Improving the thermostability of Escherichia coli phytase, appA, by enhancement of glycosylation |
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Authors: | Ming-Ze Yao Xi Wang Wei Wang Yue-Jun Fu Ai-Hua Liang |
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Institution: | 1. Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan, 030006, People’s Republic of China
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Abstract: | 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. |
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