Cloning, sequencing, and expression of an Escherichia coli acid phosphatase/phytase gene (appA2) isolated from pig colon.
Bacterial strains were isolated from the pig colon to screen for phytase and acid phosphatase activities. Among 93 colonies, Colony 88 had the highest activities for both enzymes and was identified as an Escherichia coli strain. Using primers derived from the E. coli pH 2.5 acid phosphatase appA sequence (Dassa et al. (1990), J. Bacteriol. 172, 5497-5500), we cloned a 1482 bp DNA fragment from the isolate. In spite of 95% homology between the sequenced gene and the appA, 7 amino acids were different in their deduced polypeptides. To characterize the properties and functions of the encoded protein, we expressed the coding region of the isolated DNA fragment and appA in Pichia pastoris, respectively, as r-appA2 and r-appA. The recombinant protein r-appA2, like r-appA and the r-phyA phytase expressed in Aspergillus niger, was able to hydrolyze phosphorus from sodium phytate and p-nitrophenyl phosphate. However, there were distinct differences in their pH profiles, Km and Vmax for the substrates, specific activities of the purified enzymes, and abilities to release phytate phosphorus in soybean meal. In conclusion, the DNA fragment isolated from E. coli in pig colon seems to encode for a new acid phosphatase/phytase and is designated as E. coli appA2.
Rodriguez
E
E
Department of Animal Science, Cornell University, Ithaca, New York 14853-4801, USA.
Han
Y
Y
Lei
X G
XG
D010832:6-Phytase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000135:Acid Phosphatase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003106:Colon / Q000737:chemistry* / Q000382:microbiology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005658:Fungi / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D006031:Glycosylation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010758:Phosphorus / Q000378:metabolism; D010833:Phytic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013552:Swine / Q000382:microbiology*; D013696:Temperature
D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; D010758:Phosphorus; C008644:nitrophenylphosphate; D010833:Phytic Acid; D000135:Acid Phosphatase; D010832:6-Phytase; C118716:AppA2 protein, E coli