Engineering of carbohydrate oxidoreductases for sensors and bio-fuelcells

Pyranose dehydrogenase (PDH) and pyranose 2-oxidase (POx) are flavoproteins that catalyze the oxidation of free, non-phosphorylated sugars to the corresponding ketosugars. Pyranose dehydrogenase has a broad substrate specificity for monosaccharides (and few disaccharides), but is limited to a narrow range of electron acceptors and reacts extremely slowly with dioxygen, whereas pyranose 2-oxidase shows pronounced specificity for glucose and displays high oxidase as well as dehydrogenase activity. For bio-fuelcell and sensor applications, oxygen reactivity is undesirable as it leads to electron leakage and the formation of damaging hydrogen peroxide; for biocatalytic applications, oxygen reactivity is advantageous, as oxygen is freely available and obviates downstream removal of undesired electron acceptors. Site-saturation mutagenesis libraries of eleven (POx) and twelve (PDH) residues around the active sites were screened for oxidase and dehydrogenase activities. In POx, variants T166R, Q448H, L545C, L547R and N593C displayed reduced oxidase activities (between 40% and 0.2% of the wildtype) concomitant with unaffected or even increased dehydrogenase activity, dependent on the electron acceptor used (DCPIP, 1,4-benzoquinone or ferricenium ion). Kinetic characterization showed that both affinity and turnover numbers can be affected. The switch from oxidase to dehydrogenase activity was also observed electrochemically using screen-printed electrodes. In this miniaturized set-up with a reaction volume of only 50 µL the dehydrogenase activity of variant N593C was entirely preserved in the presence of a suitable mediator, shuttling electrons from the FAD cofactor to the electrode surface. The oxidase activity, utilizing molecular oxygen as acceptor, is abolished in this variant. Of all variants of PDH that were produced by saturation mutagenesis, only variants of one position displayed increased oxygen reactivity to a minor degree. Histidine 103, carrying the covalently attached FAD cofactor, was substituted by tyrosine, phenylalanine, tryptophan and methionine. Variant H103Y displayed a five-fold increase of oxygen reactivity. Stopped flow analysis revealed that the mutation slowed down the reductive half-reaction whereas the oxidative half-reaction was affected to a minor degree. No alterations in the secondary structure were observed, but disruption of the FAD bond had negative effects on thermal and conformational stability. We also engineered PDH by systematically removing several N-glycosylation sites, in order to improve performance by reducing the distance of the active site to the electrode surface, improving accessibility for redox polymers and facilitate denser enzyme packing on the electrode. One glycosylation site, N319, was found to be indispensable for functional expression and folding of the enzyme, as no active variants could be obtained. A variant with two sites, N75 and N175 near the active site entrance, exchanged against G and Q, respectively, showed significantly improved properties when used on electrodes with Osmium-based redox polymers (Mediated Electron Transfer) and a low level of Direct Electron Transfer. The lack of two glycosylation sites results in minor negative effects on expression yield and stability. Removal of a third site, N252, on the opposite side of the active site entrance, does not bring further improvements in catalysis and electron transfer, but is detrimental to functional expression and stability. The bulk of hyperglycosylation of the recombinantly expressed enzyme (observed in both Pichia pastoris and Saccharomyces cerevisiae) is located only on this one glycosylation site. Please click Additional Files below to see the full abstract

Semi-rational engineering of cellobiose dehydrogenase for improved hydrogen peroxide production

Abstract Background The ability of fungal cellobiose dehydrogenase (CDH) to generate H2O2 in-situ is highly interesting for biotechnological applications like cotton bleaching, laundry detergents or antimicrobial functionalization of medical devices. CDH’s ability to directly use polysaccharide derived mono- and oligosaccharides as substrates is a considerable advantage compared to other oxidases such as glucose oxidase which are limited to monosaccharides. However CDH’s low activity with oxygen as electron acceptor hampers its industrial use for H2O2 production. A CDH variant with increased oxygen reactivity is therefore of high importance for biotechnological application. Uniform expression levels and an easy to use screening assay is a necessity to facilitate screening for CDH variants with increased oxygen turnover. Results A uniform production and secretion of active Myriococcum thermophilum CDH was obtained by using Saccharomyces cerevisiae as expression host. It was found that the native secretory leader sequence of the cdh gene gives a 3 times higher expression than the prepro leader of the yeast α-mating factor. The homogeneity of the expression in 96-well deep-well plates was good (variation coefficient <15%). A high-throughput screening assay was developed to explore saturation mutagenesis libraries of cdh for improved H2O2 production. A 4.5-fold increase for variant N700S over the parent enzyme was found. For production, N700S was expressed in P. pastoris and purified to homogeneity. Characterization revealed that not only the kcat for oxygen turnover was increased in N700S (4.5-fold), but also substrate turnover. A 3-fold increase of the kcat for cellobiose with alternative electron acceptors indicates that mutation N700S influences the oxidative- and reductive FAD half-reaction. Conclusions Site-directed mutagenesis and directed evolution of CDH is simplified by the use of S. cerevisiae instead of the high-yield-host P. pastoris due to easier handling and higher transformation efficiencies with autonomous plasmids. Twelve clones which exhibited an increased H2O2 production in the subsequent screening were all found to carry the same amino acid exchange in the cdh gene (N700S). The sensitive location of the five targeted amino acid positions in the active site of CDH explains the high rate of variants with decreased or entirely abolished activity. The discovery of only one beneficial exchange indicates that a dehydrogenase’s oxygen turnover is a complex phenomenon and the increase therefore not an easy target for protein engineering.The authors thank the European Commission (FP7 243529-2-COTTONBLEACH) for financial support. CKP thanks the Austrian Science Fund (FWF) for financial support (grant P22094). IK is a member of the doctoral program BioToP (Biomolecular Technology of Proteins) of the Austrian Science Fund (FWF; W1224). MA thanks the Spanish Government for financial support (BIO2010-19697).Peer Reviewe

Simple and efficient expression of Agaricus meleagris pyranose dehydrogenase in Pichia pastoris

Pyranose dehydrogenase (PDH) is a fungal flavin-dependent sugar oxidoreductase that is highly interesting for applications in organic synthesis or electrochemistry. The low expression levels of the filamentous fungus Agaricus meleagris as well as the demand for engineered PDH make heterologous expression necessary. Recently, Aspergillus species were described to efficiently secrete recombinant PDH. Here, we evaluate recombinant protein production with expression hosts more suitable for genetic engineering. Expression in Escherichia coli resulted in no soluble or active PDH. Heterologous expression in the methylotrophic yeast Pichia pastoris was investigated using two different signal sequences as well as a codon-optimized sequence. A 96-well plate activity screening for transformants of all constructs was established and the best expressing clone was used for large-scale production in 50-L scale, which gave a volumetric yield of 223 mg L−1 PDH or 1,330 U L−1 d−1 in space–time yield. Purification yielded 13.4 g of pure enzyme representing 95.8% of the initial activity. The hyperglycosylated recombinant enzyme had a 20% lower specific activity than the native enzyme; however, the kinetic properties were essentially identical. This study demonstrates the successful expression of PDH in the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, the feasibility of large-scale production of the enzyme with this expression system together with a simplified purification scheme for easy high-yield purification is shown

Characterization and engineering of pyranose dehydrogenases for applications in carbohydrate conversions

Das Flavoenzym Pyranose Dehydrogenase (PDH) wird extrazellulär von ligninolytischen Basidiomyceten der Familie Agaricaceae produziert und katalysiert die Oxidation einer Vielfalt an Zuckern. Als Elektronenakzeptor dienen Benzochinone oder komplexierte Metallionen, das Enzym reagiert nicht oder nur sehr langsam mit Sauerstoff. PDH ist ein vielversprechender Kandidat für den industriellen Einsatz in Zuckerumwandlungen, der organischen Synthese oder in Biobrennstoffzellen und -sensoren. PDHs aus Agaricus meleagris, A. xanthoderma und A. campestris wurden im Zuge dieser Arbeit rekombinant in der methylotrophen Hefe Pichia pastoris exprimiert, aufgereinigt und biochemisch charakterisiert. Mittels UV-Vis Spektroskopie, SDS-PAGE und TCA-Fällung wurden die Steady-state Kinetik und molekulare Eigenschaften der Proteine untersucht. Laktose-Umsetzungen im Labormaßstab zeigten die Eignung von PDH aus A. campestris und A. xanthoderma für die Produktion von Laktobionsäure und 2-Dehydrolaktose, einem wichtigen Intermediat für die Synthese von Laktulose. Da die physiologischen Elektronenakzeptoren von PDH nicht geeignet sind für den Einsatz in der Lebensmittelindustrie, wurden zwölf Site-saturation Mutantenbibliotheken von A. meleagris PDH in Saccharomyces cerevisiae exprimiert um Varianten mit erhöhter Sauerstoffreaktivität zu finden. Mutante H103Y wurde in größerem Maßstab in P. pastoris exprimiert und gereinigt. Die Charakterisierung ergab eine fünffache Steigerung der Sauerstoffreaktivität, trotz nicht-kovalent gebundenem FAD war die Mutante katalytisch aktiv, wenn auch in schwächerem Ausmaß. Stopped-flow Experimente zeigten dass ausschließlich die reduktive Halbreaktion von der Mutation negativ beeinflusst wurde. Die Stabilität der Mutante gegenüber thermischer und chemischer Denaturierung war im Vergleich zum Wildtyp verringert. Der angewandte Engineering-Ansatz bildet eine Grundlage für weitere Verbesserungen von PDH für den Einsatz in der Lebensmittelindustrie.Pyranose dehydrogenase (PDH), a fungal flavin-dependent oxidoreductase, catalyzes the oxidation of a broad variety of sugars substrates. The enzyme is unable to utilize dioxygen as an electron acceptor. Benzoquinones and complexed metal ions naturally present during lignocellulose degradation, the supposed biological function of PDH, are preferred. PDH represents a promising biocatalyst which can be applied in sugar conversions, organic synthesis or electrochemistry. For this purpose, PDHs from Agaricus meleagris, A. xanthoderma and A. campestris were recombinantly expressed in the methylotrophic yeast Pichia pastoris, purified and characterized biochemically. Steady-state kinetic parameters and molecular properties were investigated using UV-Vis spectroscopy, SDS-PAGE and TCA precipitation. Batch lactose conversion experiments and HPLC analysis confirmed the suitability of the enzymes from A. xanthoderma and A. campestris for the production of lactobionic acid or 2-dehydrolactose, a key intermediate for the production of lactulose. The physiological electron acceptor of PDH is undesirable for an application in food technology, therefore site-saturation mutagenesis libraries of twelve amino acids in the active site of A. meleagris PDH were expressed in Saccharomyces cerevisiae. High-throughput screening resulted in one position altering oxygen reactivity. Mutant H103Y, produced in P. pastoris, showed a five-fold increase in oxygen reactivity. Although carrying a non-covalently linked FAD-cofactor in contrast to the wild-type, mutant H103Y was still catalytically active but to a lower degree. Stopped-flow experiments revealed that only the reductive half-reaction was negatively affected by the mutation. Thermal and chemical denaturation experiments were performed and confirmed the lower stability caused by the non-covalent FAD linkage. This semi-rational approach provides a scaffold for further engineering of PDH towards a highly competent biocatalyst.submitted by Iris KrondorferAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Univ. für Bodenkultur, Diss., 2014oeBB(VLID)193128

Pyranose Dehydrogenase from Agaricus campestris and Agaricus xanthoderma: Characterization and Applications in Carbohydrate Conversions

biomolecule

Engineering Pyranose 2-Oxidase for Modified Oxygen Reactivity

<div><p>Pyranose 2-oxidase (POx), a member of the GMC family of flavoproteins, catalyzes the regioselective oxidation of aldopyranoses at position C2 to the corresponding 2-ketoaldoses. During the first half-reaction, FAD is reduced to FADH₂ and reoxidized in the second half-reaction by reducing molecular oxygen to H₂O₂. Alternative electron acceptors including quinones, radicals or chelated metal ions show significant and in some cases even higher activity. While oxygen as cheap and abundantly available electron acceptor is favored for many processes, reduced oxygen reactivity is desirable for some applications such as in biosensors/biofuel cells because of reduced oxidative damages to the biocatalyst from concomitant H₂O₂ production as well as reduced electron “leakage” to oxygen. The reactivity of flavoproteins with oxygen is of considerable scientific interest, and the determinants of oxygen activation and reactivity are the subject of numerous studies. We applied site-saturation mutagenesis on a set of eleven amino acids around the active site based on the crystal structure of the enzyme. Using microtiter plate screening assays with peroxidase/2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and 2,6-dichlorophenolindophenol, variants of POx with decreased oxidase activity and maintained dehydrogenase activity were identified. Variants T166R, Q448H, L545C, L547R and N593C were characterized with respect to their apparent steady-state constants with oxygen and the alternative electron acceptors DCPIP, 1,4-benzoquinone and ferricenium ion, and the effect of the mutations was rationalized based on structural properties.</p></div

Apparent steady-state kinetic constants of wild-type and mutant POx with D-glucose as electron donor (100 mM) and 1,4-BQ (varied from 0.01–0.5 mM) as electron acceptor.

<p>*data from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109242#pone.0109242-Spadiut2" target="_blank">[47]</a>.</p><p>Apparent steady-state kinetic constants of wild-type and mutant POx with D-glucose as electron donor (100 mM) and 1,4-BQ (varied from 0.01–0.5 mM) as electron acceptor.</p

SDS-PAGE.

<p>Recombinant <i>A. meleagris</i> PDH and variant H103Y were expressed in <i>P. pastoris</i> and purified in a two- and three-step protocol. M, molecular marker (Precision Plus Protein Standard, BioRad); 1, wild-type <i>Am</i>PDH; 2, wild-type <i>Am</i>PDH deglycosylated (PNGase F); 3, <i>Am</i>PDH variant H103Y; 4, <i>Am</i>PDH variant H103Y deglycosylated.</p

Engineering of Pyranose Dehydrogenase for Increased Oxygen Reactivity

<div><p>Pyranose dehydrogenase (PDH), a member of the GMC family of flavoproteins, shows a very broad sugar substrate specificity but is limited to a narrow range of electron acceptors and reacts extremely slowly with dioxygen as acceptor. The use of substituted quinones or (organo)metals as electron acceptors is undesirable for many production processes, especially of food ingredients. To improve the oxygen reactivity, site-saturation mutagenesis libraries of twelve amino acids around the active site of <i>Agaricus meleagris</i> PDH were expressed in <i>Saccharomyces cerevisiae</i>. We established high-throughput screening assays for oxygen reactivity and standard dehydrogenase activity using an indirect Amplex Red/horseradish peroxidase and a DCIP/D-glucose based approach. The low number of active clones confirmed the catalytic role of H512 and H556. Only one position was found to display increased oxygen reactivity. Histidine 103, carrying the covalently linked FAD cofactor in the wild-type, was substituted by tyrosine, phenylalanine, tryptophan and methionine. Variant H103Y was produced in <i>Pichia pastoris</i> and characterized and revealed a five-fold increase of the oxygen reactivity.</p></div

Apparent steady-state kinetic constants of wild-type and mutant POx with D-glucose as electron donor (100 mM) and DCPIP (varied from 0.015–1.2 mM) as electron acceptor.

<p>Apparent steady-state kinetic constants of wild-type and mutant POx with D-glucose as electron donor (100 mM) and DCPIP (varied from 0.015–1.2 mM) as electron acceptor.</p