Purification and cloning of a thermostable manganese catalase from a thermophilic bacterium.
We have purified a heat-stable catalase from a thermophilic bacterium, Thermus species strain YS 8-13. The enzyme was purified 160-fold from crude cellular extracts and possessed a specific activity of 8000 units/mg at 65 degrees C. The purified enzyme displayed the highest activity at pH 7 to 10 and temperatures around 85 degrees C. The catalase was determined to be a manganese catalase, based on results from atomic absorption spectra and inhibition experiments using sodium azide. The enzyme was composed of six identical subunits of molecular weight 36,000. Amino acid sequences determined from the purified protein were used to design oligonucleotide primers, which were in turn used to clone the coding gene. The nucleotide sequence of a 1.4-kb fragment of Thermus sp. YS 8-13 genomic DNA containing a 909-bp open reading frame was determined. The gene encoded a 302-residue polypeptide of deduced molecular weight 33,303. The deduced amino acid sequence displayed a region-specific homology with the sequences of the manganese catalase from a mesophilic organism, Lactobacillus plantarum.
Kagawa
M
M
Faculty of Engineering, Tottori University, Koyama-Minami, Tottori, 680-8552, Japan.
Murakoshi
N
N
Nishikawa
Y
Y
Matsumoto
G
G
Kurata
Y
Y
Mizobata
T
T
Kawata
Y
Y
Nagai
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002374:Catalase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008345:Manganese / Q000032:analysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames / Q000235:genetics; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D015183:Restriction Mapping; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D019810:Sodium Azide / Q000494:pharmacology; D013054:Spectrophotometry, Atomic; D013696:Temperature; D013824:Thermus / Q000201:enzymology*