Thermostable aspartase from a marine psychrophile, Cytophaga sp. KUC-1: molecular characterization and primary structure.
We found that a psychrophilic bacterium isolated from Antarctic seawater, Cytophaga sp. KUC-1, abundantly produces aspartase [EC4.3.1.1], and the enzyme was purified to homogeneity. The molecular weight of the enzyme was estimated to be 192,000, and that of the subunit was determined to be 51,000: the enzyme is a homotetramer. L-Aspartate was the exclusive substrate. The optimum pH in the absence and presence of magnesium ions was determined to be pH 7.5 and 8.5, respectively. The enzyme was activated cooperatively by the presence of L-aspartate and by magnesium ions at neutral and alkaline pHs. In the deamination reaction, the K(m) value for L-aspartate was 1.09 mM at pH 7.0, and the S(1/2) value was 2.13 mM at pH 8.5. The V(max) value were 99.2 U/mg at pH 7.0 and 326 U/mg at pH 8.5. In the amination reaction, the K(m) values for fumarate and ammonium were 0.797 and 25.2 mM, respectively, and V(max) was 604 U/mg. The optimum temperature of the enzyme was 55 degrees C. The enzyme showed higher pH and thermal stabilities than that from mesophile: the enzyme was stable in the pH range of 4.5-10.5, and about 80% of its activity remained after incubation at 50 degrees C for 60 min. The gene encoding the enzyme was cloned into Escherichia coli, and its nucleotides were sequenced. The gene consisted of an open reading frame of 1,410-bp encoding a protein of 469 amino acid residues. The amino acid sequence of the enzyme showed a high degree of identity to those of other aspartases, although these enzymes show different thermostabilities.
Kazuoka
Takayuki
T
Department of Biotechnology, Faculty of Engineering, Kansai University, Suita, Osaka 564-8680.
Masuda
Yuki
Y
Oikawa
Tadao
T
Soda
Kenji
K
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001220:Aspartate Ammonia-Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003080:Cold Temperature; D003589:Cytophaga / Q000201:enzymology*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021122:Protein Subunits; D016415:Sequence Alignment; D013696:Temperature
D000596:Amino Acids; D008670:Metals; D021122:Protein Subunits; D001220:Aspartate Ammonia-Lyase