Cells were then washed three times with PBS buffer before being resuspended in 0.5 mL PBS containing 4% formaldehyde. The presence of phytase on the P. pastoris cell surface was detected by fluorescence microscopy. Yeast cell wall was isolated according to Schreuder et al. (1993) with modifications. After induction, cells were harvested by centrifugation,

washed three times in ice-cold isolation buffer [10 mM Tris-HCl, pH 8, 1 mM phenylmethanesulfonyl fluoride (PMSF)], and resuspended in 10 mL of isolation buffer. Aliquots of 1 mL cells were lysed by glass beads (0.05 mm diameter) and the supernatant was then collected. Cell wall fractions were harvested from the supernatant by centrifugation HSP inhibitor at 1000 g, 4 °C for 5 min, and then washed three times with 1 mM PMSF. Laminarinase 10 mU (Sigma-Aldrich) was added to 100 mg (wet weight) of cell wall fraction resuspended in 200 μL reaction buffer (100 mM sodium acetate, pH 5, 1 mM PMSF). The reaction was allowed to proceed for 2 h at 37 °C, after which another 10 mU of fresh laminarinase was added to the reaction. The reaction was then continued CHIR-99021 purchase for another

2 h, for a total of 4 h. After the reaction was complete, the supernatant was collected by centrifugation at 10 000 g for 5 min before being used to test enzyme activity or analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Phytase activity was quantified according to the method described in Engelen et al. (1994). One phytase activity unit was defined as the amount of enzyme that liberates 1 μmol inorganic phosphate min−1. To determine the effect of pH on cell-surface phytase, a pH range from 2 to 10 was used with the following (100 mM) buffers: glycine-HCl (pH 2.0–4.0), acetic acid (pH 5.0–6.0), 3-(N-morpholine)propanesulfonic acid (pH 7.0–8.0) and Tris-HCl (pH 9.0–10.0). The optimal temperature was determined in the range of 30–70 °C in 100 mM acetate buffer, pH 5.5. For

the pH stability test, the enzyme was preincubated at 25 °C for 4 h in buffers with pH values of 2.0–10.0 as described above. Enzyme activity was then measured at 50 °C in 100 mM acetate buffer, pH 5.5. Temperature stability profiles Pregnenolone were determined by incubating the enzyme at temperatures of 40–80 °C for 30–120 min. The relative activity was calculated by comparing the activity remaining after each treatment with that of the untreated enzyme, which was assigned as 100%. Resistance to pepsin and trypsin was investigated following Promdonkoy et al. (2009). The in vitro digestibility test was performed according to Promdonkoy et al. (2009). For proximate analysis, cells were added to feedstuff to obtain 4 U phytase activity g–1 feedstuff (approximately 6% w/w). Then, the contents of the sample were compared with sample feedstuff without the addition of yeast cells. The analysis was completed by the Central Laboratory (Thailand) Co. Ltd. Phytase r-PhyA170 (Promdonkoy et al.