Optimization And Nature Analysis Of Athelia Rolfsii Exopolysaccharides And β-1,3-glucanase From Athelia Rolfsii

Athelia rolfsii fermentation production Athelia rolfsii exopolysaccharides as an exo-polysaccharides having immune regulation, lowering the physiological activity of cholesterol, lowering blood sugar. β-1,3-glucan enzyme has capable of specifically acting on the polysaccharide polymer with β-1,3 glycosidic linkage. This feature gives it the ability of hydrolyzing the fungal cell wall, making it not only have important applications in the production of beer, wine brewing and food preservation, but also play an important role in crop pest control fields. Our experimental group found that while this strain secreting polysaccharides in the process of corn starch and corn soak solution fermenting Athelia also to produce β-1,3 glucanase, therefore we trying to achieve both β-1,3 glucanase and exopolysaccharides through only one fermentation and obtain optimal yield. In this way, we can save energy, improve the added value of starch products, deepen and optimized processing technology food industry.In this paper, We obtain the high yield of Athelia rolfsii β-1,3-glucanase and exopolysaccharides at the same time. By separation and extraction of two products we expand β-1,3-glucanase characterization and enzymatic reaction kinetics inquiry, 50 L fermenter optimization test and sequenced.After the composite mutagenesis and protoplast fusion, we obtained AY-3 strain as fermentation strain. Using corn starch and corn soak solution as important components of culture medium, fermentation temperature(℃), fermentation time(d) and stirrer speed(r/min) as experimental factors, the optimal double response values of Athelia rolfsii β-1,3-glucanase(U/mL) and exopolysaccharides(g/L) were obtained by Box-Behnken design. The results of response surface stereogram and overlay contour analysis showed that the optimal fermentation conditions were as follows: 5% inoculum size, 28.5 ℃, 7.5 d, 180 r/min. The primary enzyme production was 39.96 U/mL, the Athelia rolfsii exopolysaccharides production was 18.11 g/L at the same time.After centrifugation, ultrafiltration, salting-out, alcohol precipitation, bleaching, dialysis, concentration, chromatography steps the polysaccharide and β-1,3-glucanase were high purity. Making polysaccharide fourier transform infrared spectroscopy to detect its structure, the result shows it is β-1,3-glucan. Making β-1,3-glucanase non-denaturing gel electrophoresis showed that the resulting enzyme has capable of hydrolyzing laminarin.Enzymatic properties showed that the optimum temperature is 30 ℃, the heat resistance is poor, and the optimum pH was 2.5, acid resistance is excellent; metal ions on the enzyme activity displayed Mn2 +, Mg2 +, Zn2 + for activity have significant role in promoting, Ba2 +, Fe3 +, Ag2 + for activity significantly inhibited; kinetics of enzymatic reactions showed enzyme Vmax is 2mg / mL ? min, Km value is 5mg / mL.50L fermenter test showed a non-ionic surfactant Tween- 80 making promote role of fermentation. Fermentation products resulting double response optimal conditions for optimum fermentation conditions β-1,3-glucanase yield was 38.32 U / mL, reached 97.14% of the predicted value, polysaccharide yield was 20.14 g / L, reached the forecast worth 111.2%. Nanotechnology- High Performance Liquid Chromatography and multi-stage series ion trap MS analysis(NanoLC-ESI-MS / MS) were identified enzyme, the enzyme showed a molecular weight of 40 203.36 Da. Database search of the peptides from our experimental production were exactly matches one peptide sequence derived from Penicillium marneffei ATCC 18224 of endo-1,3(4)-beta-glucanase. Peptide sequence is QGIWPAFWMLGDSLR.