Study On The Cloning, Expression, Enzymatic Characteristics And Physiological Functions Of Chitinase ChiB Gene From Aspergillus Nidulans

Fungal cell wall is a dynamic and rigid structure and it plays major role in maintaining the morphology of cell wall and normal physiological activity of cells..In the process of fungal cell wall growth and reproduction,the struct?re and composition of the cell has some changes,mainly phenomenon is cell wall gradually expanding.On the research achievements of Saccharomyces cerevisiae and Aspergillus cell wall,suggest that a series of enzymes involved in process of cell expansion.,chitinase is one of important enzymes.Previous studies in our laboratory have found that chitinase can promote elongation of the cell wall at the tip of the epidermis,whether the roles of chitinase can promote cell wall expansion is appropriate to the elongation growth of the cell wall of aspergillus mycelium.Therefore,this paper studies the enzymatic characteristics of chitinase ChiB and its function in the elongation of mycelia cell wall.To explore the roles of chitinase ChiB during hyphal growth and morphogenesis of Aspergillus,we take Aspergillus nidulans as materials,preliminary analysis its chitinase genes and screen the important genes for recombinant expression studies.Finally,we obtain one correct chitinase and study their enzymatic properties and physiological function.We have expressed chitinase ChiB in Escherichia coli Rosetta DE3.Recombinant strains can produce a larger number of proteins induced by IPTG for 24h ?nder 18 ?.ChiB protein can be obtained when recombinant strains was ultrasonic broken and purified by Ni-colum.The molecular mass of purified enzyme ChiB protein was estimated to be 45kDa by SDS-PAGE.The recombinant ChiB protein was purified and concentrated to test its enzymatic properties.We got the below resluts of its enzymatic properties.1)ChiB protein displayed maximal activity at pH6.0 and 50 ?,respectively;2)It exhibited maximal substrate specificity for colloidal chitin.The Km and Vmax of the enzyme is Km=1.99mg/mL,Vmax=0.155?mol·mg-1·min-1 when hydrolysis colloidal chitin.;3)The chitinase(ChiB)from Aspergillus nidulans is a member of the class 18 chitinase family and the enzyme functions by a retaining catalytic mechanism.The pattern of cleavage of N-acetygl?cosamine(GlcNAc)oligosaccharide substrates,colloidal chitin substrates and chitin powder substrates has been determined.The the products and substrates were determined by HPAC-PAD,The result of the products and substrates by HPAC-PAD suggest that the enzyme hydrolyse oligosaccharides by an exo-type cleavage pattern with transglycosylation.Nine millimeter apicial fragment of heated stipes were prepared,heated and subjected to extensometry analysis.The results s?ggest that chitinase ChiB protein can reconstruct heated-inactived apical regions of C.cinerea wall extension.We further hope to determine the lozalization of the chitinase in the process of mycelium development by fluorescence in Aspergillus nidulans.Howener,the localization of chitinase in cell was not observed,possibly due to the low expression level of the ChiB in the process of mycelium development.In spite of this,we hypothesize that the chitinase may participate in the expansion of the hyphae cell wall,by breaking the chitin chains and forming a complex network structure with the action of transglycosidases.Clearly cell wall chitinase is an important area for future research with regard to both our understanding of fungal growth and morphogenesis,and the exploitation of these highly specific enzymes as additional,novel targets for antif?ngal drugs and agricultural fungicides.It has great scientific and practical significance for controlling the proliferation of pathogenic fungus and promoting the growth of fungus in industry and agriculture.