族 19 (GH19) 几丁质酶，缺少环 I、II、IV 和 V 以及 C 末端环，这些环存在于植物 I 类和 II 类几丁质酶的催化结构域中。 BcChi-A 是一种紧凑的几丁质酶，在 GH19 几丁质酶中具有最少的环区域。使用壳寡糖的酶促实验表明，BcChi-A 对较短底物的活性比 II 类酶更高。这一特征可能是由于位于底物结合裂口末端的环区域的丢失导致的，该环区域将参与 II 类酶的底物结合。这是来自苔藓、非维管植物的几丁质酶的首次报道。
Chitinase-A (BcChi-A) was purified from a moss, Bryum coronatum, by several steps of column chromatography. The purified BcChi-A was found to be a molecular mass of 25 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 3.5. A cDNA encoding BcChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1012 nucleotides and encoded an open reading frame of 228 amino acid residues. The predicted mature BcChi-A consists of 205 amino acid residues and has a molecular weight of 22,654. Sequence analysis indicated that BcChi-A is glycoside hydrolase family-19 (GH19) chitinase lacking loops I, II, IV and V, and a C-terminal loop, which are present in the catalytic domain of plant class I and II chitinases. BcChi-A is a compact chitinase that has the fewest loop regions of the GH19 chitinases. Enzymatic experiments using chitooligosaccharides showed that BcChi-A has higher activity toward shorter substrates than class II enzymes. This characteristic is likely due to the loss of the loop regions that are located at the end of the substrate-binding cleft and would be involved in substrate binding of class II enzymes. This is the first report of a chitinase from mosses, nonvascular plants.