Document Detail

Improvement of Yersinia frederiksenii phytase performance by a single amino acid substitution.
MedLine Citation:
PMID:  19378262     Owner:  NLM     Status:  MEDLINE    
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.
Dawei Fu; Huoqing Huang; Kun Meng; Yaru Wang; Huiying Luo; Peilong Yang; Tiezheng Yuan; Bin Yao
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biotechnology and bioengineering     Volume:  103     ISSN:  1097-0290     ISO Abbreviation:  Biotechnol. Bioeng.     Publication Date:  2009 Aug 
Date Detail:
Created Date:  2009-06-22     Completed Date:  2009-08-03     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  7502021     Medline TA:  Biotechnol Bioeng     Country:  United States    
Other Details:
Languages:  eng     Pagination:  857-64     Citation Subset:  IM    
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China.
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MeSH Terms
6-Phytase / chemistry,  genetics*,  metabolism*
Amino Acid Sequence
Amino Acid Substitution*
Cloning, Molecular
Enzyme Stability
Escherichia coli / genetics
Gene Expression
Hydrogen-Ion Concentration
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Protein Structure, Tertiary
Sequence Alignment
Sequence Homology, Amino Acid
Yersinia / enzymology*
Reg. No./Substance:

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