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Detection, quantification and identification of fungal extracellular laccases using polyclonal antibody and mass spectrometry

Paper ID Volume ID Publish Year Pages File Format Full-Text
18075 42711 2007 8 PDF Available
Title
Detection, quantification and identification of fungal extracellular laccases using polyclonal antibody and mass spectrometry
Abstract

This study presents a combined method to analyze extracellular fungal laccases using a new anti-laccase antibody together with the identification of tryptic laccase peptides by mass spectrometry (nanoLC–ESI–MS/MS). The polyclonal anti-laccase antibody LccCbr2 was raised against peptides designed from the copper binding region II of fungal laccases using in silico data obtained from GenBank database. As a consequence, detection requires denaturation of the enzymes due to the stable conformation of the copper binding region II. The specificity of the antibody was shown with denatured laccase Lcc1 of Coprinopsis cinerea and laccase of Hypholoma fasciculare. LccCbr2 detected amounts as low as 5 ng of highly purified laccase, indicating a possible use of the antibody for quantification of laccase proteins. Denatured extracellular laccases from culture supernatants of the basidiomycetes C. cinerea, H. fasciculare, Lentinula edodes, Mycena sp., Piriformospora indica, Pleurotus cornucopiae, Pleurotus ostreatus, Pycnoporus cinnabarinus, Trametes versicolor and furthermore the ascomycete Verpa conica were detected with apparent molecular masses between 60 and 70 kDa by LccCbr2. The identity of extracellular laccases from C. cinerea, H. fasciculare, P. ostreatus, P. cinnabarinus and T. versicolor were verified by tryptic peptides using nanoLC–ESI–MS/MS.

Keywords
aa, amino acid; LccCbr2, polyclonal anti-laccase antibody against copper binding region II; ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); CBB, Coomassie Brilliant Blue G-250; cbr, copper binding region; MS, mass spectrometryCopper binding
First Page Preview
Detection, quantification and identification of fungal extracellular laccases using polyclonal antibody and mass spectrometry
Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 41, Issues 6–7, 1 November 2007, Pages 694–701
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering