Naslov
CGE-on-theChip (Bioanalyzer) and MALDI mass spectrometry: tools for the characterization of Streptomyces lipase-inhibitor complexes

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

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

Izvornik
25th International Symposium & Exhibit on the Separation of Proteins, Peptides & Polynucleotides Book of Abstracts / - , 2005

Skup
25th International Symposium & Exhibit on the Separation of Proteins, Peptides & Polynucleotides

Mjesto i datum
Saint Pete Beach (FL), Sjedinjene Američke Države, 06.11.2005. - 09.11.2005

Vrsta sudjelovanja
Pozvano predavanje

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
-

Sažetak
Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3), especially those from microorganisms, have found wide biotechnological applications. However, natural enzymes, including lipases, do not always show satisfactory performance for efficient use as biocatalysts. Therefore, efforts are made to optimize their enzymatic properties (activity, stability and, most importantly, enantioselectivity), e.g. by rational protein design. This requires the detailed characterization of the protein structure as well as active site of the enzyme (e.g. by means of various inhibitors). The cloning, sequencing and high-level expression of the gene encoding extracellular lipase from Streptomyces rimosus R6-554W has been recently described [1]. The gene product, which has been isolated and characterized by biochemical and bioinformatical methods, was finally analyzed by three techniques [2]: capillary gel electrophoresis on-the-chip with a laser-induced-fluorescence detector (Bioanalyzer 2100, Agilent Technologies) and MALDI linear time-of-flight (TOF) mass spectrometry (MS, AXIMA LNR/CFR+, Shimadzu Biotech) at the protein level. Afterwards (chemical and enzymatic cleavage of the enzyme) detailed sequence information was obtained by MALDI multistage/tandem MS (AXIMA QIT and AXIMA TOF/CFR prototype, Shimadzu Biotech) allowing low [3] and high energy collision-induced dissociation (CID) experiments. So a detailed comparison between the wild-type and the recombinant lipase was performed. As next step the active site was determined by incubation of the enzyme with different potential inhibitors. The resulting product (lipase-inhibitor complex) was investigated on the protein as well as peptide level by CGE on-the-chip and MALDI TOF MS and CID, which allowed the determination of stoichiometry of the complex and finally the amino acid which has bound the inhibitor was determined.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

POVEZANOST RADA