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High‐throughput screening for cellobiose dehydrogenases by Prussian Blue in situ formation
Author(s) -
Vasilchenko Liliya G.,
Ludwig Roland,
Yershevich Olga P.,
Haltrich Dietmar,
Rabinovich Mikhail L.
Publication year - 2012
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201100480
Subject(s) - cellobiose dehydrogenase , ferricyanide , cellobiose , prussian blue , dehydrogenase , chemistry , biochemistry , ferrocyanide , methylene blue , nad+ kinase , enzyme , cellulase , photocatalysis , electrochemistry , catalysis , electrode
Extracellular fungal flavocytochrome cellobiose dehydrogenase (CDH) is a promising enzyme for both bioelectronics and lignocellulose bioconversion. A selective high‐throughput screening assay for CDH in the presence of various fungal oxidoreductases was developed. It is based on Prussian Blue (PB) in situ formation in the presence of cellobiose (<0.25 mM), ferric acetate, and ferricyanide. CDH induces PB formation via both reduction of ferricyanide to ferrocyanide reacting with an excess of Fe 3+ (pathway 1) and reduction of ferric ions to Fe 2+ reacting with the excess of ferricyanide (pathway 2). Basidiomycetous and ascomycetous CDH formed PB optimally at pH 3.5 and 4.5, respectively. In contrast to the holoenzyme CDH, its FAD‐containing dehydrogenase domain lacking the cytochrome domain formed PB only via pathway 1 and was less active than the parent enzyme. The assay can be applied on active growing cultures on agar plates or on fungal culture supernatants in 96‐well plates under aerobic conditions. Neither other carbohydrate oxidoreductases (pyranose dehydrogenase, FAD‐dependent glucose dehydrogenase, glucose oxidase) nor laccase interfered with CDH activity in this assay. Applicability of the developed assay for the selection of new ascomycetous CDH producers as well as possibility of the controlled synthesis of new PB nanocomposites by CDH are discussed.