Purification and characterization of thermostable aspartase from Bacillus sp. YM55-1.
A thermostable aspartase was purified from a thermophile Bacillus sp. YM55-1 and characterized in terms of activity and stability. The enzyme was isolated by a 5-min heat treatment at 75 degrees C in the presence of 11% (w/v) ammonium sulfate and 100 mM aspartate, followed by Q-Sepharose anion-exchange and AF-Red Toyopearl chromatographies. The native molecular weight of aspartase determined by gel filtration was about 200,000, and this enzyme was composed of four identical monomers with molecular weights of 51,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Unlike Escherichia coli aspartase, the enzyme was not activated by the presence of magnesium ion at alkaline pH. At the optimum pH, the Km and Vmax were 28.5 mM and 700 units/mg at 30 degrees C and 32.0 mM and 2200 units/mg at 55 degrees C, respectively. The specific activity was four and three times higher than those of E. coli and Pseudomonas fluorescens enzymes at 30 degrees C, respectively. Eighty percent of the activity was retained after a 60-min incubation at 55 degrees C, and the enzyme was also resistant to chemical denaturants; 80% of the initial specific activity was detected in assay mixtures containing 1.0 M guanidine hydrochloride. The purified enzyme shared a high sequence homology in the N-terminal region with aspartases from other organisms.
Kawata
Y
Y
Faculty of Engineering, Tottori University, Tottori, 680-0945, Japan. kawata@bio.tottori-u.ac.jp
Tamura
K
K
Yano
S
S
Mizobata
T
T
Nagai
J
J
Esaki
N
N
Soda
K
K
Tokushige
M
M
Yumoto
N
N
D000595:Amino Acid Sequence; D001220:Aspartate Ammonia-Lyase / Q000187:drug effects / Q000302:isolation & purification*; D001407:Bacillus / Q000201:enzymology*; D004795:Enzyme Stability; D019791:Guanidine / Q000494:pharmacology; D006358:Hot Temperature; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D001220:Aspartate Ammonia-Lyase; D019791:Guanidine