Studies on the protein-carbohydrate interaction of the Trichoderma harzianum chitinase Chit42

Trichoderma harzianum is a filamentous fungus that can secrete several chitinases (EC 3.2.1.14) to the culture medium under inductive conditions. T. harzianum Chit42 is among the best studied Trichoderma chitinases hydrolysing polymeric chitin mainly into chitobiose, a disaccharide. Chit42 is a single catalytic module enzyme, which belongs to the glycosyl hydrolase family 18 having a (beta/alfa)8 barrel fold. A three-dimensional structural model of T. harzianum Chit42 was built based on the 53% amino acid sequence identity with another fungal chitinase CiX1 from Coccidioides immitis, whose crystal structure has been determined (1). Our modelling studies suggested that Chit42 has at least 7 subsites (-5-4-3-2-1+1+2) for binding the polymeric substrate, and the substrate binding groove is formed by loops positioned between the carboxy-terminal end of the beta-strands and the amino-terminal end of the alfa-helices (2). We have additionally shown that the wild-type Chit42 can recognize animal like, beta-1,4-galactosylated and alfa-1,3-fucosylated chito-oligosaccharides (2). Our aim is to investigate the interactions of the T. harzianum Chit42 with natural and modified chito-oligosaccharides using wild-type and different engineered chitinase variants. The work will involve heterologous expression of the chitinase cDNA in E.coli, protein engineering by rational design or by directed evolution methods, and characterising the wild-type and mutant chitinases for their activity and binding specificity. The characterisation of the protein-carbohydrate interactions will include the usage of atomic force microscopy force-distance curves.