The extracellular proteases of<i>Myxococcus xanthus</i>

25 psychrotrophic protease-forming bacteria isolated from glaciers were identified as follows: Pseudomonas spp. (1 8 strains), Fluvobucterium spp. (3 strains), Xunthomonas multophilia (2 strains), Aeromonus hydrophila ( 1 strain) and Bacillus sp. (1 strain). Characteristics of proteolytic activity in cell-free culture supernatants were studied. Proteases produced by 40% of the bacteria showed a maximum azocaseinolytic activity around 40 "C and pH 7. Enzymes were rapidly inactivated after heating at 50 "C. pI-values of proteases ranged from > 9.5 to 7.0. Sensitivity to EDTA indicates that most proteases are metalloproteases. Proteases of psychrotrophs could be differentiated from proteases of mesophiles by a lower temperature optimum, a lower activation energy and a higher sensitivity against heat treatment.

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“…Multiple bands may arise from post-translational modification or partial proteolysis (COLETTA andMILLER 1986, EGGEN etul. 1990).…”

Section: Isoelectric Pointmentioning

confidence: 99%

Proteases of psychrotrophic bacteria isolated from glaciers

Margesin¹,

Palma²,

Knauseder³

et al. 1991

J. Basic Microbiol.

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“…x-Casein agarose gel was stained for 15 min with Coomassie blue and destained in a solution containing methanol, acetic acid and water (3 : 1: 6, v/v/v). Hydrolyzed x-casein appeared as a non-stained band allowing location of protease activity [9].…”

Section: Determination Of Clotting Activitymentioning

confidence: 99%

A chymosin‐like extracellular acidic endoprotease from Myxococcus xanthus DK101

Carias¹,

Raingeaud

Mazaud³

et al. 1990

FEBS Letters

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SDS-PAGE and N-terminal sequence analysis of hydrolysis products from 3 substrates containing a unique sensitive bond usually recognized by chymosin (rc-casein, a synthetic hexapeptide and a recombinant tripartite protein) revealed that a 45 kDa endoprotease of Myxococcus xanrhus DKlOl cleaved the same characteristic Phe-Met bond with high specificity. Such an enzyme, easy to obtain from culture supematant and to use in acidic conditions, could be a new tool for protein engineering.

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“…xunthus [3-61. Eight caseinolytic activities have been demonstrated by electrophoresis of M . xanthus FB culture supernatant [7]. These proteases may have different functions.…”

mentioning

confidence: 99%

Purification and characterization of an alkaline elastase from Myxococcus xanthus

Dumont¹,

Verneuil²,

Wallach

et al. 1994

European Journal of Biochemistry

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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...

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The extracellular proteases ofMyxococcus xanthus

Cited by 20 publications

References 17 publications

Proteases of psychrotrophic bacteria isolated from glaciers

Proteases of psychrotrophic bacteria isolated from glaciers

A chymosin‐like extracellular acidic endoprotease from Myxococcus xanthus DK101

Purification and characterization of an alkaline elastase from Myxococcus xanthus

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