For high-level expression of heterologous protein using a methylotrophic yeast, P. pastoris, in an air lift bioreactor, the initial methanol concentration and inoculation time were investigated. When 30 g/L of methanol was used, α-amylase activity was highest (235 U/mL). When the inoculation was carried out at 18 hr of culture, the maximum α-amylase activity was obtained (270 U/mL) and the consumed methanol concentration was 24.8 g/L. The α-amylase activity expressed in P. pasteris was stable up to 50℃ and showed 86.4% of residual activity at 60℃. However, in the case of S. cerevisiae, it remained only 57.3% under the same conditions. Between pH 6 and 7, the mouse α-amylase activity expressed in P. pasteris was stable. However, in the case of S. cerevisiae, it was stable between pH 7 and 8. Using the optimum culture conditions, the fed cultures were carried out for 4 days. The methanol consumption increased upon culture time up to 3 days, but after 4 days it began to decrease. The cell concentration was similar to that of S. cerevisiae. The maximum α-amylase activity was 610 U/mL at 4 days of culture; this value was about 6.0-fold higher than that of S. cerevisiae. In the case of product yield from methanol, it was 15.2 U/mL/g the consumed carbon source, which was about 11.5-fold higher than that of S. cerevisiae using glucose.