One-step purification and characterization of a β-1,4-glucosidase from a newly isolated strain of Stereum hirsutum

Abstract

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.

Hydrolytic and phosphorolytic metabolism of cellobiose by the marine aerobic bacterium Saccharophagus degradans 2-40T

“…This transglycosylation activity mirrored the catalytic specificity of the enzymes. This phenomenon is more common with fungal and archaeal b-glucosidases [6,14,23]. Transglycosylation indicates the formation of an enzyme-substrate intermediate during catalysis that appears to involve the non-reducing end glucosyl unit.…”

Characterization of a recombinant aryl β-glucosidase from Neosartorya fischeri NRRL181

“…The evidence from enzymology and MD simulation studies strongly suggests that NfBGL1 is a member of the GH1 family of glycoside hydrolases. Although experiments have been conducted on the characterization of recombinant BGLs from various fungal strains (Table 3), NfBGL1 catalyzes the hydrolysis of pNPG with a high level of V max , demonstrating its potential for use in industrial applications including flavor enhancement during the wine fermentation process (Barbagallo et al 2004;Nguyen et al 2010). The successful identification and over-expression of the NfBGL1 allows us to characterize a BGL showing high specificity towards pNPG and now sets the stage for more detailed investigations of this enzyme such as X-ray crystallography and protein engineering studies to figure out the molecular determinant of the high V max and unique substrate specificity.…”

“…In addition, there is an increasing demand for the production of BGL in the conversion of cellulose to glucose for the subsequent production of fuel ethanol (Saha et al 1994). BGL has been the focus of much research recently because of their important roles in a variety of fundamental biological processes Joo et al 2009;Nguyen et al 2010;Singh et al 2011).…”

A novel thermophilic β-glucosidase from Caldicellulosiruptor bescii: Characterization and its synergistic catalysis with other cellulases

“…Moreover, the use of higher temperatures allows for the hydrolysis to be more consistent due to the lower viscosity at elevated temperatures, thus ultimately leading to improved performance and lower production costs [5,6]. A number of thermostable BGLs have been investigated, with some studies focusing on the biochemical and kinetic properties of the enzyme [7,8], mode of action [9,10], and its use in the conversion of cellulose to glucose [11,12]. However, major barriers to the commercial development of cellulose enzymatic hydrolysis still exist, such as thermal inactivation, substrate inhibition, product inhibition and the high cost of BGL [13].…”