Alkaline phosphatase from the Antarctic strain TAB5 - Properties and psychrophilic adaptations

The gene encoding alkaline phosphatase (AP) from the psychrophilic strain T ABS was cloned, and its nucleotide sequence was determined. A single open r eading frame consisting of 1125 base pairs which encodes a polypeptide cons isting of signal peptide of 22 amino acids and a mature protein of 353 amin o acids was identified. The deduced protein sequence of AP exhibits a 38% i dentity to the AP III and AP IV sequences of Bacillus subtilis and conserve s the typical sequence motifs of the core structure and active sites of APs from various sources. Based on the crystal structure of the mutated Escher ichia coli AP D153H, a homology-based 3D model of the TABS AP was construct ed on the basis of which various features of the enzyme amino-acid sequence can be interpreted in terms of potential psychrophilic adaptations. The AP gene was expressed in E. coli BL21(DE3) cells, the recombinant prote in was isolated to homogeneity from the membrane fraction of the cells and its properties were examined. The purified TABS AP shows typical features o f a cold enzyme: high catalytic activity at low temperature and a remarkabl e thermosensitivity. The use of this heat-labile enzyme, for dephosphorylation of nucleic acids, simplifies dephosphorylation protocols.