Optimization Of Culture Conditions Of Hypoxylon Sp. On Lectin Production, Isolation And Partial Characterization Of The Hypoxylon Sp. Lectin

Fungal lectins are proteins（or glycoproteins） from fungi,other than antibodies and enzymes,which bind specifically and reversibly to carbohydrates,resulting in cell agglutination or precipitation of glycoconjugates.In recent years,the research on fungal lectins mainly focused on its physiological,biochemical characterizations and biological functions,such as defense insects in plant,antitumor,antivirus and gene engineering.The research presented in this paper was to optimize the submerged culture conditions for the mycelial lectin by Hypoxylon sp..The lectin was purified from isolated mycelium of Hypoxylon sp..Then its physiological,biochemical characterizations and biological functions were studied preliminarily,to lay the foundation for the further development and utilization.The optimal fermentation conditions of Hypoxylon sp.were studied by means of single factor.The optimal fermentation medium was:lactose as a source of carbon,yeast extract as a source of nitrogen and C:N ratio of 2:1.With the optimal fermentation medium and condition,the titer of the mycelial lectin could be up to 1600 U/mL.Hypoxylon sp.lectin（HSL） was isolated from the mycelium of Hypoxylon sp.by extraction,precipitation by（NH₄）₂SO₄,ion-exchange chromatography on DEAE-Cellulose,and gel filtration on Sephedex G-100,which was purified by 9.8-fold with a recovery of 13%.The purified HSL showed a single protein band,and the molecular weight of subunit was 15.7 kDa when submitted to SDS-polyacrylamide gel electrophoresis.The gel was dyed by Schiff reagent and only a faint pink band was noticed,which suggested that the lectin was glycoprotein.The carbonhydrate content of 15.5%detected by the anthrone-sulfuric acid method.β-Elimination revealed the bond between the polysaccharide and the protein of HSL was O-type glucopeptide one.The maximum absorption wavelength of HSL was 280.4 nm.HSL could agglutinate rabbit erythrocytes,mouse erythrocytes and human regardless of blood type and animal species,and the minimum hemagglutinating concentration was 0.02μg/mL for rabbit erythrocytes.The carbohydrate inhibition experiments of hemagglutinating activity indicated the activity was inhibited by lactose and D-galactose, and the minimum inhibitory concentration of D-galactose was much lower than the lactose.The hemagglutinating activity was relatively insensitive to temperatures at 20-40℃,but declined obviously at 50℃or the higher temperature,which indicated HSL was a less thermostable glycoprotein.After exposure to 100mmol/L HCl,slightly only 12.5%of the activity remained but the activity was not affected in the presence of 100mmol/L NaOH,and the activity reduced to 50%in 3 mmol/L NaOH solution.The hemagglutinating activity of HSL was affected by demetalization with EDTA,which indicated that its activity was dependent on metal cations.Al³⁺ and Fe³⁺ were able to potentiate the hemagglutinating activity of HSL,while Mg²⁺ and K⁺ were devoid of any effect.The inhibition activities to fungi and bacteria showed that HSL only exerted antifungal activity toward Colletotrichum graminicola（Cesati） Wilson,but did not against Fusarium oxysporium f.sp.uasinfectum,Fusarium oxysporum Schl.F.sp.Dioscoreae Wellman,Fulvia fulva（Cooke） Cifferri,Fusarium oxysporium f.sp. cinerea,Aspergillus niger and Fusarium moniliforme Berk.at the concenteation up to 0.1 mg/mL.HSL also had no inhibitive effect on Staphylococcus aureus,Escherichia coli and Shigella dysenteriae.