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MALDI TOF- and MALDI low energy CID multistage MS-based characterization of an extracellular lipase from Streptomyces rimosus before and after covalent inhibition by 3, 4-dichloroisocoumarin


Zehl, Martin; Leščić, Ivana; Abramić, Marija; Kojić-Prodić, Biserka; Allmaier, Günter
MALDI TOF- and MALDI low energy CID multistage MS-based characterization of an extracellular lipase from Streptomyces rimosus before and after covalent inhibition by 3, 4-dichloroisocoumarin // 22nd Informal Meeting on Mass Spectrometry
Tokaj, 2004. (poster, međunarodna recenzija, sažetak, znanstveni)


Naslov
MALDI TOF- and MALDI low energy CID multistage MS-based characterization of an extracellular lipase from Streptomyces rimosus before and after covalent inhibition by 3, 4-dichloroisocoumarin

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

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

Skup
22nd Informal Meeting on Mass Spectrometry

Mjesto i datum
Tokaj, Mađarska, 02-06.05.2004

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
Streptomyces rimosus; GDS(L) lipase; MALDI-TOF mass spectrometry; 3; 4-dichloroisocoumarin; protein sequence; disulfide bridges; catalytic serine

Sažetak
Lipases catalyze both, the hydrolysis and synthesis of esters obtained from glycerol and fatty acids. In addition to their biological function, particularly microbial lipases have found wide application as biocatalysts in biotechnology and organic synthesis. However, natural enzymes do not always exhibit desired properties (e.g. enantioselectivity) and further steps to optimize them have to be performed. The prerequisite for such an optimization is the detailed characterization of the primary structure as well as the enzyme active site. A novel extracellular GDS(L)-lipase from Streptomyces rimosus has been purified and biochemically characterized. Cloning and high-level expression yielded sufficient amount of lipase for MS characterization on the protein level. An optimized MALDI preparation procedure allowed accurate molecular mass determination of the intact lipase (Δ m = +0.26 Da to the value 24165.76 Da calculated from nucleotide sequence) using a linear TOF instrument. The primary structure derived from the nucleotide sequence was verified by means of tryptic digestion and formic acid cleavage of the protein followed by peptide mass fingerprinting (combined sequence coverage was 100%). Comparison of the peptide mass fingerprints from the reduced and non-reduced protein yielded the complete disulfide bond pattern. The proteolytic peptides were further partial sequenced by low energy CID multistage MS using a novel MALDI-QIT-rTOF hybrid instrument. Finally, the lipase was site-specific inhibited by the serine protease inhibitor 3, 4-dichloroisocoumarin. The modification site was localized on the serine-rich N-terminal tryptic peptide and the exact position of the enzymatically active serine could be determined by CID experiments. This work was supported by the Austrian Science Foundation (grant P14181 to G.A.), the Croatian Ministry of Science and Technology (grants 0098036 and 0098055) and a bilateral cooperation grant Croatia-Austria (1/2002 and 1/2004 to G.A. and B.K.-P.).

Izvorni jezik
Engleski

Znanstvena područja
Kemija



POVEZANOST RADA


Projekt / tema
0098036
0098055

Ustanove
Institut "Ruđer Bošković", Zagreb