Cloning of cold-active alkaline phosphatase gene of a psychrophile, Shewanella sp., and expression of the recombinant enzyme.
A psychrophilic alkaline phosphatase (EC 3.1.3.1) from Shewanella sp. is a cold-active enzyme that has high catalytic activity at low temperature [Ishida et al. (1998) Biosci. Biotechnol. Biochem., 62, 2246-2250]. Here, we identified the nucleotide sequence of a gene encoding the enzyme after cloning with the polymerase chain reaction (PCR) and inverted PCR techniques. The deduced amino acid sequence of the enzyme contained conserved amino acids found among mesophilic alkaline phosphatases and showed some structural characteristics including a high content of hydrophobic amino acid residues and the lack of single alpha-helix compared with the alkaline phosphatase of Escherichia coli, which were possibly efficient for catalytic reaction at low temperatures. The recombinant enzyme expressed in E. coli was purified to homogeneity with the molecular mass of 41 kDa. The recombinant enzyme had a specific activity of 1,500 units/mg and had high catalytic activity at low temperatures.
Murakawa
Takeshi
T
Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Hyogo, Japan.
Yamagata
Hiroshi
H
Tsuruta
Hiroki
H
Aizono
Yasuo
Y
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D020592:Shewanella / Q000201:enzymology* / Q000235:genetics; D013816:Thermodynamics
D010446:Peptide Fragments; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase