Studies On Soluble Bioactive Substance Of G. Lucidum By Submerged Fermentation

The research mainly focused on the following aspects about proteinase A inhibitor from Ganoderma lucidum by submerged fermentation, including extracting, purification, some properties, primary structure, the relationship of structure and function, procreant condition, application for increasing draft beer foam stabilization.First, this research discovered G.lucidum by Submergeded fermentation can too producing SOD, it mainly exsits in mycelium.The half life is about seven days at tempreture 4℃, the molecular mass is 35000.Two kind of proteinase inhibitor GLPIA₁ and GLPIA₂ was purified to homogeneity from G.lucidum by submerged fermentation. The purification was carried out by ethanol fractionation (30～80%), UltroGel ACA44 gel-filtration chromatogram and Source 30Q anion exchange chromatogram, respectively.The molecular mass of GLPIA₁was 38000 as estimated via SDS–PAGE and gel filtration. This inhibitor showed a remarkable heat stability. It retained more than 95% of activity at up to 80℃for 30min , and there was only a 9% loss of avtivity at 98℃. The pH stability range of GLPIA₁ is about 3.0～7.5. By investigating the interaction between this inhibitor and a variety of proteinases, it is indicated that the inhibitor was more specific against yeast proteinase A than other proteinases. The dissociation constants (Ki) and concentration required for 50% inhibition (IC₅₀) for proteinase A were 2.2×10^-6 mol/L and 0.16 mg/ml, respectively. Kinetic studies(Lineweaver-Burk method)showed GLPIA₁ is a kind of noncompetitive inhibitor to yeast proteinase A.After GLPIA₁ was hydrolyzed by neutral proteinase and trypsin or byβ1,3 glucanase, the inhibitory activity decrease obviously. Amino acid composition date indicate that GLPIA₁ is rich in proline and acidic amino acid(about 11% and 27%, respectively). There are four kinds monosaccharide in GLPIA₁ determined by gas chromatography, including Glucose ,Mannose, Arabinose, Rhamnose, the percentage of glucose is about 96%.β-Elimination revealed that the linkage between the glycan and the core protein backbone might be O-linkage. Analysis by IR Spectrum ,¹H -NMR and ¹³C- NMR , we consider that GLPIA₁ is a typical glucoprotein, Its backbone includedβ1,3 glucosidic linkage , the branch ofβ1,6 glucosidic linkage exist rarely.To explore the dimensional structure and the relationship between dimensional structure and inhibitor activity of GLPIA₁, the chemical reaction of Congo red was applied to forecast the dimentional structure of GLPIA₁ and to abserve the changes of dimentional structure. The result displays that GLPIA₁ is triple helical conformation in water. After being treated with different concentrations of sodium, the inhibitor activities vary with the change of dimentional structure. Obviously, the dimentional structure of GLPIA₁ is tightly related with biological activities.The other proteinase A inhibitor GLPIA₂ only has absorption at 215nm. Amino acid...