Production, Purification And Characterization Of β-mannanase By Aspergillus Niger In Solid State Fermentation

Endo-1,4-β-D-mannanase was one of the glycosidase hydrolases, which was widely usedin food, feed, biological degumming, papermaking and petroleum industry. It was mainlyderived from microbial fermentation. Especially, acidic β-mannanase secreted by fungi could beused for preparation of functional oligosaccharides which had high economic value. In thisdissertation, the optimum medium condition of β-mannanase from Aspergillus niger NL02, theprocess condition for koji plate, purification and characterization of β-mannanase was studied.The main results are as follows:As the lignocellulosic materials are extensive and have low prices, five different rawmaterials (corn stover, corn cobs, bamboos, wheat bran, XOS residues) to produce β-mannanasein solid state fermentation were compared. Residues of xylo-oligosaccharides production wasinstead of wheat bran for β-mannanase preparation with solid state fermentation and konjacflour was employed to induce β-mannanase production. Multivariant statistical approachescontaining Plackett-Burman design and response surface methodology wre employed toevaluate several variables in flask with the strain Aspergillus niger NL02. The optimummedium ingredients were as follows: the proportion of XOS waste residue to wheat bran4:6,17.49%konjac powder,4%（NH4）2SO4,0.30%KH2PO4,0.5%gSO47H2O,0.4%CaCl2,0.3%Tween80,0.05%FeSO47H2O, initial moisture74.85%, initial pH natural(all related to drymedium). The cultivation was carried out at30℃for5d. With these conditions,β-mannanaseactivity, β-mannosidase and protein content were341.8U/g,7.02U/g,8.40mg/g.β-mannanase scale-up production was carried out in koji plate(20cm×26cm) based onflask conditions. The optimal koji plate fermentation conditions were as follows: initialmoisture content80%, initial natural pH, drymedium content100g/koji plate, inoculum size10%(related to dry medium). At the same time, β-mannanase activity and β-mannosidaserespectively reached355.27U/g,3.33U/g.The endo-β-mannanase of Aspergillus niger NL02was purified by30~80%ammoniumsulfate precipitation, Source15Q anion-exchange chromatography, Superdex75Prep Grade gelfiltration and DEAE Sepharose Fast Flow anion-exchange chromatography in sequence. Theelectrophretially pure component was obtained and the activity recovery was0.56%, butcompared with crude enzyme purified19.54-fold. And the pure β-mannanase molecular weightwas （40±1）kDa.The optimum reaction temperature and pH value of endo-β-mannanase was60℃and2.5,respectively. The thermal stsbility range and pH stability range of endo-β-mannanase was40~70℃and pH2.5~8.0. This enzyme hadhigh activity within the range of pH2.5~5.5, so itbelonged to acidic enzyme. The Kmand Vmaxvalues of endo-β-mannanase were6.5mg/mL and9.17μmol·min-1·ml-1, respectively.