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.
Purification of beta-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 beta-1,3-1,4-glucanase J1 and pJ2 had optimum pH values of 6-6.5 and an optimum temperature of 60 degrees C. Both enzymes were not inhibited by Li(2+) but were inhibited significantly by Ca(2+), Cu(2+), Mn(2+) and Zn(2+). However, J1 was slightly inhibited by Fe(2+), while pJ2 was inhibited by Mg(2+) as well. They were highly specific to only barley beta-glucan. K (m) and V (max) values of J1 were 1.53 mg/ml and 8,511 microU/ml.min, respectively, while those for pJ2 were 4.36 mg/ml and 7,397 microU/ml.min, respectively. Degradation of barley beta-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.
Apiraksakorn
Jirawan
J
Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, 50 Paholyothin Rd., Jatujak, Bangkok, 10900, Thailand.
Nitisinprasert
Sunee
S
Levin
Robert E
RE
D001412:Bacillus subtilis / Q000187:drug effects / Q000201:enzymology*; D002413:Cations, Divalent / Q000494:pharmacology; D002846:Chromatography, Affinity; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D043326:Glucan 1,3-beta-Glucosidase / Q000302:isolation & purification*; D001467:Hordeum / Q000378:metabolism; D006868:Hydrolysis / Q000187:drug effects; D007526:Isoelectric Point; D007700:Kinetics; D009844:Oligosaccharides / Q000378:metabolism; D006109:Poaceae / Q000187:drug effects / Q000378:metabolism*; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature; D047071:beta-Glucans / Q000378:metabolism
D002413:Cations, Divalent; D009844:Oligosaccharides; D047071:beta-Glucans; C067858:beta-glucan, (1-3)(1-4)-; D043326:Glucan 1,3-beta-Glucosidase