A codon-optimized Escherichia coli appA phytase gene was synthesized and expressed in Pichia pastoris. Two residue substitutions (Q258N, Q349N) were sequentially introduced to enhance its glycosylation activity. Secretion of appA-Q258N/Q349N was approx. 0.3 mg ml(-1) and enzyme activity reached 1,030 U ml(-1). Purified appA-Q258N/Q349N had a specific activity of 3,137 U mg(-1) with an MW of approx. 53 kDa. Compared with appA-WT, appA-Q258N/Q349N showed over 40 % enhancement in thermostability (85 °C for 10 min) and 4-5 °C increases in the melting temperatures (Tm). The Km and Kcat of appA-Q258N/Q349N were 0.43 mM and 3,058 s(-1), respectively, which are similar with that of appA-WT. The mutant appA-Q258N/Q349N obtained in this study could be used for the large-scale commercial production of phytase.