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Elements of Streptomyces rimosus extracellular lipase stability : Assignment of disulfide bridges


Leščić, Ivana; Zehl, Martin; Abramić, Marija; Allmaier, Günter; Kojić-Prodić, Biserka
Elements of Streptomyces rimosus extracellular lipase stability : Assignment of disulfide bridges // 6th International Conference on Protein Stabilization ProtStab2004
Bratislava, 2004. (poster, međunarodna recenzija, sažetak, znanstveni)


Naslov
Elements of Streptomyces rimosus extracellular lipase stability : Assignment of disulfide bridges

Autori
Leščić, Ivana ; Zehl, Martin ; Abramić, Marija ; Allmaier, Günter ; Kojić-Prodić, Biserka

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Skup
6th International Conference on Protein Stabilization ProtStab2004

Mjesto i datum
Bratislava, Slovačka, 26.-29.09.2004

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
Streptomyces rimosus; lipase; enzyme stability; MALDI-MS; disulfide bridges

Sažetak
Lipases (E.C. 3.1.1.3) are enzymes of substantial physiological and biotechnological importance. They catalyze both the hydrolysis and synthesis of esters. The latter reaction is enabled by the fact that lipases are active also in organic solvents. Stability in organic solvents and at wide pH and temperature range, broad substrate specificity and high enantioselectivity are among the properties that make lipases biocatalysts of choice for many technological and industrial applications. Streptomycetes are Gram-positive bacteria that produce a variety of secondary metabolites (e.g. antibiotics) and employ numerous extracellular hydrolytic enzymes, including lipases, to degrade organic material in their natural environment. However, only five lipases from this genus were characterized so far. The extracellular lipase from Streptomyces rimosus R6-554W was isolated and biochemically characterized. Its molecular mass was determined to be 27.5 kDa with electrophoresis and 24166 Da with mass spectrometry. Isoelectric point of this enzyme is at pH=8.5, as estimated from isoelectric focusing. It was shown to be a true lipase, by the substrate specificity and interfacial activation. Potential applicability of this enzyme in biotechnology was predicted, based on lipase’ s relatively high temperature- and pH-optima, pronounced thermal stability and preservation of activity in a broad pH range, and ruggedness towards solvent mixtures. The gene encoding this protein was sequenced, and deduced amino acid sequence confirmed with peptide mass fingerprints. It was shown to contain 6 cysteines. The sensitivity of S. rimosus lipase to reducing agent dithiothreitol and its insensitivity to thiol blocking agent p-hydroxymercuribenzoate indicated the presence of disulfide bonds in this enzyme. In order to confirm this, and to reveal the S-S bridges pattern (since disulfide bonds are known to contribute to enzyme’ s stability) we used mass spectrometry. Comparison of the peptide mass fingerprints from the reduced and non-reduced enzyme unequivocally revealed three intramolecular disulfide bonds with following linkages: C27-C52, C93-C101 and C151-C198.

Izvorni jezik
Engleski

Znanstvena područja
Kemija



POVEZANOST RADA


Projekt / tema
0098036
0098055

Ustanove
Institut "Ruđer Bošković", Zagreb