Extremely high alkaline protease from a deep-subsurface bacterium, Alkaliphilus transvaalensis.
A new high-alkaline protease (ALTP) was purified to homogeneity from a culture of the strictly anaerobic and extremely alkaliphilic Alkaliphilus transvaalensis. The molecular mass was 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme showed the maximal caseinolytic activity higher than pH 12.6 in KCl-NaOH buffer at 40 degrees C. Hydrolysis of the oxidized insulin B-chain followed by mass spectrometric analysis of the cleaved products revealed that as many as 24 of the total 29 peptide bonds are hydrolyzed in a block-cutting manner, suggesting that ALTP has a widespread proteolytic functions. Calcium ion had no effect on the activity and stability of ALTP, unlike known subtilisins. The deduced amino acid sequence of the enzyme comprised 279 amino acids plus 97 prepropeptide amino acids. The amino acid sequence of mature ALTP was confirmed by capillary liquid chromatography coupled to tandem mass spectrometry, which was the 93% coverage of the deduced amino acid sequence. The mature enzyme showed moderate homology to subtilisin LD1 from the alkaliphilic Bacillus sp. strain KSM-LD1 with 64% identity, and both enzymes formed a new subcluster at an intermediate position among true subtilisins and high-alkaline proteases in a phylogenetic tree of subtilase family A. ALTP is the first high-alkaline protease reported from a strict anaerobe in this family.
Kobayashi
Tohru
T
Extremobiosphere Research Center of Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15, Natsushima, Yokosuka, Japan. kobayashit@jamstec.go.jp
Lu
Jie
J
Li
Zhijun
Z
Hung
Vo Si
VS
Kurata
Atsushi
A
Hatada
Yuji
Y
Takai
Ken
K
Ito
Susumu
S
Horikoshi
Koki
K
D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005618:Fresh Water / Q000382:microbiology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D010450:Endopeptidases; C087822:alkaline protease