Purification and characterization of thermostable beta-glucosidase from the brown-rot basidiomycete Fomitopsis palustris grown on microcrystalline cellulose.
An extracellular beta-glucosidase was purified 154-fold to electrophoretic homogeneity from the brown-rot basidiomycete Fomitopsis palustris grown on 2.0% microcrystalline cellulose. SDS-polyacrylamide gel electrophoresis gel gave a single protein band and the molecular mass of purified enzyme was estimated to be approximately 138 kDa. The amino acid sequences of the proteolytic fragments determined by nano-LC-MS/MS suggested that the protein has high homology with fungal beta-glucosidases that belong to glycosyl hydrolase family 3. The Kms for p-nitorophenyl-beta-D-glucoside (p-NPG) and cellobiose hydrolyses were 0.117 and 4.81 mM, and the Kcat values were 721 and 101.8 per sec, respectively. The enzyme was competitively inhibited by both glucose (Ki= 0.35 mM) and gluconolactone (Ki= 0.008 mM), when p-NPG was used as substrate. The optimal activity of the purified beta-glucosidase was observed at pH 4.5 and 70 degrees. The F. palustris protein exhibited half-lives of 97 h at 55 degrees and 15 h at 65 degrees, indicating some degree of thermostability. The enzyme has high activity against p-NPG and cellobiose but has very little or no activity against p-nitrophenyl-beta-lactoside, p-nitrophenyl-beta-xyloside, p-nitrophenyl-alpha-arabinofuranoside, xylan, and carboxymethyl cellulose. Thus, our results revealed that the beta-glucosidase from F. palustris can be classified as an aryl-beta-glucosidase with cellobiase activity.
Yoon
Jeong-Jun
JJ
Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea.
Kim
Ki-Yeon
KY
Cha
Chang-Jun
CJ
D000595:Amino Acid Sequence; D001487:Basidiomycota / Q000201:enzymology*; D002482:Cellulose / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry* / Q000302:isolation & purification*
D010455:Peptides; D002482:Cellulose; D001617:beta-Glucosidase; C109691:microcrystalline cellulose