Purification and characterization of a beta-1,4-glucosidase from a newly isolated strain of Fomitopsis pinicola.
An efficient beta-1,4-glucosidase (BGL) producing strain, Fomitopsis pinicola KMJ812, was isolated and identified based on morphological features and sequence analysis of internal transcribed spacer rDNA. An extracellular BGL was purified to homogeneity by sequential chromatography of F. pinicola culture supernatants on a DEAE-sepharose column, a gel filtration column, and then on a Mono Q column with fast protein liquid chromatography. The relative molecular weight of F. pinicola BGL was determined to be 105 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis, or 110 kDa by size exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the BGL had a pH optimum of 4.5 and a temperature optimum of 50 degrees C. The enzyme showed high substrate specificity and high catalytic efficiency (kcat=2,990 s-1, Km=1.76mM, kcat/Km=1,700 mM-1 s-1) for p-nitrophenyl-beta-d-glucopyranoside. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase family 3, indicating that the F. pinicola BGL is a member of glycoside hydrolase family 3. Although BGLs have been purified and characterized from several other sources, F. pinicola BGL is distinguished from other BGLs by its high catalytic efficiency and strict substrate specificity.
Joo
Ah-Reum
AR
Department of Chemical Engineering, Konkuk University, Seoul, 143-701, Korea.
Jeya
Marimuthu
M
Lee
Kyoung-Mi
KM
Sim
Won-Il
WI
Kim
Jung-Soo
JS
Kim
In-Won
IW
Kim
Yeong-Suk
YS
Oh
Deok-Kun
DK
Gunasekaran
Paramasamy
P
Lee
Jung-Kul
JK
D044602:Cellulases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D055453:Coriolaceae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012988:Soil Microbiology; D013379:Substrate Specificity
D005656:Fungal Proteins; D044602:Cellulases