One-step purification and characterization of a beta-1,4-glucosidase from a newly isolated strain of Stereum hirsutum.
A highly efficient beta-1,4-glucosidase (BGL) secreting strain, Stereum hirsutum SKU512, was isolated and identified based on morphological features and sequence analysis of internal transcribed spacer rDNA. A BGL containing a carbohydrate moiety was purified to homogeneity from S. hirsutum culture supernatants using only a single chromatography step on a gel filtration column. The relative molecular weight of S. hirsutum BGL was determined as 98 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis or 780 kDa by size exclusion chromatography, indicating that the enzyme is an octamer. S. hirsutum BGL showed the highest activity toward p-nitrophenyl-beta-D-glucopyranoside (V (max) = 3,028 U mg-protein(-1), k (cat) = 4,945 s(-1)) ever reported. The enzyme also showed good stability at an acidic pH ranging from 3.0 to 5.5. The BGL was able to promote transglycosylation with an activity of 42.9 U mg-protein(-1) using methanol as an acceptor and glucose as a donor. The internal amino acid sequences of the isolated enzyme showed significant homology with hydrolases from glycoside hydrolase family 1 (GH1), indicating that the S. hirsutum BGL is a member of GH1 family. The characteristics of S. hirsutum BGL could prove to be of interest in several potential applications, especially in enhancing flavor release during the wine fermentation process.
Department of Chemical Engineering, Konkuk University, Seoul, Republic of Korea.
Ngoc-Phuong-Thao
NP
Nguyen
Kyoung-Mi
KM
Lee
Kyoung-Min
KM
Lee
In-Won
IW
Kim
Yeong-Suk
YS
Kim
Marimuthu
M
Jeya
Jung-Kul
JK
Lee
D001487:Basidiomycota / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000302:isolation & purification*; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D012988:Soil Microbiology; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D005656:Fungal Proteins; D001617:beta-Glucosidase