Expression, purification, and characterization of galactose oxidase of Fusarium sambucinum in E. coli

AbstractA gene encoding a galactose oxidase (GalOx) was isolated from Fusarium sambucinum cultures and overexpressed in Escherichia coli yielding 4.4mg enzyme per L of growth culture with a specific activity of 159Umg−1. By adding a C-terminal His-tag the enzyme could be easily purified with a single affinity chromatography step with high recovery rate (90%). The enzyme showed a single band on SDS–PAGE with an apparent molecular mass of 68.5kDa. The pH optimum for the oxidation of galactose was in the range of pH 6–7.5. Optimum temperature for the enzyme activity was 35°C, with a half-life of 11.2min, 5.3min, and 2.7min for incubation at 40°C, 50°C, and 60°C, respectively. From all tested substrates, the highest relative activity was found for 1-methyl-β-galactopyranoside (226Umg−1) and the highest catalytic efficiency (kcat/Km) for melibiose (2700mM−1s−1). The enzyme was highly specific for molecular oxygen as an electron acceptor, and showed no appreciable activity with a range of alternative acceptors investigated. Different chemicals were tested for their effect on GalOx activity. The activity was significantly reduced by EDTA, NaN3, and KCN

Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris

<p>Abstract</p> <p>Background</p> <p>FAD dependent glucose dehydrogenase (GDH) currently raises enormous interest in the field of glucose biosensors. Due to its superior properties such as high turnover rate, substrate specificity and oxygen independence, GDH makes its way into glucose biosensing. The recently discovered GDH from the ascomycete <it>Glomerella cingulata </it>is a novel candidate for such an electrochemical application, but also of interest to study the plant-pathogen interaction of a family of wide-spread, crop destroying fungi. Heterologous expression is a necessity to facilitate the production of GDH for biotechnological applications and to study its physiological role in the outbreak of anthracnose caused by <it>Glomerella </it>(<it>anamorph Colletotrichum) spp</it>.</p> <p>Results</p> <p>Heterologous expression of active <it>G. cingulata </it>GDH has been achieved in both <it>Escherichia coli </it>and <it>Pichia pastoris</it>, however, the expressed volumetric activity was about 4800-fold higher in <it>P. pastoris</it>. Expression in <it>E. coli </it>resulted mainly in the formation of inclusion bodies and only after co-expression with molecular chaperones enzymatic activity was detected. The fed-batch cultivation of a <it>P. pastoris </it>transformant resulted in an expression of 48,000 U L^-1of GDH activity (57 mg L^-1). Recombinant GDH was purified by a two-step purification procedure with a yield of 71%. Comparative characterization of molecular and catalytic properties shows identical features for the GDH expressed in <it>P. pastoris </it>and the wild-type enzyme from its natural fungal source.</p> <p>Conclusions</p> <p>The heterologous expression of active GDH was greatly favoured in the eukaryotic host. The efficient expression in <it>P. pastoris </it>facilitates the production of genetically engineered GDH variants for electrochemical-, physiological- and structural studies.</p

Beiträge zur Geschichte des Landkreises Regensburg 24

24 Marginalien von 18 Autoren; darin: Werner, Hannsjürgen: Ein endneolithisches Körpergrab bei Moosham (S. 4); Fischer, T.: Die vor 110 Jahren erwähnt Burg Langenerling (S. 8); Bößl, Karl: Als Kareth zur freien Reichsstadt Regensburg gehörte (S. 10); Hummel, Franz: Der Hermann-, German- und Gerhof in der Gemeinde Wolfsegg (S. 12); Hemrich, Hans: Die letzte Ritterschlacht wurde bei Wenzenbach geschlagen (S. 14); Unterstöger, A.: Die Wallfahrtskirche St. Salvator in Donaustauf (S. 16); Daxl, Petra: Die Besiedlung der Hohengebrachinger Heide (S. 18); Neumann, Wenzel: Die Mötzinger Votivtafeln von 1775 und 1815 (S. 20); Schön, M.: Napoleon in Alteglofsheim (S. 22); Geser, Werner: Zum Alter der Wolfgangskapelle Klausen bei Thalmassing (S. 24); Jörgl, Fritz: Interessantes aus alten Geislinger Rechnungen (S. 25); Bösl, Hans-Josef: Die Heilung eines Taubstummen in der Wallfahrtskirche Mariae Schnee (S. 28); Fendl, Josef: Soll die Egglfinger Kirche verfallen? ( S. 30); Ebner, Karl: Aus der Geschichte des Schützenvereins Pfatter (S. 32); Staudigl, Franz Xaver: Beratzhausen in der Literatur (S. 34); Besenreiter, J.: Handwerk und Gewerbe in Aufhausen im Wandel der Zeit (S. 36); Forster, Fritz: Der Thurn- und Taxis´sche Wildpark (S. 38

"Leo in fabula"

Analyse der Figur des Löwen in La Fontaines Fabeln unter Bezugnahme auf König Ludwig XIV.Analysis of the lion's figure in La Fontaine's Fables and its interrelation with King Louis XIV

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

biomolecule

l‑Arabinose Isomerase and d‑Xylose Isomerase from Lactobacillus reuteri: Characterization, Coexpression in the Food Grade Host Lactobacillus plantarum, and Application in the Conversion of d‑Galactose and d‑Glucose

The l-arabinose isomerase (l-AI) and the d-xylose isomerase (d-XI) encoding genes from Lactobacillus reuteri (DSMZ 17509) were cloned and overexpressed in Escherichia coli BL21 (DE3). The proteins were purified to homogeneity by one-step affinity chromatography and characterized biochemically. l-AI displayed maximum activity at 65 °C and pH 6.0, whereas d-XI showed maximum activity at 65 °C and pH 5.0. Both enzymes require divalent metal ions. The genes were also ligated into the inducible lactobacillal expression vectors pSIP409 and pSIP609, the latter containing a food grade auxotrophy marker instead of an antibiotic resistance marker, and the l-AI- and d-XI-encoding sequences/genes were coexpressed in the food grade host Lactobacillus plantarum. The recombinant enzymes were tested for applications in carbohydrate conversion reactions of industrial relevance. The purified l-AI converted d-galactose to d-tagatose with a maximum conversion rate of 35%, and the d-XI isomerized d-glucose to d-fructose with a maximum conversion rate of 48% at 60 °C

Galactose oxidase from Fusarium oxysporum--expression in E. coli and P. pastoris and biochemical characterization.

A gene coding for galactose 6-oxidase from Fusarium oxysporum G12 was cloned together with its native preprosequence and a C-terminal His-tag, and successfully expressed both in Escherichia coli and Pichia pastoris. The enzyme was subsequently purified and characterized. Among all tested substrates, the highest catalytic efficiency (kcat/Km) was found with 1-methyl-β-D-galactopyranoside (2.2 mM(-1) s(-1)). The Michaelis constant (Km) for D-galactose was determined to be 47 mM. Optimal pH and temperature for the enzyme activity were 7.0 and 40°C, respectively, and the enzyme was thermoinactivated at temperatures above 50°C. GalOx contains a unique metalloradical complex consisting of a copper atom and a tyrosine residue covalently attached to the sulphur of a cysteine. The correct formation of this thioether bond during the heterologous expression in E. coli and P. pastoris could be unequivocally confirmed by MALDI mass spectrometry, which offers a convenient alternative to prove this Tyr-Cys crosslink, which is essential for the catalytic activity of GalOx

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