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- Title
Improving the thermostability of Escherichia coli phytase, appA, by enhancement of glycosylation.
- Authors
Yao, Ming-Ze; Wang, Xi; Wang, Wei; Fu, Yue-Jun; Liang, Ai-Hua
- 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 and enzyme activity reached 1,030 U ml. Purified appA-Q258N/Q349N had a specific activity of 3,137 U mg 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 (T). The K and K of appA-Q258N/Q349N were 0.43 mM and 3,058 s, 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.
- Subjects
ESCHERICHIA coli; GLYCOSYLATION; PICHIA pastoris; PHYTASES; ENZYMES
- Publication
Biotechnology Letters, 2013, Vol 35, Issue 10, p1669
- ISSN
0141-5492
- Publication type
Article
- DOI
10.1007/s10529-013-1255-x