Cloning, purification and characterisation of a recombinant purine nucleoside phosphorylase from Bacillus halodurans Alk36.
A purine nucleoside phosphorylase from the alkaliphile Bacillus halodurans Alk36 was cloned and overexpressed in Escherichia coli. The enzyme was purified fivefold by membrane filtration and ion exchange. The purified enzyme had a V (max) of 2.03 x 10(-9) s (-1) and a K (m) of 206 microM on guanosine. The optimal pH range was between 5.7 and 8.4 with a maximum at pH 7.0. The optimal temperature for activity was 70 degrees C and the enzyme had a half life at 60 degrees C of 20.8 h.
Visser
Daniel F
DF
Enzyme Technologies Group, CSIR Biosciences, Private Bag X2, Modderfontein, Johannesburg, 1645, South Africa.
Hennessy
Fritha
F
Rashamuse
Konanani
K
Louw
Maureen E
ME
Brady
Dean
D
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011683:Purine-Nucleoside Phosphorylase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein; D013816:Thermodynamics
D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D011683:Purine-Nucleoside Phosphorylase