Browsing by Author "Porkka, Kaija"

Chitinolytic enzymes belong to a group of pathogenesis-related proteins, which are induced in plants by a pathogenic attack. They have also been shown to function in abiotic stresses and related to signalling. Chitinases catalyze the degradation of β-1,4-N-acetyl glucosamine units of chitin. Chitin is not found in plants, but for example the shells of invertebrates, cell walls of certain fungi, algae and bacteria contain it abundantly. Lately chitinases have gathered attention because of their potential utilization possibilities in medicine, agriculture, food industry and biofuels. Strong chitinase gene expression and also chitinolytic activity has been found in a spruce (Picea abies L. Karst.) cell culture, which produces apoplastic lignin into the medium. Plant chitinases are known to be induced also in developmental processes. Chitinase-like genes have been shown to be essential in cellulose synthesis and in development of cell walls and tracheary elements and also affected the localisation of lignin in tissues. Therefore, the secretion of chitinases into the culture medium has not necessarily been caused by stress and chitinases might use also some endogenic substrate in addition to chitin. Because chitin is a structural component of plant pathogens but not present in plants and the growing circumstances of the studied aceptic spruce cell culture were stable, the research hypothesis was, that the secreted chitinases in the culture medium were not a stress reaction but related to development and besides chitin of the pathogens they utilize some own polysaccharide substrate of spruce. Additional hypothesis was connection of the strong expression of chitinases and chitinase-like genes in the culture medium and the production of apoplastic lignin by the spruce cell line. The aim of the study was to characterize the chitinases and chitinase-like enzymes of the spruce cell suspension culture medium and developing xylem by isolating, purifying and cloning them and by producing them heterologously. The aim was also to identify the putative endogenous substrates for the chitinases and chitinase-like proteins to reach better understanding about the meaning of the chitinases in the development of the spruce tissues. Two chitinases of the spruce cell suspension culture were isolated and purified by chromatographic methods. One chitinase gene in spruce cell culture and one chitinase-like gene in spruce xylem were cloned, after which the chitinases and chitinase-like proteins were produced in Pichia pastoris yeast. The total proteins of the culture medium, the purified chitinases isolated from the medium and the chitinases and chitinase-like proteins produced in P. pastoris were used to examine their substrate alternatives with the carbohydrate microarray method. The connection between apoplastic lignin and the chitinolytic enzymes in culture medium was not found and obvious candidates for the endogenous substrates were not detected. However, the mannan degradation in the presence of the chitinases was strong enough to give reason to further analyses.