Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10233157 | Enzyme and Microbial Technology | 2005 | 8 Pages |
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 S2, S1 and Sâ²1 subsites: Abz-KXRFSKQ-EDDnp, Abz-KLXFSKQ-EDDnp and Abz-KLRXSKQ-EDDnp (X are natural amino acids). The results indicated that S1 subsite has a broad specificity, being Gly the preferred amino acid for this subsite followed by positively charged residues (Arg and His). The S2 and Sâ²1 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 °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.
Keywords
MALDI-TOFFPLCp-HMBCbzMatrix assisted laser desorption ionization time of flightSerratia proteamaculansEDDnpTLCKTPCKPMSFABZE-64DMSOEDTAEthylenediaminetetraacetic acidortho-aminobenzoic acidFluorescence resonance energy transferFRETdimethyl sulphoxideRetention timeSerralysinfast protein liquid chromatographyphenylmethylsulphonyl fluoridetosyl lysyl chloromethyl ketoneFluorescent peptides
Related Topics
Physical Sciences and Engineering
Chemical Engineering
Bioengineering
Authors
PatrÃcia A. Bersanetti, Ho-Yong Park, Kyung Sook Bae, Kwang-Hee Son, Dong-Ha Shin, Izaura Y. Hirata, Maria A. Juliano, Adriana K. Carmona, Luiz Juliano,