Friedrich Giffhorn

Bio: Friedrich Giffhorn is an academic researcher from Saarland University. The author has contributed to research in topics: Pyranose oxidase & Rhodobacter sphaeroides. The author has an hindex of 25, co-authored 50 publications receiving 1463 citations.

Fungal pyranose oxidases : occurrence, properties and biotechnical applications in carbohydrate chemistry

Abstract: Pyranose oxidases are widespread among lignin-degrading white rot fungi and are localized in the hyphal periplasmic space. They are relatively large flavoproteins which oxidize a number of common monosaccharides on carbon-2 in the presence of oxygen to yield the corresponding 2-keto sugars and hydrogen peroxide. The preferred substrate of pyranose oxidases is d-glucose which is converted to 2-keto-d-glucose. While hydrogen peroxide is a cosubstrate in ligninolytic reactions, 2-keto-d-glucose is the key intermediate of a secondary metabolic pathway leading to the antibiotic cortalcerone. The finding that 2-keto-d-glucose can serve as an intermediate in an industrial process for the conversion of d-glucose into d-fructose has stimulated research on the use of pyranose oxidases in biotechnical applications. Unique catalytic potentials of pyranose oxidases have been discovered which make these enzymes efficient tools in carbohydrate chemistry. Converting common sugars and sugar derivatives with pyranose oxidases provides a pool of sugar-derived intermediates for the synthesis of a variety of rare sugars, fine chemicals and drugs.

Rare Keto‐Aldoses from Enzymatic Oxidation: Substrates and Oxidation Products of Pyranose 2‐Oxidase

Abstract: Pyranose oxidases are known to oxidise o-glucose, o-xylose and l- sorbose to keto-aldoses, biochemically interesting compounds that may also be used for synthetic purposes in a variety of reactions. In this study pyranose oxidase from the basidiomycete Penio- phora gigonteo was investigated, and it was found that this enzyme is able to oxidise a broad variety of substrates very effectively. ln analogy to its natural mode of action. most substrates are oxidised regioselectively in position 2. Certain compounds, however, are con- verted into 3-keto derivatives. and the enzyme even exhibits transfer potential, that is, disaccharides are formed from /j- glycosides of higher alcohols. Substrates that may be oxidised at C-2 in yields between 40-98"A are n-allose, o-galac- tose, 6-deoxy-o-glucose, D-gentiobiose, a-o-glucopyranosyl fluoride and the very interesting 3-deoxy-o-glucose. 1,5- Anhydro-o-glucitol ( 1 -deoxy-o-glucose) is very effectively oxidised in position 2 in 98 % yield and additionally gives a product of dioxidation at C-2 and C-3 upon prolonged reaction time. Selective oxidation at C-3 was found for 2-deoxv-

Purification and characterization of a pyranose oxidase from the basidiomycete Peniophora gigantea and chemical analyses of its reaction products.

Abstract: A pyranose oxidase was isolated from mycelium extracts of the basidiomycete Peniophora gigantea. This enzyme was purified 104-fold to apparent homogeneity with a yield of about 75% by steps involving fractionated ammonium sulphate precipitation, chromatography on DEAE-Sephacel, Sephacryl S 300, S Sepharose and Q Sepharose. The native pyranose oxidase has a relative molecular mass (Mr) of 322800 ± 18300 as determined on the basis of its Stokes' radius (rs= 6.2 nm) and sedimentation coefficient (s20,w= 10.6), dynamic light-scattering experiments, gradient-gel electrophoresis and cross-linking studies. SDS/PAGE resulted in one single polypeptide band of Mr76000 indicating that the enzyme consists of four subunits of identical size. The pyranose oxidase was shown to be an extremely stable glycoprotein with an isoelectric point of pH 5.3. It contains covalently bound FAD with an estimated stoichiometry of 3.6 molecules FAD/molecule enzyme. Pyranose oxidase was active with the substrates d-glucose, d-xylose, l-sorbose, d-galactose, methyl β-d-glucoside, maltose and d-fucose. Regioselective oxidation of d-glucose, l-sorbose and d-xylose to 2-keto-d-glucose, 5-keto-d-fructose and 2-keto-d-xylose, was demonstrated by identifying the reaction products by mass spectroscopy 13C-NMR spectroscopy and 1H-NMR spectroscopy after purification and derivatization. The pH optimum of the pyranose oxidase was in the range pH 6.0–6.5 in 0.1 M potassium phosphate, and its activation energy ( H°) for the conversion of d-glucose was 34.6 kJ/mol. The reactions with the sugars exhibited Michaelis-Menten kinetics, and the Km values determined for d-glucose, l-sorbose, d-xylose and oxygen were 1.1 mM, 50.0 mM, 29.4 mM and 0.65 mM, respectively. The activity of pyranose oxidase was only slightly affected by chelating reagents, thiol reagents, reducing reagents and bivalent cations each at 1 mM.

Production, purification and characterization of glucose oxidase from a newly isolated strain of Penicillium pinophilum

Abstract: A number of nutritional factors influencing growth and glucose oxidase (EC 1.1.3.4) production by a newly isolated strain of Penicillium pinophilum were investigated. The most important factors for glucose oxidase production were the use of sucrose as the carbon source, and growth of the fungus at non-optimal pH 6.5. The enzyme was purified to apparent homogeneity with a yield of 74%, including an efficient extraction step of the mycelium mass at pH 3.0, cation-exchange chromatography and gel filtration. The relative molecular mass (M r) of native glucose oxidase was determined to be 154 700 ± 4970, and 77 700 for the denatured subunit. Electron-microscopic examinations revealed a sandwich-shaped dimeric molecule with subunit dimensions of 5.0 × 8.0 nm. Glucose oxidase is a glycoprotein that contains tightly bound FAD with an estimated stoichiometry of 1.76 mol/mol enzyme. The enzyme is specific for d-glucose, for which a K m value of 6.2 mM was determined. The pH optimum was determined in the range pH 4.0–6.0. Glucose oxidase showed high stability on storage in sodium citrate (pH 5.0) and in potassium phosphate (pH 6.0), each 100 mM. The half-life of the activity was considerably more than 305 days at 4 °C and 30 °C, and 213 days at 40 °C. The enzyme was unstable at temperatures above 40 °C in the range pH 2.0–4.0 and at a pH above 7.0.

Enhanced hydrogen production from aromatic acids by immobilized cells of Rhodopseudomonas palustris

Abstract: Cells of the purple non-sulphur bacterium Rhodopseudomonas palustris DSM 131 were immobilized in agar, agarose, κ-carrageenan or sodium alginate gel. With alginate beads, prepared by an emulsion technique, and an optimal cell load of 10 mg dry weight/ml gel, the hydrogen production from aromatic acids was doubled as compared to that resulting from liquid cultures. Hydrogen yields of 60%, 57%, 86% or 88% of the maximal theoretical value were obtained from mandelate, benzoylformate, cinnamate or benzoate respectively. Benzoate concentrations above 16.5 mM were inhibitory. During a period of 55 days the process of hydrogen evolution with immobilized cells was repeated in five cycles with slowly decreasing efficiency.

Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes

Abstract: Since its inception, the carbohydrate-active enzymes database (CAZy; http://www.cazy.org ) has described the families of enzymes that cleave or build complex carbohydrates, namely the glycoside hydrolases (GH), the polysaccharide lyases (PL), the carbohydrate esterases (CE), the glycosyltransferases (GT) and their appended non-catalytic carbohydrate-binding modules (CBM). The recent discovery that members of families CBM33 and family GH61 are in fact lytic polysaccharide monooxygenases (LPMO), demands a reclassification of these families into a suitable category. Because lignin is invariably found together with polysaccharides in the plant cell wall and because lignin fragments are likely to act in concert with (LPMO), we have decided to join the families of lignin degradation enzymes to the LPMO families and launch a new CAZy class that we name “Auxiliary Activities” in order to accommodate a range of enzyme mechanisms and substrates related to lignocellulose conversion. Comparative analyses of these auxiliary activities in 41 fungal genomes reveal a pertinent division of several fungal groups and subgroups combining their phylogenetic origin and their nutritional mode (white vs. brown rot). The new class introduced in the CAZy database extends the traditional CAZy families, and provides a better coverage of the full extent of the lignocellulose breakdown machinery.

Microbial production of hydrogen : an overview

Abstract: Production of hydrogen by anaerobes, facultative anaerobes, aerobes, methylotrophs, and photosynthetic bacteria is possible. Anaerobic Clostridia are potential producers and immobilized C. butyricum produces 2 mol H2/mol glucose at 50% efficiency. Spontaneous production of H2 from formate and glucose by immobilized Escherichia coli showed 100% and 60% efficiencies, respectively. Enterobactericiae produces H2 at similar efficiency from different monosaccharides during growth. Among methylotrophs, methanogenes, rumen bacteria, and thermophilic archae, Ruminococcus albus, is promising (2.37 mol/mol glucose). Immobilized aerobic Bacillus licheniformis optimally produces 0.7 mol H2/mol glucose. Photosynthetic Rhodospirillum rubrum produces 4, 7, and 6 mol of H2 from acetate, succinate, and malate, respectively. Excellent productivity (6.2 mol H2/mol glucose) by co-cultures of Cellulomonas with a hydrogenase uptake (Hup) mutant of R. capsulata on cellulose was found. Cyanobacteria, viz., Anabaena, Synechococcus, and Oscillatoria sp., have been studied for photoproduction of H2. Immobilized A. cylindrica produces H2 (20 ml/g dry wt/h) continually for 1 year. Increased H2 productivity was found for Hup mutant of A. variabilis. Synechococcus sp. has a high potential for H2 production in fermentors and outdoor cultures. Simultaneous productions of oxychemicals and H2 by Klebseilla sp. and by enzymatic methods were also attempted. The fate of H2 biotechnology is presumed to be dictated by the stock of fossil fuel and state of pollution in future.