Study On Heterologous Recombinant Expression Of β - 1, 3 - Glucanase From

Fungal cell wall is a rigid structure and it provides protective function for protoplast. In the process of fungal cell growth and reproduction, the structure and composition of the cell wall has some changes, mainly phenomenon is cell wall gradually expanding. On the research achievements of Saccharomyces cerevisiae and Aspergillus think is in a series of enzymes involved in the process of cell expansion. β-1,3-glucanase is one of important enzymes, which relax the cell walls through hydrolytic action, and transfer new synthesis glucan chains to the walls of the original structure. Under the series of action, the cell wall expand.To explore in basidiomycete edible fungus β-1,3-glucanase role to changes of the cell wall, we take Coprinus cinereus as materials, preliminary analysis its β-1,3-glucanase genes and screen the important genes for recombinant expression studies. Finally, we obtain two correct β-1,3-glucanases and study their enzymatic properties.we have expressed exo-β-1,3-glucanase (EXG1) in Escherichia coli Rosetta DE 3. Recombinant strains can produce a large number of proteins induced by IPTG for 48 h under 20 ℃. EXG1 can be obtained when Recombinant strains was ultrasonic broken and purified by Ni-column. The molecular size of EXG1 is 85kDa. The study of enzymatic properties shows that the enzyme can behave maximum activity at the conditon of 60 ℃ and pH6.0. It hydrolysis glucan from the non-reducing end and without glucosyltransferases activity. The Km and Vmax of the enzyme is 2.23 mg ml-1 and 232.56 μmol mg-1 min-1 when hydrolysis laminarin.We expressed endo-β-1,3(4)-glucanase (engl6A) in pichia pastoris GS115. Recombinant strains can produce a large number of proteins induced by methyl alcohol for four days or more. The Recombinant protein was synthesised and secreted to culture medium. So we can purified the culture medium by Ni-column and obtain eng16A. The molecular size of eng16A is 60 kDa. The study of enzymatic properties shows that the enzyme can behave maximum activity at the conditon of 60 ℃ and pH6.0. It can hydrolysis glucan contain β-1,3-glucosidic bond at random site. The Km and Vmax of the enzyme is 6.49 mg ml-1 mg ml-1 and 57.47 μmol mg-1 min-1 when hydrolysis laminarin. If hydrolysis barley P-glucan, The Km and Vmax is 20.84 mg ml-1 and 384.62 μmol mg-1min-1.With Escherichia and Pichia as host, we expressed two β-1,3-glucanase gene and obtained the proteins has correct enzyme activity. Besides, we get large number of enzymes by Ni-column rapidly. we avoid many problems such as cultivation process is complicated, fruitbody growth take some time and the enzymes obtained is relatively less. When we study the properties of the two enzymes, we find that the exo-β-1,3-glucanase is mainly used to hydrolysis β-1,3-glucan in cell wall with other enzyme, and the endo-β-1,3(4)-glucanase is mainly used to hydrolysis β-1,3-glucan in culture medium. I think the two β-1,3-glucanase would become great tools to research the cell wall of basidiomycete fungi.