Studies On The Production, Purification And Characterization Of Acidic β-Mannanase

Systematic studies on solid-state fermentation, purification, and characterizationof the acidic β-mannanase(β-1,4-D-mannan mannohydrolase, EC 3.2.1.78) producedby Aspergillus niger WM20-11 were undertaken. Several important results wereobtained in these studies and were presented below.The effects of the nutrition ingredients and cultural conditions on theβ-mannanase production were studied. The optimal fermentation medium contained9.0 g of wheat bran, 1.0 g of soybean flour, 0.98 g of konjac powder, 0.375 mL ofsteep liquor, 1.25% of (NH4)2SO4, 0.1% of CaCl2, 0.1% of MgSO4, 0.25% of KH2PO4,and 11.7 mL of H2O. The highest β-mannanase activity reached 1337 IU/g dry matterwith solid-state fermentation at 30℃ for 96 h. The optimum temperature and pH ofthe cross β-mannanase were 70℃ and 3.5.The β-mannanase from solid-state fermentation culture was purified byextracting with water, ammonium sulfate precipitation, and DEAE Sepharose fastflow and Sephadex G-100 column chromatographies. The purified β-mannanase washomogeneous on Native-PAGE and SDS-PAGE. After these steps the enzyme waspurified by 15.8-fold with a recovery of 2.2%.Molecular weight of the β-mannanase was determined as 39 kD on SephadexG-100 gel filtration and 40 kD on SDS-PAGE, indicating that it was a monomer. Theisoelectric point was estimated to be pI 4.0 by IEF-PAGE. The optimum temperatureand pH were 70℃and 3.5, respectively. It contained 19.6% carbohydrate byphenol-vitriol method. The activity of the enzyme was stimulated by Mg2+,Ca2+,Li+,Na+,K+ ions, but inhibited by Pb2+,Co2+,Fe3+,Mn2+ and Hg2+ ions. Kineticscoefficients Km and Vmax were 6.67% and 333 μmol·min-1·mg-1 with locust bean gumas substrate. Analysis of amino acid composition showed that content of acidic aminoacid Asp and Glu was high. Enzyme hydrolysis products from locust bean gum wereoligosaccharides by HPLC analysis, estimating that it may be endo-mannanase.