Improvement of Yersinia frederiksenii phytase performance by a single amino acid substitution.
A new phytase (APPA) with optimum pH 2.5--substantially lower than that of most of microbial phytases (pH 4.5-6.0)--was cloned from Yersinia frederiksenii and heterologously expressed in Escherichia coli. Containing the highly conserved motifs typical of histidine acid phosphatases, APPA has the highest identity (84%) to the Yersinia intermedia phytase (optimal pH 4.5), a member of histidine acid phosphatase family. Based on sequence alignment and molecular modeling of APPA and related phytases, APPA has only one divergent residue, Ser51, in close proximity to the catalytic site. To understand the acidic adaptation of APPA, five mutants (S51A, S51T, S51D, S51K, and S51I) were constructed by site-directed mutagenesis, expressed in E. coli, purified, and characterized. Mutants S51T and S51I exhibited a shift in the optimal pH from 2.5 to 4.5 and 5.0, respectively, confirming the role of Ser51 in defining the optimal pH. Thus, a previously unrecognized factor other than electrostatics--presumably the side-chain structure near the active site--contributes to the optimal pH for APPA activity. Compared with wild-type APPA, mutant S51T showed higher specific activity, greater activity over pH 2.0-5.5, and increased thermal and acid stability. These properties make S51T a better candidate than the wild-type APPA for use in animal feed.
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China.
Dawei
D
Fu
Huoqing
H
Huang
Kun
K
Meng
Yaru
Y
Wang
Huiying
H
Luo
Peilong
P
Yang
Tiezheng
T
Yuan
Bin
B
Yao
D010832:6-Phytase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D015007:Yersinia / Q000201:enzymology*
D010832:6-Phytase