title: Improving the thermostability of Escherichia coli phytase, appA, by enhancement of glycosylation.
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.
authors.affiliation: Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan, 030006, People's Republic of China.
authors.forename: Ming-Ze
authors.initials: MZ
authors.lastname: Yao
authors.forename: Xi
authors.initials: X
authors.lastname: Wang
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Yue-Jun
authors.initials: YJ
authors.lastname: Fu
authors.forename: Ai-Hua
authors.initials: AH
authors.lastname: Liang
mesh_terms: D010832:6-Phytase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000135:Acid Phosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D007700:Kinetics; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000201:enzymology; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D000135:Acid Phosphatase; D010832:6-Phytase; C406788:appA protein, E coli