Characterization of arazyme, an exocellular metalloprotease isolated from Serratia proteamaculans culture medium

Characterization of arazyme, an exocellular metalloprotease isolated from Serratia proteamaculans culture medium

Author Bersanetti, P. A. Google Scholar
Park, H. Y. Google Scholar
Bae, K. S. Google Scholar
Son, K. H. Google Scholar
Shin, D. H. Google Scholar
Hirata, I. Y. Google Scholar
Juliano, M. A. Google Scholar
Carmona, A. K. Google Scholar
Juliano, L. Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Korea Res Inst Biosci & Biotechnol
Insect Biotech Co Ltd
Abstract We investigated the biochemical properties of a 51.5 kDa metalloprotease (arazyme, 3.4.24.40) secreted into the culture medium by Aranicola proteolyticus, a symbiotic bacterium of the spider Nephila clavata. the enzyme was purified to apparent homogeneity by ion exchange chromatography in a Resource Q column (FPLC system). the substrate specificity requirements of purified arazyme were examined using fluorescence resonance energy transfer (FRET) peptides derived from the lead sequence Abz-KLRFSKQ-EDDnp (Abz = ortho-aminobenzoic acid; EDDnp=ethylenediaminedinitrophenyl). Three series of peptides were assayed to map the S-2, S-1 and S'(1) subsites: Abz-KXRFSKQ-EDDnp, Abz-KLXFSKQ-EDDnp and Abz-KLRXSKQ-EDDnp (X are natural amino acids). the results indicated that S-1 subsite has a broad specificity, being Gly the preferred amino acid for this subsite followed by positively charged residues (Arg and His). the S-2 and S'(1) subsites accommodated better hydrophobic residues with aliphatic or aromatic side chains (Leu, Phe). the pH effect on hydrolysis of Abz-KLFFSKQ-EDDnp indicated that optimal hydrolysis occurred at pH 8.0 or higher. the effect of NaCl on the arazyme activity depends on the substrate, but in general the activity was reduced with this salt. the temperature did not affect the enzyme from 10 to 45 degrees C, after which activity decreased sharply. Arazyme presented high hydrolytic activity on substance P and peptides related to bradykinin. in addition, arazyme activity was resistant to the treatment by pepsin, trypsin and chymotrypsin. in conclusion, arazyme has a broad hydrolytic profile and works in very aggressive conditions, which justify its potential use in therapeutics and biotechnological applications. (c) 2005 Elsevier Inc. All rights reserved.
Keywords serralysin
Serratia proteamaculans
fluorescent peptides
Language English
Date 2005-11-01
Published in Enzyme and Microbial Technology. New York: Elsevier B.V., v. 37, n. 6, p. 574-581, 2005.
ISSN 0141-0229 (Sherpa/Romeo, impact factor)
Publisher Elsevier B.V.
Extent 574-581
Origin http://dx.doi.org/10.1016/j.enzmictec.2005.01.041
Access rights Closed access
Type Article
Web of Science ID WOS:000232363400002
URI http://repositorio.unifesp.br/handle/11600/28527

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