Collagenolytic subtilisin-like protease from the deep-sea bacterium Alkalimonas collagenimarina AC40T.
A new alkaline protease (AcpII) was purified from a culture of the deep-sea bacterium Alkalimonas collagenimarina AC40(T). AcpII degraded collagen three times faster than it degraded casein. The optimal pH was 8.5-9, and the optimal temperature was 45 degrees C for the degradation of collagen. AcpII was completely inhibited by phenylmethylsulfonyl fluoride and partially by EDTA. Cloning and sequencing the gene for AcpII revealed a 2,283-bp open reading frame encoding a protein of 760 amino acids. AcpII comprises a prepropeptide, a catalytic domain that includes a protease-associated domain (PA domain), and tandem repeat prepeptidase C-terminal domains. To elucidate the role of the PA domain of AcpII, we constructed genes for two enzyme derivatives that possessed the catalytic domains with or without the PA domain and expressed them in Escherichia coli. The derivative without the PA domain showed increased specific activities toward all proteinaceous substrates tested, including gelatin, casein, and collagen, compared with those of the derivative with the PA domain.
Kurata
Atsushi
A
Institute of Biogeoscience, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima, Yokosuka, 237-0061, Japan. kurata090401@nara.kindai.ac.jp
Uchimura
Kohsuke
K
Kobayashi
Tohru
T
Horikoshi
Koki
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001483:Base Sequence; D003094:Collagen / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004492:Edetic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli; D020563:Gammaproteobacteria / Q000201:enzymology* / Q000302:isolation & purification; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011994:Recombinant Proteins; D012623:Seawater / Q000382:microbiology*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D010664:Phenylmethylsulfonyl Fluoride; D003094:Collagen; D004492:Edetic Acid; D010447:Peptide Hydrolases