Purification and characterization of an alkaline elastase from Myxococcus xanthus

Abstract

An extracellular elastase, termed Myxococcus xanthus alkaline protease 1 (MAPl), has been purified from M. xanthus DK1622 culture supernatants by a combination of ion-exchange and affinity chromatographies. It consists of a single peptide chain of 39 kDa. The elastolytic activity was totally suppressed by 10 mM 1,lO-phenanthroline and the enzyme may then be classified as a metalloprotease. Its pH optimum was estimated to be 8.2 with both elastin-orcein and succinyl-Ala, p-nitroanilide as substrates. Despite its low PI (5.2), MAPl was adsorbed on elastin at 80%, a result which privileges hydrophobic interactions between MAPl and elastin rather than salt bridges, as for known basic elastases. About 80% of the original amidasic and elastolytic activities were conserved after a 30-min prior incubation of the enzyme at 40°C; however, 70% of the amidasic activity is measured, instead of 15% for the elastolytic activity, after 30 min at 50°C. Thermal denaturation at this temperature may prevent adsorption of the enzyme on elastin without any important change of the elastase structure. MAPl readily hydrolyzes the Gly23-Phe24 bond in the oxidized insulin B chain; the peptide bonds Alal4-LeulS, Leul5-Tyrl6, Phe24-Phe25, Phe25-Tyr26 are also cleaved, suggesting a primary specificity of the enzyme for hydrophobic or aromatic residues at the first amino acid towards the C-terminus from the cleavage site (P'l position) [Schechter, I. & Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-1621. This hypothesis is consistent with the fact that Ala,-Phe-Ala and Ala,-Phe-Ala are hydrolyzed even though tri-alanine to hexa-alanine oligomers are not.The evidence of an elastase with the same molecular mass and PI as MAPl is given during fruiting body development in submerged culture of M. xanthus. The fact that aromatic amino acids have been found to be the most representative of A-signal [Kuspa, A., Plamann, L. & Kaiser, D.(1992) J. Bacteriol. 174, is consistent with the hypothesis that, regarding its specificity, MAPl is likely to play a role in development of myxobacteria.Myxococcus xanthus, a Gram-negative bacterium is found in nature living in the soil. The cells feed on other organisms and they secrete a wide range of proteases to use their nitrogenous compounds. Extracellular proteases have been previously detected in the culture fluid during growth of M . xunthus [3-61. Eight caseinolytic activities have been demonstrated by electrophoresis of M . xanthus FB culture supernatant [7]. These proteases may have different functions. First, they are likely to play a nutritional role because myxobacteria used proteins as both carbon and energy sources. The growth rate of M . xanthus on previously hydrolyzed casein has been proved to be independent of cell density but increased with the inoculum cell density when using casein. This suggests that the higher local concentration of enzymes might enhance the splitting of macromolecu- avenue Albert Thomas, F-87060 Limoges Cedex, France pancreatic elastase ; Suc-Ala,-NH-Np, succinyl-Al...

Ecological Function of Myroilysin, a Novel Bacterial M12 Metalloprotease with Elastinolytic Activity and a Synergistic Role in Collagen Hydrolysis, in Biodegradation of Deep-Sea High-Molecular-Weight Organic Nitrogen

“…To our knowledge, this enzyme is the first reported elastase in family M12. The cleavage pattern of myroilysin with the oxidized insulin B chain also showed that it had broad specificity and that it shared some, but not all, cleavage sites with pseudolysin and porcine pancreatic elastase (8). These results show the difference between myroilysin and pseudolysin in terms of substrate specificity.…”

“…It has been reported that animal elastase has a synergistic effect with collagenase in collagen degradation (27,34). Although some bacterial elastases have been studied (8,18,31), there has been no report that a bacterial elastase has a synergistic effect with collagenase in collagen degradation. Myroilysin did not readily hydrolyze type I collagen fiber, yet its collagen-swelling activity exceeded that of 6 M urea.…”

Purification and Characterization of a Unique Alkaline Elastase from Micrococcus luteus

“…Cleavage at two sites in the oxidized insulin ␤-chain predominated at 1 and 30 min following the addition of low amounts of alkaline protease (24,41,46,47). At 1 min, cleavage of the Leu 15 -Tyr 16 and Phe 24 -Phe 25 bonds generated peptides containing residues 16 -30 and 25-30 with a yield of 15 and 12%, respectively.…”

“…Mammalian elastase is characterized by its cleavage specificity for small hydrophobic amino acids such as alanine. In contrast, bacteria produce a wide variety of proteases with elastolytic activity including metalloproteases (24,25) and members of both the bacterial serine enzyme superfamilies (26 -28). These proteases have cleavage specificities that include bulky, as well as small, hydrophobic amino acids.…”

An endo-N-acetyl-beta-D-glucosaminidase, acting on the di-N-acetylchitobiosyl part of N-linked glycans, is secreted during sporulation of Myxococcus xanthus

“…Although it is known that numerous extracellular proteins are produced during vegetative growth, few have been characterized or purified so far, and these are mainly lytic enzymes such as cell wall lytic enzymes (35) and proteases (4,6,9,21,29). Recently, it has been established (31) that some secreted trypsin-like proteases from M. xanthus were a component of the above-mentioned A factor, a complex mixture of amino acids, peptides, and proteases which induces the differentiation pathway of this bacterium after starvation.…”