The reactions were stopped by freezing the flasks at −80 °C and the

hydrolyzed samples were lyophilised. Isoflavones were extracted from the lyophilised samples (1 g) with 5 mL of 80% methanol by stirring for 2 h at room temperature. The mixtures were centrifuged at 16,100g for 10 min and the supernatants were filtered through a 0.45 μm filter for analysis of the isoflavones via HPLC. The contents and compositions of isoflavones were determined PD-0332991 purchase quantitatively by HPLC. The HPLC system used was a Shimadzu HPLC (Kyoto, Japan), consisting of an LC-10AD pump, a UV detector (SPD-10AV) and a Shim-pack CLC-ODS (M) column (4.6 × 250 mm) (Shimadzu Co., Kyoto, Japan). The mobile phase consisted of solvent (A) composed of 0.1% (v/v) acetic acid in filtered MilliQ water, and (B) solvent consisting of 0.1% (v/v) acetic acid in acetonitrile. The following gradient for solvent B was applied: 15–25% from 0 to 35 min, 25–26.5% over the next 12 min and 26.5–50% over 30 s followed by isocratic elution for 14.5 min. The flow rate was 1.0 mL/min, column temperature was 40 °C and the absorbance was LY294002 molecular weight measured at 254 nm. Isoflavone content of the samples was calculated by interpolation of the calibration curves prepared using

varying concentrations of the 12 isoflavone standards. D. Hansenii UFV-1 grown in YP medium containing cellobiose as carbon source presented expressive biomass production and intracellular β-glucosidase activity (data not shown). The yeast exhibited intracellular β-glucosidase activity and biomass production of 0.016 U/mL and 4.36 mg/mL, respectively, when cultivated during 12 h in the YP medium with cellobiose. Cellobiose was the most effective sugar tested for induction of growth and intracellular β-glucosidase

activity in D. hansenii UFV-1. Extracellular β-glucosidase production induced by cellobiose was reported for Debaryomyces vanrijiae and Debaryomyces pseudopolymorphus ( Belancic et al., 2003 and Villena et al., 2006). Different from the others, D. hansenii UFV-1 did not secrete β-glucosidase when grown on cellobiose. The presence of this intracellular Terminal deoxynucleotidyl transferase enzyme could suggest that D. hansenii presents a cellobiose transporter. Several yeast species including Clavispora lusitaniae, Candida wickerhamii, Debaryomyces polymorphus and Pichia guillermondii have the ability to transport cellobiose across the plasma membrane ( Freer, 1991 and Freer and Greene, 1990). Kluyveromyces lactis produces an intracellular β-glucosidase, implying that this yeast also has the ability to transport cellobiose into the cell ( Tingle & Halvorson, 1972). Results of D. hansenii UFV-1 β-glucosidase purification are summarised in Table 1. After dialysis, the enzymatic extract was subjected to ion exchange chromatography, resulting in the separation of one protein fraction with β-glucosidase activity, which was eluted with 0.1 M NaCl. This step promoted considerable specific activity enrichment ( Table 1).