A thesis submitted in partial fulfillment of the requirements for the degree of Master of Engineering, School of Environment, Resources and Development

Thesis (M.Eng.) - Asian Institute of Technology, 2005

Trichoderina harziaiuun BCC5828, a mycoparasitic fungus and an enzyme of the chitinase enzyme complex, N- acetylglucosaminidase were tested as a biocontrol agent to control the development of three three pathogens, Fusarium oxysporum, Rizoctonia solani,Pythium aphaniderinatum. Initially Trichoderina strain was grown in liquid medium in shake flasks culture was induced to produce of N- acetylglucosaminidase enzyme in the presence of chitin. In a bioreactor the enzyme production was doubled with the same level of Chitin 1% as compared to shake flask cultures. The enzyme was purified by ammonium sulphate precipitation followed by CM cellulose chromatography, DEAE chromatography and Sephadex G-50 gel filtration. Overall a 32 fold increased in enzyme purity was obtained. In order to characterize the molecular weight of the N-acetylglucosaminidase enzyme was determined by SDS PAGE analysis and the was found to have a molecular weight of 35 KDa Maximal activities were determined at pH 6 and optimum temperature range of 50 °C to 60°C. The enzyme is stable at 25°C and 37°C and enhanced activity was obtained with iron salts. The K,,, and V max value were 4.15mM and 2.O4 mol/min of substrates. The enzyme was not found to be very effected as a biocontrol agent in well diffusion test done on three plant pathogens. However the fungi itself was found be very active and can be used as a good biocontrol against for the three plant pathogens studied