Stabilization of modified glucose oxidase from Penicillium vitale incorporated into the polymeric chains of gels

Glucose oxidase from Penicillium vitale was modified by unsaturated compounds, e.g. acrolein, allylisothiocyanate, acryloyl chloride and maleic anhydride. The degree of modification for the respective agents made up to 27.2, 8.3, 11.1 and 35.0% of the amine residues; the enzymatic activity was thereby retained by 98, 100, 0.03 and 58%, respectively. The thermal stability of modified enzymes was decreased 2-7 times. The modified preparations were copolymerized with acrylamide or 2-hydroxyethyl methacrylate, spatially cross-linked by N,N'-methylenebisacrylamide or ethyleneglycol dimethacrylate, respectively. The rate constants for thermal inactivation of immobilized glucose oxidase were 2-31 times lower as compared to the inactivation constants of the unmodified enzyme, incorporated into the same matrices.     

Study of the subunit structure of catalase from Penicillium vitale

A molecule of Penicillium vitale catalase is shown to dissociate into subunits with the molecular weight 75-80 kDalton. When hemin is splitted off the molecule also disintegrates into subunits equalling 1/4 of the enzyme molecule. The amino acid composition and fingerprints of the catalase subunits were studied. It is supposed that N-terminal residue of the subunit is blocked.     

Three-dimensional structure of catalase from Penicillium vitale at 2.0 A resolution

The three-dimensional structure analysis of crystalline fungal catalase from Penicillium vitale has been extended to 2.0 A resolution. The crystals belong to space group P3(1)21, with the unit cell parameters of a = b = 144.4 A and c = 133.8 A. The asymmetric unit contains half a tetrameric molecule of 222 symmetry. Each subunit is a single polypeptide chain of approximately 670 amino acid residues and binds one heme group. The amino acid sequence has been tentatively determined by computer graphics model building (using the FRODO system) and comparison with the known sequence of beef liver catalase. The atomic model has been refined by the Hendrickson & Konnert (1981) restrained least-squares program against 68,000 reflections between 5 A and 2 A resolution. The final R-factor is 0.31 after 24 refinement cycles. The secondary and tertiary structure of the catalase has been analyzed.     

Intracellular glucose oxidase from Penicillium funiculosum 46.1

A method for isolating extracellular glucose oxidase from the fungus Penicillium funiculosum 46.1, using ultrafiltration membranes, was developed. Two samples of the enzyme with a specific activity of 914-956 IU were obtained. The enzyme exhibited a high catalytic activity at pH above 6.0. The effective rate constant of glucose oxidase inactivation at pH 2.6 and 16 degrees C was 2.74 x 10(-6) s-1. This constant decreased significantly as pH of the medium increased (4.0-10.0). The temperature optimum for glucose oxidase-catalyzed beta-D-glucose oxidation was in the range 30-65 degrees C. At temperatures below 30 degrees C, the activation energy for beta-D-glucose oxidation was 6.42 kcal/mol; at higher temperatures, this parameter was equal to 0.61 kcal/mol. Kinetic parameters of glucose oxidase-catalyzed delta-D-glucose oxidation depended on the initial concentration of the enzyme in the solution. Glucose oxidase also catalyzed the oxidation of 2-deoxy-D-glucose, maltose, and galactose.     

Comparison of beef liver and Penicillium vitale catalases

The structures of Penicillium vitale and beef liver catalase have been determined to atomic resolution. Both catalases are tetrameric proteins with deeply buried heme groups. The amino acid sequence of beef liver catalase is known and contains (at least) 506 amino acid residues. Although the sequence of P. vitale catalase has not yet been determined chemically, 670 residues have been built into the 2 A resolution electron density map and have been given tentative assignments. A large portion of each catalase molecule (91% of residues in beef liver catalase and 68% of residues in P. vitale catalase) shows structural homology. The root-mean-square deviation between 458 equivalenced C alpha atoms is 1.17 A. The dissimilar parts include a small fragment of the N-terminal arm and an additional "flavodoxin-like" domain at the carboxy end of the polypeptide chain of P. vitale catalase. In contrast, beef liver catalase contains one bound NADP molecule per subunit in a position equivalent to the chain region, leading to the flavodoxin-like domain, of P. vitale catalase. The position and orientation of the buried heme group in the two catalases, relative to the mutually perpendicular molecular dyad axes, are identical within experimental error. A mostly hydrophobic channel leads to the buried heme group. The surface opening to the channel differs due to the different disposition of the amino-terminal arm and the presence of the additional flavodoxin-like domain in P. vitale catalase. Possible functional implications of these comparisons are discussed.     

Structural and biochemical properties of glycosylated and deglycosylated glucose oxidase from Penicillium amagasakiense

Glucose oxidase from Penicillium amagasakiense was purified to homogeneity by ion-exchange chromatography and deglycosylated with endoglycosidase H. On the basis of gas chromatography and sodium dodecyl sulphate/polyacrylamide gel electrophoretic (SDS-PAGE) analyses, the protein-bound high-mannose-type carbohydrate moiety corresponded to 13% of the molecular mass of glycosylated glucose oxidase. A total of six N-glycosylation sites per dimer were determined from the N-acetylglucosamine content. The enzymatically deglycosylated enzyme contained less than 5% of the original carbohydrate moiety. A molecular mass of 130 kDa (gel filtration) and 133 kDa (native PAGE) was determined for the dimer and 67 kDa (SDS-PAGE) for the monomer of the deglycosylated enzyme. The N-terminal sequence, which has not been published for glucose oxidase from P. amagasakiense to date and which showed less than 50% homology to the N terminus of glucose oxidase from Aspergillus niger, and the amino acid composition were not altered by the deglycosylation. Deglycosylation also did not affect the kinetics of glucose oxidation or the pH and temperature optima. It also did not increase the susceptibility of the enzyme to proteolytic degradation. However, deglycosylated glucose oxidase exhibited decreased pH and thermal stability. The thermal stability of both enzymes was shown to be dependent on the buffer concentration and was enhanced by certain additives, particularly 1 M (NH₄)₂SO₄, which stabilised glucose oxidase 100- to 300-fold at 50 °C and pH 7–8, and 2 M KF, which stabilised the enzyme up to 36-fold at 60 °C and pH 6. In sodium acetate buffer, changes in pH (4–6) affected the affinity for glucose but had no effect on the V _max of the reaction. In contrast, in TRIS buffer, pH 8, a 10-fold decrease in V _max and a 2-fold decrease in K _m were observed. Received: 8 October 1996 / Received revision: 14 January 1997 / Accepted: 17 January 1997     

Thermal stability of Penicillium adametzii glucose oxidase]

The thermal stability of glucose oxidase was studied at temperatures between 50 and 70 degrees C by kinetic and spectroscopic (circular dichroism) methods. The stability of glucose oxidase was shown to depend on the medium pH, protein concentration, and the presence of protectors in the solution. At low protein concentrations (< 15 micrograms/ml) and pH > 5.5, the rate constants kin (s-1) for thermal inactivation of glucose oxidase were high. Circular dichroic spectra suggested an essential role of beta structures in stabilizing the protein globule. At a concentration of 15 micrograms protein/ml, the activation energy Ea of thermal inactivation of glucose oxidase in aqueous solution was estimated at 79.1 kcal/mol. Other thermodynamic activation parameters estimated at 60 degrees C had the following values: delta H = 78.4 kcal/mol, delta G = 25.5 kcal/mol, and delta S = 161.9 entropy units. The thermal inactivation of glucose oxidase was inhibited by KCl, polyethylene glycols, and polyols. Among polyols, the best was sorbitol, which stabilized glucose oxidase without affecting its activity. Ethanol, phenol, and citrate exerted destabilizing effects.     

Isolation and characterization of extracellular glucose oxidase from Penicillium adametzii LF F-2044.1

Hydroxides of magnesium and zinc, aluminum oxide, zinc phosphate, and co-precipitated Ca₃(PO₄)₂ and Mg(OH)₂ were efficient in binding extracellular glucose oxidase (GO) of P. adametzii LF F-2044.1 in a culture liquid filtrate (CLF). Basic Al₂O₃ was the most appropriate adsorbent for GO isolation from the CLF of the fungus. A GO isolation method was developed, which allowed for obtaining an enzyme with a high degree of purification. Spectral properties of the enzyme, its catalytic activity, and stability were characterized. The GO of P. adametzii LF F-2044.1 exhibited high pH stability, retaining activity within the range 4.5–9.0. The rate that GO-catalyzed D-glucose oxidation increased as the temperature increased (up to approximately 60°C). The catalytic activity and thermal stability of GO depended on its concentration in the medium. Under optimum conditions, the fractions GO-1 and GO-2 were characterized by K _M values of 1.56 × 10^?2 and 2.19 × 10^?2 M, respectively; the corresponding values of k _cat equaled 235.1 and 318.2 s^?1.     

Isolation and characterization of extracellular glucose oxidase from Penicillium adametzii LF F-2044.1

Hydroxides of magnesium and zinc, aluminum oxide, zinc phosphate, and co-precipitated Ca3(PO4)2 and Mg(OH)2 were efficient in binding extracellular glucose oxidase (GO) of P. adametzii LF F-2044.1 in a culture liquid filtrate (CLF). Basic Al2O3 was the most appropriate adsorbent for GO isolation from the CLF of the fungus. A GO isolation method was developed, which allowed for obtaining an enzyme with a high degree of purification. Spectral properties of the enzyme, its catalytic activity, and stability were characterized. The GO of P. adametzii LF F-2044.1 exhibited high pH stability, retaining activity within the range 4.5-9.0. The rate that GO-catalyzed D-glucose oxidation increased as the temperature increased (up to approximately 60 degrees C). The catalytic activity and thermal stability of GO depended on its concentration in the medium. Under optimum conditions, the fractions GO-1 and GO-2 were characterized by KM values of 1.56 x 10(-2) and 2.19 x 10(-2) M, respectively; the corresponding values of kcat equaled 235.1 and 318.2 s(-1).     

Recombinant glucose oxidase from Penicillium amagasakiense for efficient bioelectrochemical applications in physiological conditions

GOX is the most widely used enzyme for the development of electrochemical glucose biosensors and biofuel cell in physiological conditions. The present work describes the production of a recombinant glucose oxidase from Penicillium amagasakiense (yGOXpenag) displaying a more efficient glucose catalysis (k_cat/K_M(glucose) = 93 μM⁻¹ s⁻¹) than the native GOX from Aspergillus niger (nGOXaspng), which is the most industrially used (k_cat/K_M(glucose) = 27 μM⁻¹ s⁻¹). Expression in Pichia pastoris allowed easy production and purification of the recombinant active enzyme, without overglycosylation. Its biotechnological interest was further evaluated by measuring kinetics of ferrocinium-methanol (FM_ox) reduction, which is commonly used for electron transfer to the electrode surface. Despite their homologies in sequence and structure, pH-dependant FM_ox reduction was different between the two enzymes. At physiological pH and temperature, we observed that electron transfer to the redox mediator is also more efficient for yGOXpenag than for nGOXaspng(k_cat/K_M(FM_ox) = 27 μM⁻¹ s⁻¹ and 17 μM⁻¹ s⁻¹ respectively). In our model system, the catalytic current observed in the presence of blood glucose concentration (5 mM) was two times higher with yGOXpenag than with nGOXaspng. All our results indicated that yGOXpenag is a better candidate for industrial development of efficient bioelectrochemical devices used in physiological conditions.     

Distribution and typology of glucose oxidase activity in the genus Penicillium

Glucose oxidase (GOD) production by 84 strains of the genus Penicillium was studied in order to define the distribution of the activity among and within the species. Plate screening and sub-screening in shake culture showed a rather homogeneous behaviour of strains belonging to the same species. Penicillium chrysogenum, P. expansum, P. italicum and P. variabile had a concomitant good production of GOD and catalase activity; peroxidase and polyphenol oxidase were never present. The capability of the culture filtrates to oxidase substrates other than glucose was also studied.     

Selection of Penicillium funiculosum strains with high glucose oxidase activity

Metabolic inhibitors, riboflavin, and end products of glucose oxidation were shown to hold much promise for the selection of Penicillium funiculosum mutant strains with a high glucose oxidase activity. The incidence of positive mutations was highest in clones resistant to sodium azide, riboflavin, and beta-D-glucono-delta-lactone. Enzyme activity in Penicillium funiculosum mutants was studied under conditions of submerged cultivation. The intensity of glucose oxidase synthesis in seven cultures was 24-56% higher than that in the parent strain of Penicillium funiculosum NMM95.132.     

The mechanism of inactivation of glucose oxidase from Penicillium amagasakiense under ambient storage conditions

Glucose oxidase (GOx) from Penicillium amagasakiense has a higher specific activity than the more commonly studied Aspergillus niger enzyme, and may therefore be preferred in many medical and industrial applications. The enzyme rapidly inactivates on storage at pH 7.0-7.6 at temperatures between 30 and 40 °C. Results of fluorimetry and circular dichroism spectroscopy indicate that GOx inactivation under these conditions is associated with release of the cofactor FAD and molten globule formation, indicated by major loss of tertiary structure but almost complete retention of secondary structure. Inactivation of GOx at pH < 7 leads to precipitation, but at pH ≥ 7 it leads to non-specific formation of small soluble aggregates detectable by PAGE and size-exclusion chromatography (SEC). Inactivation of P. amagasakiense GOx differs from that of A. niger GOx in displaying complete rather than partial retention of secondary structure and in being promoted rather than prevented by NaCl. The contrasting salt effects may reflect differences in the nature of the interface between subunits in the native dimers and/or the quantity of secondary structure loss upon inactivation.     

Crystallization and preliminary X-ray diffraction studies of a deglycosylated glucose oxidase from Penicillium amagasakiense.

The dimeric glucose oxidase from Penicillium amagasakiense was deglycosylated, purified and crystallized as a complex with its coenzyme FAD. Deglycosylation and purification to isoelectric homogeneity were shown to be an important prerequisite step to obtain crystals suitable for X-ray investigations. Crystals of the deglycosylated enzyme were reproducibly grown using ammonium sulfate as precipitant at pH 7.4 to 7.5. Crystals diffract to at least 2.0 A resolution and belong to the orthorhombic space group P2(1)2(1)2(1), with refined lattice constants of a = 59.3 A, b = 136.3 A and c = 156.7 A. Assuming two monomers (approximately 135 kDa) per asymmetric unit the Vm value is 2.3 A3/Da.     

Growth characteristics and glucose oxidase production by mutant Penicillium funiculosum strains

The main parameters of growth and glucose oxidase production by the mutant Penicillium funiculosum strains BIM F-15.3, NMM95.132, and 46.1 were studied. The synthesis of extracellular glucose oxidase by these strains was constitutive and occurred following the phase of exponential growth. The mutant strains also synthesized extracellular invertase and cell-associated catalase and glucose oxidase. The syntheses of invertase, the cell-associated enzymes, and extracellular glucose oxidase were found to be maximum between 14 and 18 h, between 48 and 52 h, and by the 96th h of cultivation, respectively. Among the mutants studied, P. funiculosum 46.1 showed the maximal rates of growth and glucose oxidase synthesis.