Singh Arpita, Kirubakaran S Isaac, Sakthivel N
Department of Biotechnology, School of Life Sciences, Pondicherry University, Kalapet, Puducherry 605 014, India.
Protein Expr Purif. 2007 Nov;56(1):100-9. doi: 10.1016/j.pep.2007.06.013. Epub 2007 Jul 12.
Chitinases (EC 3.2.1.14) have been grouped into seven classes (class I-VII) on the basis of their structural properties. Chitinases expressed during plant-microbe interaction are involved in defense responses of host plant against pathogens. In the present investigation, chitinase gene from wheat has been subcloned and overexpressed in Escherichia coli BL-21 (DE3). Molecular phylogeny analyses of wheat chitinase indicated that it belongs to an acidic form of class VII chitinase (glycosyl hydrolase family 19) and shows 77% identity with other wheat chitinase of class IV and low level identity to other plant chitinases. The three-dimensional structural model of wheat chitinase showed the presence of 10 alpha-helices, 3 beta-strands, 21 loop turns and the presence of 6 cysteine residues that are responsible for the formation of 3 disulphide bridges. The active site residues (Glu94 and Glu103) may be suggested for its antifungal activity. Expression of chitinase (33 kDa) was confirmed by SDS-PAGE and Western hybridization analyses. The yield of purified chitinase was 20 mg/L with chitinase activity of 1.9 U/mg. Purified chitinase exerted a broad-spectrum antifungal activity against Colletotrichum falcatum (red rot of sugarcane) Pestalotia theae (leaf spot of tea), Rhizoctonia solani (sheath blight of rice), Sarocladium oryzae (sheath rot of rice) Alternaria sp. (grain discoloration of rice) and Fusarium sp. (scab of rye). Due to its innate antifungal potential wheat chitinase can be used to enhance fungal-resistance in crop plants.
几丁质酶（EC 3.2.1.14）已根据其结构特性被分为七类（I - VII类）。在植物与微生物相互作用过程中表达的几丁质酶参与宿主植物对病原体的防御反应。在本研究中，从小麦中克隆了几丁质酶基因，并在大肠杆菌BL - 21（DE3）中进行了过表达。对小麦几丁质酶的分子系统发育分析表明，它属于VII类几丁质酶的酸性形式（糖基水解酶家族19），与IV类的其他小麦几丁质酶有77%的同一性，与其他植物几丁质酶的同一性较低。小麦几丁质酶的三维结构模型显示存在10个α - 螺旋、3个β - 链、21个环转角，以及6个半胱氨酸残基，这些残基负责形成3个二硫键。活性位点残基（Glu94和Glu103）可能与其抗真菌活性有关。通过SDS - PAGE和Western杂交分析证实了几丁质酶（33 kDa）的表达。纯化的几丁质酶产量为20 mg/L，几丁质酶活性为1.9 U/mg。纯化的几丁质酶对甘蔗炭疽菌（甘蔗赤腐病）、茶轮斑病菌（茶叶斑病）、立枯丝核菌（水稻纹枯病）、稻小核菌（水稻鞘腐病）、链格孢属（水稻粒变色病）和镰刀菌属（黑麦赤霉病）具有广谱抗真菌活性。由于其固有的抗真菌潜力，小麦几丁质酶可用于增强作物对真菌的抗性。
