Purification and characterization of a β-1,4-glucosidase from a newly isolated strain of Fomitopsis pinicola

Abstract

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.

Heterologous Expression and Characterization of a GH3 β-Glucosidase from Thermophilic Fungi Myceliophthora thermophila in Pichia pastoris

“…Usually, fungal β-glucosidases of family GH3 have an optimal pH at the acid range of pH 4.0-6.0 [5,24,25]; however, the MtBgl3b investigated in this study has a maximum activity at pH 5.0 and remained >70 % relative activity at pH 3.5-6.0. Besides the acidity, working temperature is another key character for industrial enzymes; thermophilic and thermostable enzymes have many overwhelming advantages in applications, because high reaction temperature cannot only increase the reaction rate but also reduce the enzyme dosage.…”

“…(60°C, [30]), and Ceriporiopsis subvermispora (60°C, [31]), lower than that of the β-glucosidases from Thermoascus aurantiacus (70°C, [28]), Aspergillus niger (70°C, [32]), and Fomitopsis palustris (70°C, [33]), but higher than the β-glucosidases from Stachybotrys sp. (50°C, [34]), Fomitopsis pinicola (50°C, [24]), and A. niger (55°C, [25]). Interestingly, the MtBgl3b we have cloned could be active at low temperature, the relative activity is 51 % at 40°C, which is better than β-glucosidase from Penicillium purpurogenum KJS506 (5.0 and 65°C, 20 % relative activity is 20 % of that at 40°C, [5]); this property might make the MtBgl3b having some application in the industry which needs low reaction temperature.…”

“…When using the pNPG as substrate, the K m and V max values of MtBgl3b were 2.78 mM and 927.9 μM mg −1 min −1 , respectively. Penicillium purpurogenum GH3, [5] Aspergillus fumigatus GH3, [9] Paecilomyces thermophila GH3, [14] Myceliophthora thermophila GH3, [22] Fomitopsis pinicola GH3, [24] Aspergillus niger GH1, [25] Thermoascus aurantiacus GH3, [28] Paenibacillus xylanilyticus GH3, [39] Serratia sp. TN49 GH3, [40] Phialophora sp.…”

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

“…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).…”

Two-dimensional zymography in detection of proteolytic enzymes in wheat leaves

“…Furthermore, the system has been successfully used for detection of glycohydrolases: glucanases and glucosidases, xylanases, mannases, hemicellulases, cellulases and ligninases (Joo et al 2009;Royer and Nakas 1990;Sakamoto and Toyohara 2009) as well as esterases, lipases (Kwon et al 2011) and nucleases (Cazenave and Toulme 2001). A crucial role in detection of enzymes activities using zymography play inhibitors.…”