High‐throughput screening for cellobiose dehydrogenases by Prussian Blue in situ formation

Abstract

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 Fe³⁺ (pathway 1) and reduction of ferric ions to Fe²⁺ 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.

Protein engineering of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae InvSc1 for increased activity and stability

“…YNB-CAA /Gal plates with grown colonies were used and 20mL of molten 2% agar containing 0.15mM cellobiose, 0.78mM K3[Fe(CN)6] and 0.16mM NH4Fe(SO4)2 in 0.1 M Na-acetate buffer (pH 4.0), cooled to 40ᵒC was added [22]. After cooling, the plates were incubated 4h at room temperature and monitored for CDH producing transformants were identified by the presence of colorless halo around the S.cerevisiae colonies on plates overlaid with DCIP, resulting from CDH activity.…”

Predominant Nonproductive Substrate Binding by Fungal Cellobiohydrolase I and Implications for Activity Improvement

“…Acceleration of heterosidic bond hydrolysis in MUG 4 can be monitored by fluorophore liberation with high accuracy in both continuous and discontinuous assays, whereas hydrolysis of holosidic bonds can be quantified by coupled CDH‐based high troughput assay with an appropriate one‐electron acceptor to increase sensitivity, as described here or in refs. . Just a gentle decrease in the MUG 4 nonproductive binding by fine tuning of A1 loop structure is expected to increase the turnover number of 1,4‐β‐glucosidic bond hydrolysis by an order of magnitude making Cel7A more close to Cel7B that shares the same catalytic mechanism, same structural and dynamic behavior at site −2, and strictly conserved amino acid residues involved in the formation of glycosyl‐enzyme at site −1 …”

Convenient microtiter plate‐based, oxygen‐independent activity assays for flavin‐dependent oxidoreductases based on different redox dyes

“…Furthermore, quinones can give confounded results due to their spontaneous oxidative side reactions in the alkaline pH range [18]. Recently, ferricyanide was used in a high-throughput screening assay that is based on the formation of Prussian Blue, but it was shown that this assay is highly specific for CDH while other flavin-dependent carbohydrate oxidoreductases (glucose oxidase, PDH) do not show similar reactions [25]. …”