A novel metalloproteinase, almelysin, from a marine bacterium, Alteromonas sp. No. 3696: purification and characterization.
We have discovered a novel metalloproteinase, which has high activity at low temperatures, from the culture supernatant of a marine bacterium. The strain was identified as Alteromonas sp. No. 3696. The metalloproteinase, named almelysin, was purified to homogeneity from the cultured supernatant at 10 degrees C by two column chromatographies. About 20 mg of purified almelysin was obtained from 18.4 liters of the culture supernatant. The molecular mass of almelysin was estimated to be 28 kDa by SDS-PAGE and the isoelectric point was 4.3. The optimum pH for activity of almelysin was pH 8.5-9.0 and 6.5 using casein and (7-methoxycoumarin-4-yl)acetyl(MOCAc)-Pro-Leu-Gly-Leu-(N3- [2,4-dinitrophenyl]-L-2,3-diaminopropionyl) [A2pr(Dnp)]-Ala-Arg-NH2 as substrates, respectively. Almelysin was stable between pH 7.5-8.0 and below 40 degrees C. The optimum temperature for the activity was observed to be 40 degrees C using both casein and MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2 as substrates. The activity of almelysin was inhibited by such metallo chelators as EDTA and o-phenanthroline, while talopeptin, phosphoramidon, and SMPI, typical metalloproteinase inhibitors, had no effect. Almelysin primarily cleaved the Ala14-Leu15 bond and Phe24-Phe25 bond, and secondarily the Tyr16-Leu17 bond in oxidized insulin B-chain. However, almelysin could not cleave the His5-Leu6, His10-Leu11, and Gly23-Phe24 bonds, which were cleaved by other metalloproteinases. These results indicate that the substrate specificity of almelysin is different from other metalloproteinases. Interestingly, Alteromonas sp. No. 3696 strain produced another proteinase as well as almelysin at 25 degrees C.
Shibata
M
M
Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Japan.
Takahashi
S
S
Sato
R
R
Oda
K
K
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006090:Gram-Negative Bacteria / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
D011480:Protease Inhibitors; D008666:Metalloendopeptidases; C105831:almelysin