Heterologous expression of new antifungal chitinase from wheat

• Published in: Protein expression and purification 2007-11, Vol.56 (1), p.100-109
• Main Authors: Singh, Arpita, Isaac Kirubakaran, S., Sakthivel, N.
• Format: Article
• Language: English
• Subjects: Alternaria; Amino Acid Sequence; Antifungal Agents - metabolism; Antifungal Agents - pharmacology; Antifungal protein; Ascomycota - drug effects; Chitinases - biosynthesis; Cloning, Molecular - methods; Colletotrichum falcatum; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli - metabolism; Escherichia coli expression; Fusarium; Fusarium - drug effects; Models, Molecular; Molecular phylogeny; Molecular Sequence Data; Oryza sativa; Pestalotia; Phylogeny; Rhizoctonia - drug effects; Rhizoctonia solani; Sarocladium oryzae; Sequence Alignment; Triticum - enzymology; Triticum aestivum; Wheat chitinase
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2007.06.013

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 α-helices, 3 β-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.