Grass Degrading [beta]-1,3-1,4-d-glucanases from Bacillus subtilis GN156: Purification and Characterization of Glucanase J1 and pJ2 Possessing Extremely Acidic pI

• Published in: Applied biochemistry and biotechnology 2008-04, Vol.149 (1), p.53
• Main Authors: Apiraksakorn, Jirawan, Nitisinprasert, Sunee, Levin, Robert E
• Format: Article
• Language: English
• Subjects: Bacteria; Barley; Biotechnology; Carbohydrates; Enzymes; Grasses; Studies; Ultrafiltration
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-007-8058-2

Purification of β-1,3-1,4-glucanase from the cell-free culture fluid of Bacillus subtilis GN156 by affinity chromatography of epoxy-activated sepharose 6B and ultrafiltration technique resulted in homogeneous J1 and partially purified pJ2 enzymes. The molecular weight and pI of J1 were 25 kDa and 3.5, respectively, while those for J2 were 90 kDa and 3.6, respectively. Both β-1,3-1,4-glucanase J1 and pJ2 had optimum pH values of 6-6.5 and an optimum temperature of 60°C. Both enzymes were not inhibited by Li2+ but were inhibited significantly by Ca2+, Cu2+, Mn2+ and Zn2+. However, J1 was slightly inhibited by Fe2+, while pJ2 was inhibited by Mg2+ as well. They were highly specific to only barley β-glucan. Km and Vmax values of J1 were 1.53 mg/ml and 8,511 μU/ml.min, respectively, while those for pJ2 were 4.36 mg/ml and 7,397 μU/ml.min, respectively. Degradation of barley β-1, 3-1,4-glucan resulted in four different oligosaccharides with 1,3 linkages triose, tetrose, pentose and a high molecular weight (HMW) with 1,3 linkage estimated from their mobilities. The quantitative degradation by the crude enzyme after of incubation yielded in descending order: triose, pentose and tetrose, while that of J1 in descending order yielded: pentose, triose and tetrose. The pJ2 showed low activity yielding a degradation pattern in descending order: pentose, triose, tetraose and a HMW polysaccharide. (PUBLICATION ABSTRACT)