D. PCI32765 hansenii

UFV-1 cells (15 g) were ground with liquid nitrogen and resuspended in 40 mL of 0.1 M sodium acetate buffer, pH 5.0, containing 0.25% (by weight) Triton X-100. This mixture was submitted to a series of nitrogen freezing and thawing at 40 °C in a water bath (Branson, USA). It was then submitted to an ultrasonic bath for 10 min and centrifuged (25,000g for 20 min at 4 °C). The supernatant was used as the source of intracellular enzyme. The enzymatic extract was submitted to dialysis against 4 L of 10 mM sodium phosphate buffer for 15 h at 4 °C. A dialysis membrane with 3 kDa of pore exclusion was used. After this procedure, the sample was loaded onto a DEAE-Sepharose anion exchange column (6.8 × 2.0 cm), equilibrated with 50 mM sodium phosphate buffer, pH 7 at 4 °C. Elution was performed at the flow rate of 60 mL/h, with a linear gradient formed with 150 mL of 50 mM sodium phosphate buffer and 150 mL of the same buffer containing 0.8 M NaCl. Fractions containing β-glucosidase activity were pooled and concentrated by Amicon ultrafiltration with a 3 kDa

molecular membrane cut off at 4 °C, 3500g for 1 h. The concentrated sample was subjected to FPLC (Fast Protein Liquid Chromatography) with a Sephacryl S-300 gel filtration column (26 × 60 cm), equilibrated with 25 mM sodium phosphate buffer, pH 7. The proteins were http://www.selleckchem.com/products/RO4929097.html eluted at a flow rate of 60 mL/h. Fractions containing β-glucosidase activity were pooled and loaded onto a phenyl-sepharose column (2.5 × 1.6 cm) coupled to the FPLC, equilibrated with 0.5 M ammonium sulfate in 25 mM sodium phosphate buffer, pH 7. The proteins were eluted at a flow rate of 240 mL/h with a linear gradient of ammonium sulfate (0.5–0 M) in 25 mM sodium phosphate buffer, pH 7. The active fractions were pooled and analysed for purity by SDS–PAGE. β-Glucosidase activity was assayed by measuring the Methane monooxygenase amount of p-nitrophenol (pNP) released from the hydrolysis of p-nitrophenyl-β-d-glucopyranoside (pNPβGlc) as substrate. Both, pNP and pNPβGlc, were purchased from Sigma Aldrich (USA). The standard reaction mixture contained 2 mM pNPβGlc, 50 mM sodium phosphate buffer (pH 6.0) and the enzyme preparation

in a final volume of 1.0 mL. After incubation at 40 °C for 15 min, 1.0 mL of 0.5 M sodium carbonate was added to the mixture to stop the reaction. The absorbance of the mixture was then measured at 410 nm. The amount of pNP released was calculated according to a standard curve, and one unit of enzyme activity (U) was defined as the amount of enzyme that releases 1.0 μmol of ρNP per min under the assay conditions. For β-glucosidase activity determination in immobilised cells, the assays were conducted with the same reagents but replacing the enzyme preparation with 4 alginate beads and modifying the pH of the phosphate buffer from 6.0 to 5.5. The activities against cellobiose, maltose, gentiobiose, lactose and melibiose were determined by the glucose oxidase method.