The production of reducing sugar was determined using RG7420 cost the 3.5-dinitrosalicylate reagent where sucrose and cellulose were used as substrates (Miller, 1959). One

unit of enzyme activity (U) was defined as the amount of enzyme that releases 1.0 μmol of product per min under the assay conditions. Data presented for β-glucosidase activity is the mean of assays performed in triplicate. Protein concentration in the enzymatic extracts was determined by the BCA (bicinchoninic acid) method (Smith et al., 1985) with bovine serum albumin (BSA) as the standard. The molecular weight (MW) of the purified enzyme was estimated by SDS–PAGE using a 12.5% (w/v) polyacrylamide gel (Laemmli, 1970). The molecular mass standards were obtained from Sigma Aldrich (Sigma Markers Wide Range MW 6500–200,000 Da, St. Louis, MO, USA). After electrophoresis, the proteins were visualised by silver staining (Blum, Beier, & Gross, 1987). The protocol used for permeabilisation of D. hansenii UFV-1 cells

was the same as that reported by Junior et al., 2009, with some alterations. Yeast culture samples were centrifuged (25,900g for 5 min at 4 °C) and the pellet was resuspended in a 50% (v/v) ethanol solution at the proportion of 450 μL of this solvent selleck screening library to 0.2 g of cells. After agitation for 5 min at room temperature, the suspension was centrifuged (4000g for 5 min at 4 °C) and the permeabilised cells were dried for 1 h at 37 °C. The protocol used for immobilisation of permeabilised D. hansenii UFV-1 cells was the same as that reported by Junior et al., 2009, with some alterations. The dry permeabilised cells were mixed with a 2% (w/v) sodium alginate solution, in a proportion

of 4 g of cells to 1 g of alginate. This suspension was extruded through a hypodermic needle using a peristaltic pump to obtain a uniform particle size. The droplets eluted from the hypodermic needle were collected in a flask, containing 0.1 M CaCl2 solution to form alginate beads. The beads were maintained in a 0.1 M CaCl2 solution for 12 h at 4 °C. They were subsequently washed three times with 0.1 M sodium phosphate buffer pH 5.5 and kept at 4 °C in the same buffer until utilisation. The assay of PAK6 re-use of the alginate beads was performed using pNPβGlc or isoflavones as substrates. Ten millilitres of 2 mM pNPβGlc in 50 mM sodium phosphate buffer pH 5.5 and 40 alginate beads were added to 25 mL Erlenmeyer flasks and incubated under agitation (100 rpm) at 50 °C. After 15 min incubation time, an aliquot (100 μL) of solution was taken and the amount of pNP was determined. The isoflavones hydrolysis assay was performed according item 2.12, except that the temperature was 50 °C. After this first cycle, the beads were separated by filtration, washed with 50 mM sodium phosphate buffer pH 5.