The optimal culture conditions for attaining high levels of expression of mouse alpha-amylase in the methylotrophic yeast Pichia pastoris were investigated. Cell growth and enzyme production were found to be optimal at a temperature of 28 degrees C and a pH of 6.0. Yeast extract and peptone were the most appropriate nitrogen sources for the expression of alpha-amylase. When a mixture of 20 g/l peptone and 20 g/l yeast extract was used as the nitrogen source, alpha-amylase activity reached 300 U/ml. The expression of alpha-amylase in a fed-batch culture using methanol as the sole carbon source reached 720 U/ml at 3 days of culture. However, when a methanol-to-glycerol mixture was used at ratio of 1:0.5 at a feed rate of 3 g/h, the maximum a-amylase activity was 1.14 kU/ml. Under the optimal culture conditions, when 40 g/l of a mixture of yeast extract and peptone (1:1) was added to the culture at 3 and 5 days the final alpha-amylase activity increased to 2.4 kU/ml, which was 5.8- and 2.1- fold higher than the activity in the batch culture and in the fed-batch culture with feeding a mixture of methanol and glycerol at a ratio of 1:0.5, respectively. This study demonstrates that an addition of the appropriate amount of an organic nitrogen source to the fed-batch culture is effective to improve high level of expression of mouse alpha-amylase in the methylotrophic yeast Pichia pastoris. (c) 2005 Elsevier Ltd. All rights reserved.