2.3 Expression of AGL in P. pastoris
Well-grown colonies were incubated in 5 mL of YPD medium (10 g/L yeast extract, 20 g /L peptone, and 20 g/L glucose) and grew overnight at 30°C and 200 rpm. Then, 0.5 mL of the culture was inoculated into 50 ml of BMGY medium (2% glycerol, 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base w/o amino acids, 4 x 10-5 % biotin and 100 mM sodium phosphate buffer, pH 6.0) in a 250-mL flask and incubated at 30°C and 200 rpm. When the culture turbidity (OD600) reached approximately 18–20, it was centrifuged (2000 x g ) for 10 min at 4 °C and the cell pellet was resuspended in 200 mL of BMGY[42]. The cells were cultured at 30 °C and 200 rpm for another 72 h. Glucose was added every 12 h to a final concentration of 1%. The cell culture (1 mL) was separated by centrifugation at 6,000 x g for 10 min and the supernatant was kept at 4 °C for AGL activity assay.
The recombinant P. pastoris with the highest yield of AGL was used for scale-up fermentation in a 5-L fermenter. The fermentation began in 2 L Basal Salt Medium (4% glycerol, 26.7 mL 85% H3PO4, 0.93 g CaSO4, 18.2 g K2SO4, 14.9 g MgSO4·7H2O, 4.13 g KOH and 2 mL 5% antifoam) containing 4.35 mL/L PTM1 salt solution (2 g/L CuSO4·5H2O, 7 g/L ZnCl2, 0.08 g/L NaI, 22.0 g/L FeSO4.7H2O, 3.0 g/L MnSO4·7H2O, 0.2 g/L biotin, 0.2 g/L Na2MoO4.2H2O, 0.02 g/L boric acid, 0.5 g/L CoCl2, 2 mL H2SO4) and lasted for about 16 h. Using 50% glycerol (w/v) containing 12 mL/L of PTM1 as carbon sources, the fed-batch fermentation of glycerol was initiated at 16 mL/L/h for 3 h. In the subsequent fermentation, the feeding rate was kept at 10 mL/L/h for 90 h. The temperature and pH during fermentation were maintained at 30 °C and 5.0, respectively. The dissolved oxygen was kept at around 20% and was automatically adjusted by cascading agitation speed (600–1000 rpm) [42]. The cell cultures (10 mL) were collected at the same time intervals, and the supernatants were obtained by centrifugation at 6,000 x g and 4 °C for 20 min for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis [43] and AGL activity determinations.