The Escherichia coli phytase gene appA was highly expressed in the methylotrophic yeast Pichia pastoris under the control of the AOX1 promoter. Replacement of culture medium with fresh medium in order to remove repressing glycerol and metabolic wastes prior to methanol induction significantly improved phytase expression. The phytase activity level was enhanced from 118 to 204U/ml at the flask scale and 1880-4946 U/ml for high cell-density fermentation, respectively, by appropriately modifying the medium composition and fermentation strategy. Most of the protein in the culture supernatant was recombinant phytase, the enzyme characteristics of which were similar to native E. coli phytase. (C) 2004 Elsevier Inc. All rights reserved.