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

    Research output: Contribution to conferenceConference AbstractScientific

    Abstract

    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.
    Original languageEnglish
    Number of pages1
    Publication statusPublished - 2006
    MoE publication typeNot Eligible
    Event9th European Training Course on Carbohydrates - Wageningen, Netherlands
    Duration: 6 Jun 20069 Jun 2006
    Conference number: 9

    Course

    Course9th European Training Course on Carbohydrates
    CountryNetherlands
    CityWageningen
    Period6/06/069/06/06

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