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Immunoassays of fungal laccases for screening of natural enzymes and control of recombinant enzyme production
Author(s) -
Loginov Dmitry S.,
Vavilova Ekaterina A.,
Savinova Оlga S.,
Abyanova Alfia R.,
Chulkin Andrey M.,
Vasina Daria V.,
Zherdev Anatoly V.,
Koroleva Olga V.
Publication year - 2014
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1160
Subject(s) - laccase , recombinant dna , polyclonal antibodies , escherichia coli , enzyme , biochemistry , antibody , biology , epitope , monoclonal antibody , chemistry , microbiology and biotechnology , gene , immunology
Because of the wide application of laccases in different biotechnological processes and intense studies of the enzymes from different sources, the development of efficient techniques for monitoring laccase level is a task of significant importance. Enzyme‐linked immunosorbent assay ( ELISA ) and W estern blotting techniques were developed to control total content and isoform composition of laccases, including their recombinant preparations. Because glycosylated and nonglycosylated forms have different structures and sets of epitopes, two kinds of polyclonal antibodies were obtained and applied. The first antibody recognized the native (glycosylated) laccase purified from Trametes hirsuta and the second one reacted with recombinant (nonglycosylated) laccase expressed in Escherichia coli . Titers of the antibodies were analyzed by indirect ELISA with laccases isolated from several strains of basidiomycetes. The obtained cross‐reactivity data for both antibodies demonstrated a correspondence with sequence homology of the laccases. The antibodies raised against recombinant (nonglycosylated) laccase had higher titers and thus were preferable for screening of recombinant laccase in cultural media. Thus, optimal antibody preparations were selected for screening of laccase‐producing strains, and the control of recombinant enzymes and the efficiency of their use in immunochemical control of laccase levels were confirmed.