Christian Leitner

Purification and Characterization of Pyranose Oxidase from the White Rot Fungus Trametes multicolor

ABSTRACT We purified an intracellular pyranose oxidase from mycelial extracts of the white rot fungus Trametes multicolor by using ammonium sulfate fractionation, hydrophobic interaction, ion-exchange chromatography, and gel filtration. The native enzyme has a molecular mass of 270 kDa as determined by equilibrium ultracentrifugation and is composed of four identical 68-kDa subunits as determined by matrix-assisted laser desorption ionization mass spectrometry. Each subunit contains one covalently bound flavin adenine dinucleotide as its prosthetic group. The enzyme oxidizes several aldopyranoses specifically at position C-2, and its preferred electron donor substrates are d-glucose,d-xylose, and l-sorbose. During this oxidation reaction electrons are transferred to oxygen, yielding hydrogen peroxide. In addition, the enzyme catalyzes the two-electron reduction of 1,4-benzoquinone, several substituted benzoquinones, and 2,6-dichloroindophenol, as well as the one-electron reduction of the ABTS [2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)] cation radical. As judged by the catalytic efficiencies (kcat/Km), some of these quinone electron acceptors are much better substrates for pyranose oxidase than oxygen. The optimum pH of the pyranose oxidase-catalyzed reaction depends strongly on the electron acceptor employed and varies from 4 to 8. It has been proposed that the main metabolic function of pyranose oxidase is as a constituent of the ligninolytic system of white rot fungi that provides peroxidases with H2O2. An additional function could be reduction of quinones, key intermediates that are formed during mineralization of lignin.

Structural basis for substrate binding and regioselective oxidation of monosaccharides at c3 by pyranose 2-oxidase.

Pyranose 2-oxidase (P2Ox) participates in fungal lignin degradation by producing the H2O2 needed for lignin-degrading peroxidases. The enzyme oxidizes cellulose- and hemicellulose-derived aldopyranoses at C2 preferentially, but also on C3, to the corresponding ketoaldoses. To investigate the structural determinants of catalysis, covalent flavinylation, substrate binding, and regioselectivity, wild-type and mutant P2Ox enzymes were produced and characterized biochemically and structurally. Removal of the histidyl-FAD linkage resulted in a catalytically competent enzyme containing tightly, but noncovalently bound FAD. This mutant (H167A) is characterized by a 5-fold lower kcat, and a 35-mV lower redox potential, although no significant structural changes were seen in its crystal structure. In previous structures of P2Ox, the substrate loop (residues 452-457) covering the active site has been either disordered or in a conformation incompatible with carbohydrate binding. We present here the crystal structure of H167A in complex with a slow substrate, 2-fluoro-2-deoxy-d-glucose. Based on the details of 2-fluoro-2-deoxy-d-glucose binding in position for oxidation at C3, we also outline a probable binding mode for d-glucose positioned for regioselective oxidation at C2. The tentative determinant for discriminating between the two binding modes is the position of the O6 hydroxyl group, which in the C2-oxidation mode can make favorable interactions with Asp452 in the substrate loop and, possibly, a nearby arginine residue (Arg472). We also substantiate our hypothesis with steady-state kinetics data for the alanine replacements of Asp452 and Arg472 as well as the double alanine 452/472 mutant.

Continuous enzymatic regeneration of redox mediators used in biotransformation reactions employing flavoproteins

Abstract Oxidoreductases are a group of enzymes that have been regarded uneconomical for industrial processes due to their dependence on cofactors or prosthetic groups for activity and the difficulties of regenerating these. Especially, flavoproteins have long been neglected for biocatalytical applications. The prosthetic group of some of these enzymes, but not all, can be regenerated by oxygen, resulting in hydrogen peroxide formation, which is detrimental to enzyme stability. As a contribution to alleviating this problem, a novel concept for the regeneration of electron acceptors (redox mediators) for flavoenzymes is described. Flavin-containing enzymes such as cellobiose dehydrogenase (CDH) or pyranose oxidase (P2O) are used in conjunction with laccases and a redox mediator. The flavin of the synthetic enzyme is reduced while the oxidized product of interest is formed, in turn, the flavin is reoxidized with the help of an electron acceptor, which then is regenerated using a laccase. Laccases are copper containing phenol oxidases that can transfer four electrons to oxygen, producing two molecules of water. Preliminary screening experiments with different redox mediators, and a coupled enzyme system of CDH and laccase, showed that a wide variety of different substances can efficiently shuttle electrons between these two enzymes. Among them are substituted and unsubstituted ortho - and para -quinones, benzoquinone imines, cation radicals such as 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), redox dyes such as phenothiazines or phenoxazines, as well as iron complexes. Experiments in which CDH completely oxidizes lactose to lactobionic acid and P2O entirely converts glucose to 2-keto-glucose are presented. Catalytic amounts of redox mediators are used and continuously regenerated by a laccase. Specific productivities of up to 19.3 g·(h·kU) −1 and 72 g·(h·kU) −1 for CDH and P2O, respectively, were found. The total turnover numbers (TTNs) for the two enzymes used were in the range of 10 5 –10 6 . Oxygen supply for the laccase is a crucial factor in avoiding rate limitation. Undeniably, this system facilitates the efficient use of a hitherto underexploited group of enzymes for preparative purposes.

Enhanced Formation of Extracellular Laccase Activity by the White-Rot Fungus Trametes multicolor

The white-rot fungus Trametes multicolor MB 49 has been identified as an excellent producer of the industrially important enzyme laccase. The formation of extracellular laccase could be considerably stimulated by the addition of Cu(II) to a simple, glycerol-based culture medium. In this study, optimal concentrations of copper were found to be 0.5-1 mM, which were added during the growth phase of the fungus. Other medium components important for laccase production are the carbon and nitrogen sources employed. When using an optimized medium containing glycerol (40 g/L), peptone from meat (15 g/L), and MgSO4 x 7H2O and stimulating enzyme formation by the addition of 1.0 mM Cu, maximal laccase activities obtained in shake-flask cultures were approx 85 U/mL. These results, however, could not be scaled up to a laboratory fermentor cultivation. Laccase production by T. multicolor decreased considerably when the fungus was grown in a stirred-tank reactor, presumably because of damage of the mycelia caused by shear stress and/or changes in the morphology of the fungus.

Identification of the covalent flavin adenine dinucleotide-binding region in pyranose 2-oxidase from Trametes multicolor.

We present the first report on characterization of the covalent flavinylation site in flavoprotein pyranose 2-oxidase. Pyranose 2-oxidase from the basidiomycete fungus Trametes multicolor, catalyzing C-2/C-3 oxidation of several monosaccharides, shows typical absorption maxima of flavoproteins at 456, 345, and 275 nm. No release of flavin was observed after protein denaturation, indicating covalent attachment of the cofactor. The flavopeptide fragment resulting from tryptic/chymotryptic digestion of the purified enzyme was isolated by anion-exchange and reversed-phase high-performance liquid chromatography. The flavin type, attachment site, and mode of its linkage were determined by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy of the intact flavopeptide, without its prior enzymatic degradation to the central aminoacyl moiety. Mass spectrometry identified the attached flavin as flavin adenine dinucleotide (FAD). Post-source decay analysis revealed that the flavin is covalently bound to histidine residue in the peptide STHW, consistent with the results of N-terminal amino acid sequencing by Edman degradation. The type of the aminoacyl flavin covalent link was determined by NMR spectroscopy, resulting in the structure 8alpha-(N(3)-histidyl)-FAD.

Properties of pyranose dehydrogenase purified from the litter‐degrading fungus Agaricus xanthoderma

We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion‐exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose–methanol–choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi‐covalent flavinylation in PDH, 9‐S‐cysteinyl, 8‐α‐N3‐histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are d‐glucose, d‐galactose, l‐arabinose, and d‐xylose. As shown by in situ NMR analysis, d‐glucose and d‐galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino‐bis‐(3‐ethylbenzthiazolin‐6‐sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (kcat/Km). The enzyme may play a role in lignocellulose degradation.

The Cetus Process Revisited: A Novel Enzymatic Alternative for the Production of Aldose-Free D-Fructose

In the Cetus process crystalline D-fructose is produced from D-glucose via the intermediate 2-keto-D-glucose. Whereas the first step in the traditional process is catalyzed by the immobilized enzyme pyranose 2-oxidase, the ensuing reduction is performed by catalytic hydrogenation. In an entirely enzymatic variation of this process, soluble pyranose 2-oxidase from Trametes multicolor was employed. This biocatalyst could be efficiently stabilized under operational conditions by the addition of bovine serum albumin (BSA) together with catalase which decomposes hydrogen peroxide formed as a by-product. D-Glucose could be converted into 2-keto-D-glucose in yields above 98%. When the biocatalyst together with both stabilizing agents was separated from the product solution by ultrafiltration, it could be reutilized for several subsequent batch operation cycles. 2-Keto-D-glucose thus obtained was quantitatively reduced to D-fructose by NAD(P)-dependent aldose reductase from Candida tenuis. Two different enzymatic...

Pichia pastoris secretes recombinant proteins less efficiently than Chinese hamster ovary cells but allows higher space-time yields for less complex proteins.

Chinese hamster ovary (CHO) cells are currently the workhorse of the biopharmaceutical industry. However, yeasts such as Pichia pastoris are about to enter this field. To compare their capability for recombinant protein secretion, P. pastoris strains and CHO cell lines producing human serum albumin (HSA) and the 3D6 single chain Fv-Fc anti-HIV-1 antibody (3D6scFv-Fc) were cultivated in comparable fed batch processes. In P. pastoris, the mean biomass-specific secretion rate (qp) was 40-fold lower for 3D6scFv-Fc compared to HSA. On the contrary, qp was similar for both proteins in CHO cells. When comparing both organisms, the mean qp of the CHO cell lines was 1011-fold higher for 3D6scFv-Fc and 26-fold higher for HSA. Due to the low qp of the 3D6scFv-Fc producing strain, the space-time yield (STY) was 9.6-fold lower for P. pastoris. In contrast, the STY of the HSA producer was 9.2-fold higher compared to CHO cells because of the shorter process time and higher biomass density. The results indicate that the protein secretion machinery of P. pastoris is much less efficient and the secretion rate strongly depends on the complexity of the recombinant protein. However, process efficiency of the yeast system allows higher STYs for less complex proteins.

Growth, productivity and protein glycosylation in a CHO EpoFc producer cell line adapted to glutamine-free growth

A primary objective of cell line development and process optimisation in animal cell culture is the improvement of culture performance as indicated by desirable properties such as high cell concentration, viability, productivity and product quality. The inefficient energy metabolism of mammalian cells in culture is still a major limiting factor for improvements in process performance. It results in high uptake rates of glucose and glutamine and the concomitant accumulation of waste products which in turn limits final cell concentrations and growth. To avoid these negative side effects, a CHO host cell line was established recently which is able to grow in completely glutamine free medium (Hernandez Bort et al., 2010). To determine the influence of this adaptation on productivity and product quality, the same procedure was repeated with a recombinant CHO cell line producing an erythropoietin-Fc fusion protein (CHO-EpoFc) for this publication. After adaptation to higher cell densities and glutamine free medium, culture performance was monitored in batch bioprocesses and revealed comparable growth properties and EpoFc product formation in both cell lines. The level of reactive oxygen species was elevated in the adapted cells, reflecting a higher level of oxidative stress, however, at the same time the level of the oxido-protective glutathione was also higher, so that cells seem adequately protected against cellular damage. Analysis of nucleotides and nucleotide sugars revealed elevated UDP-sugars in cells grown in the absence of glutamine. Furthermore, the antennarity of N-glycans was moderately higher on the Epo part of the protein produced by the adapted cell line compared to the parental cell line. Except for this, the glycosylation, with respect to site occupancy, degree of sialylation and glycoform structure, was highly comparable, both for the Epo and the Fc part of the protein.

Production of a Novel Pyranose 2-Oxidase by Basidiomycete Trametes multicolor

During a screening for the enzyme pyranose 2-oxidase (P2O) which has a great potential as a biocatalyst for carbohydrate transformations, Trametes multicolor was identified as a promising, not-yet-described producer of this particular enzyme activity. Furthermore, it was found in this screening that the enzyme frequently occurs in basidiomycetes. Intracellular P2O was produced in a growth-associated manner by T. multicolor during growth on various substrates, including mono-, oligo-, and polysaccharides. Highest levels of this enzyme activity were formed when lactose or whey were used as substrates. Peptones from casein and other casein hydrolysates were found to be the most favorable nitrogen sources for the formation of P2O. By applying an appropriate feeding strategy for the substrate lactose, which ensured an elevated concentration of the carbon source during the entire cultivation, levels of P2O activity obtained in laboratory fermentations, as well as the productivity of these bioprocess experiments, could be enhanced more than 2.5-fold.