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

Expression and characterization of carbohydrate oxidizing enzymes

Galaktose Oxidase (GalOx) ist ein sekretorisches, von Pilzen produziertes Enzym mit einem breiten Substratspektrum. Das Enzym katalysiert die Oxidation von primären Alkoholen an der C6-Position zu den entsprechenden Aldehyden unter der Reduktion von molekularem Sauerstoff zu Wasserstoffperoxid. Die dafür notwendige zwei-Elektronen Redox-Chemie wird mit Hilfe eines mononuklearen Kupfer-Ions im aktiven Zentrum und einem Tyrosin-Radikal, welches als zweiter Redox-Cofaktor fungiert, bewerkstelligt. Das Kupfer wird von vier Aminosäure-Seitenketten koordiniert: zwei Tyrosinen (Tyr272 und Tyr495) und zwei Histidinen (His496 und His581). Der Kupferligand Tyr272 ist außerdem über sein C- Atom kovalent mit dem Schwefel-Atom von Cys228 unter Bildung einer Thioether-Bindung verknüpft. Die für Galaktose Oxidase kodierenden Gene von Fusarium oxysporum und Fusarium sambucinum wurden erfolgreich heterolog in Escherichia coli und jenes von F. oxysporum auch in Pichia pastoris exprimiert. Die produzierten Enzyme wurden mittels IMAC und einer Gelpermeationschromatographie gereinigt. Anschließend wurden die beiden Galaktose Oxidasen biochemisch charakterisiert. Der Einfluss des pH-Wertes und der Temperatur (Temperaturoptimum, Thermostabilität, dynamische Differenzkalorimetrie) auf die Enzymaktivität wurde untersucht. Für verschiedene Elektronen-Donoren, wie z.B. D-Galaktose, 1-Methyl--D-Galaktopyranosid und D-Melibiose wurden steady-state kinetische Konstanten gemessen. GalOx weist eine hohe Spezifität gegenüber molekularem Sauerstoff als Elektronen-Akzeptor auf. Es konnten keine alternativen Elektronen-Akzeptor gefunden werden. Der Effekt von verschiedenen Komponenten, wie z.B. monovalenten und divalenten Kationen, nicht-ionischen Detergenzien, EDTA, Azid und Cyanid, auf die Aktivität von GalOx wurde untersucht. Die korrekte Bildung der einzigartigen Thioether Bindung konnte mittels Massenspektrometrie für beide Expressionssysteme bestätigt werden.Galactose oxidase (GalOx) is a secretory fungal enzyme with a broad substrate spectrum. The enzyme catalyzes the oxidation of primary alcohols at the C6-position to the corresponding aldehydes with concomitant reduction of molecular oxygen to hydrogen peroxide. Therefore, a two-electron redox chemistry is provided by a mononuclear copper ion active site and a tyrosine radical serving as the second redox cofactor. The copper is coordinated by four amino acid side chains: two tyrosines (Tyr272 and Tyr495) and two histidines (His496 and His581). Furthermore, Tyr272 is covalently linked at C to the sulphur atom of Cys228 under formation of a thioether bond. The genes encoding for GalOx from Fusarium oxysporum and F. sambucinum were successfully heterologously expressed both in Escherichia coli, and the F. oxysporum gene in Pichia pastoris as well. The corresponding enzymes were purified based on the His-tag, which is provided by the expression vectors, by IMAC followed by a polishing step of size-exclusion chromatography. Furthermore, the biochemical properties of the two galactose oxidases produced by E. coli were investigated. Amongst others, the pH optima and the effect of temperature (temperature optimum, thermal stability, differential scanning calorimetry) on GalOx activity were determined. Steady-state kinetic constants were measured for different electron donor substrates including D-galactose, 1-methyl--D-galactopyranoside and D-melibiose. GalOx is highly specific for molecular oxygen as an electron acceptor and no alternative acceptor could be found. The effect of various compounds (e. g. monovalent and divalent cations, nonionic detergents, EDTA, azide and cyanide) on GalOx activity was determined. The formation of the unique thioether bond in both expression systems was confirmed directly for the first time using mass spectrometry.Regina PauknerZsfassung in dt. SpracheWien, Univ. für Bodenkultur, Diss., 2015OeBB(VLID)193194

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

Recombinantly produced cellobiose dehydrogenase from Corynascus thermophilus for glucose biosensors and biofuel cells

Cellobiose dehydrogenase (CDH) is an emerging enzyme in the field of bioelectrocatalysis. Due to its flexible cytochrome domain, which acts as a built-in redox mediator, CDH is capable of direct electron transfer (DET) to electrode surfaces. This rare property is employed in mediatorless "third generation" biosensors. The ability of Corynascus thermophilus CDH to oxidize glucose under physiological conditions makes it a promising candidate for miniaturized glucose biosensors or glucose powered biofuel cell anodes. We report for the first time the electrochemical application and characterization of a recombinantly produced CDH in a glucose biosensor. Recombinant CDH from C. thermophilus (rCtCDH) was expressed by the methylotrophic yeast Pichia pastoris (376 U L-1, 132 mg L-1). A comparative characterization of rCtCDH and CtCDH shows identical pH optima, KM values and heme b midpoint potentials. In contrast, the specific activity of rCtCDH (2.84 U mg(-1)) and consequently the turnover numbers were similar to five-times lower than for CtCDH, which was caused by a sub-stoichiometric occupation of catalytic sites with flavin-adenin-dinukleotid (FAD). The performance of rCtCDH-modified electrodes demonstrates the suitability for electrochemical studies. This opens the possibility to engineer the substrate specificity of C. thermophilus CDH for specific carbohydrates by rational engineering or directed evolution

Alignment of GalOx from <i>F. oxysporum</i> and <i>F. graminearum</i>.

<p>The prepro sequence is underlined. Amino acid residues involved in copper binding are highlighted.</p

MALDI-TOF peptide mass map of GalOx expressed in <i>E. coli</i>.

<p>The peptides were generated by sequential digestion using trypsin and Asp-N. The peak labels correspond to the [M+H]⁺ ions of the obtained peptide fragments and their positions in the GalOx sequence. The spectrum also shows two intense signals (marked with asterisk) at m/z 2237.9 and 2374.9 related to the cross-linked peptides 266–274/312–323 and 265–274/312–323, respectively. The identity of the cross-linked peptides was verified by MS/MS fragmentation.</p

Temperature dependence of the activity of GalOx expressed in <i>E. coli.</i>

<p>Temperature dependence of the activity of GalOx expressed in <i>E. coli.</i></p

Effect of the pH on the activity of GalOx expressed in <i>E. coli</i>.

<p>The buffers used were 50(♦), 50 mM phosphate (_▀) and 50 mM Tris (▴).</p

Purification of recombinant GalOx expressed in <i>E. coli.</i>

<p>Purification of recombinant GalOx expressed in <i>E. coli.</i></p

Electrophoresis analysis of crude extract and purified GalOx expressed in <i>E. coli</i>.

<p>Lane 1, Precision Plus Protein Standard (BioRad); lane 2, GalOx crude extract; lane 3, GalOx after IMAC; lane 4, GalOx after size-exclusion chromatography.</p