Structural and Kinetic Properties of Nonglycosylated Recombinant Penicillium amagasakiense Glucose Oxidase Expressed in Escherichia coli

The gene coding for Penicillium amagasakiense glucose oxidase (GOX; β-d-glucose; oxygen 1-oxidoreductase [EC 1.1.3.4]) has been cloned by PCR amplification with genomic DNA as template with oligonucleotide probes derived from amino acid sequences of N- and C-terminal peptide fragments of the enzyme. Recombinant Escherichia coli expression plasmids have been constructed from the heat-induced pCYTEXP1 expression vector containing the mature GOX coding sequence. When transformed into E. coli TG2, the plasmid directed the synthesis of 0.25 mg of protein in insoluble inclusion bodies per ml of E. coli culture containing more than 60% inactive GOX. Enzyme activity was reconstituted by treatment with 8 M urea and 30 mM dithiothreitol and subsequent 100-fold dilution to a final protein concentration of 0.05 to 0.1 mg ml⁻¹ in a buffer containing reduced glutathione-oxidized glutathione, flavin adenine dinucleotide, and glycerol. Reactivation followed first-order kinetics and was optimal at 10°C. The reactivated recombinant GOX was purified to homogeneity by mild acidification and anion-exchange chromatography. Up to 12 mg of active GOX could be purified from a 1-liter E. coli culture. Circular dichroism demonstrated similar conformations for recombinant and native P. amagasakiense GOXs. The purified enzyme has a specific activity of 968 U mg⁻¹ and exhibits kinetics of glucose oxidation similar to those of, but lower pH and thermal stabilities than, native GOX from P. amagasakiense. In contrast to the native enzyme, recombinant GOX is nonglycosylated and contains a single isoform of pI 4.5. This is the first reported expression of a fully active, nonglycosylated form of a eukaryotic, glycosylated GOX in E. coli.Glucose oxidase (GOX; β-d-glucose; oxygen 1-oxidoreductase [EC 1.1.3.4]) is a hydrogen peroxide-generating flavoprotein catalyzing the oxidation of β-d-glucose to d-glucono-1,5-lactone. GOX is used in the food industry for the removal of glucose from powdered eggs, as a source of hydrogen peroxide in food preservation, for gluconic acid production, and in the production of beer and soft drinks, in which its reaction serves an antioxidant function (10, 39, 42). GOX is also used extensively for the quantitative determination of d-glucose in samples such as blood, food, and fermentation products (10, 39, 49). The enzyme has been purified from both Aspergillus niger (45) and Penicillium spp. (33), with A. niger NRRL3 being the most widely used strain for industrial-scale production (11). A problem with utilizing GOX from its native source is the presence of impurities such as catalase, cellulase, and amylase, which may impair some of its applications. To overcome these difficulties and to simplify the stringent purification procedures, which are relatively expensive, A. niger GOX has been cloned and expressed in Saccharomyces cerevisiae as a highly glycosylated form (17).The most frequent employment of GOX has been in biosensors, in which the biochemical event of glucose oxidation is detected by electrochemical, thermometric, or optical techniques. The most interesting possibilities appear to lie in electron transfer reactions, with artificial electron acceptors or mediators being used to transfer information from the enzyme to the electrode (49). The electrical communication between GOX and the electrode and thereby its biosensor performance are hampered by the protein-bound carbohydrate moiety of the enzyme (1, 15), which most probably impedes electron tunneling through the enzyme (32). Almost complete (24, 27) or partial (15, 32) deglycosylation of GOX is possible, but the procedure is expensive and complicated. A more efficient and effective way of obtaining nonglycosylated GOX would be to express the enzyme in a prokaryotic host. This would also enable the properties and efficiency of GOX to be improved for its use in biosensors by protein engineering techniques (49). As a first step towards this objective, GOX from Penicillium amagasakiense was cloned and expressed in Escherichia coli. GOX from P. amagasakiense was selected since the enzyme has a higher turnover rate and a better affinity for β-d-glucose than its A. niger counterpart (30, 33).In this study, we describe the cloning and expression of the gene encoding P. amagasakiense GOX and the refolding, purification, and characterization of the nonglycosylated recombinant enzyme. The activity of the recombinant GOX, expressed in the form of insoluble inclusion bodies, was reconstituted, and the active enzyme was shown to possess properties and secondary structure composition similar to those of native P. amagasakiense GOX. This is the first reported expression of a fully active nonglycosylated form of a eukaryotic glycosylated GOX in a prokaryote, which enabled us to demonstrate that in contrast to previous assumptions (4, 9, 47) the protein-bound carbohydrate moiety is not essential for the correct folding of GOX.