Cloning of Cold-active Alkaline Phosphatase Gene of a Psychrophile,Shewanellasp., and Expression of the Recombinant Enzyme

Abstract

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.

Adenosine monophosphate degradation and inosinic acid accumulation in the shrimp Penaeus japonicus

“…The species of Shewanella identified in our study had different activity, but as there are many different species within the genus, multiple phosphatases are likely. At present, the type(s) of phosphatase(s) in the three species identified in this study are not known, although several studies Murakawa et al 2002;Yasuda et al 2002;Yamaguchi et al 2006;Tsuruta et al 2010) have examined phosphatase production in Shewanella spp. The species of bacteria identified in our study probably produce a phosphatase that degrades AMP to AdR in shrimp.…”

Gene Cloning, Overproduction, and Characterization of Thermolabile Alkaline Phosphatase from a Psychrotrophic Bacterium

“…[17][18][19] Previously, we reported that RNase HI 22) and FKBP22 23) from Shewanella sp. SIB1 show the highest amino acid sequence identities, of 78% and 85% respectively, to those from Shewanella oneidensis MR-1.…”

“…30) It shows amino acid sequence identities of 62% to SCAPase, 19) 33% to TAB5 APase, 17) 37% to E. coli APase, 31) 42% to B. subtilis APase III, 32) 35% to Thermus sp. FD3041 APase (unpublished, accession no.…”

“…18) The recombinant protein of SCAPase did not show typical features of a cold enzyme. Its optimal temperature for activity is 70 C, 19) which is shifted upward by 30 C compared to that of the native enzyme.…”

“…G15-21 (Vibrio APase), 18) and the psycrophile Shewanella sp. (SCAPase) 19) were cloned and overexpressed in E. coli. These proteins show amino acid sequence identities of 32-34% to E. coli APase but these proteins may not be suitable as a partner of E. coli APase to analyze the cold-adaptation mechanism of enzymes for the following reasons.…”