Purification and thermodynamic characterization of glucose oxidase ...

519

Purification and thermodynamic characterization of glucose oxidase from a newly isolated strain of Aspergillus niger H.N. Bhatti, M. Madeeha, M. Asgher, and N. Batool

Abstract: An intracellular glucose oxidase (GOD) was isolated from the mycelium extract of a locally isolated strain of Aspergillus niger NFCCP. The enzyme was partially purified to a yield of 28.43% and specific activity of 135 U mg–1 through ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The enzyme showed high specificity for D-glucose, with a Km value of 25 mmol L–1. The enzyme exhibited optimum catalytic activity at pH 5.5. Optimum temperature for GOD-catalyzed D-glucose oxidation was 40 °C. The enzyme displayed a high thermostability having a half-life (t1/2) of 30 min, enthalpy of denaturation (H*) of 99.66 kJ mol–1, and free energy of denaturation (G*) of 103.63 kJ mol–1. These characteristics suggest that GOD from A. niger NFCCP can be used as an analytical reagent and in the design of biosensors for clinical, biochemical, and diagnostic assays. Key words: glucose oxidase, Aspergillus niger, kinetics, thermodynamics, thermal stability. Résumé : Une glucose oxydase (GOD) extracellulaire fut isolée de l’extrait du mycélium d’une souche isolée localement de Aspergillus niger NFCCP. L’enzyme fut partiellement purifiée avec un rendement de 28,43 % et une activité spécifique de 135 U mg–1 par précipitation au sulfate d’ammonium, chromatographie à échange anionique et filtration sur gel. L’enzyme a démontré une spécificité élevée au D-glucose, avec une valeur Km de 25 mmol L–1. L’enzyme a démontré une activité catalytique optimale à un pH de 5,5. La température optimale pour l’oxydation du D-glucose catalysée par la GOD était de 40 °C. L’enzyme a manifesté une thermostabilité élevée avec une demi-vie (t1/2) de 30 min, une enthalpie de dénaturation (H*) de 99,66 kJ mol–1 est une énergie libre de dénaturation (G*) de 103,63 kJ mol–1. Ces caractéristiques encouragent l’utilisation de la GOD de A. niger NFCCP comme réactif analytique et dans la conception des biosenseurs pour des analyses cliniques, biochimiques et diagnostiques. Mots clés : glucose oxydase, Aspergillus niger, cinétique, thermodynamique, stabilité thermique. [Traduit par la Rédaction]

Bhatti et al.

524

Introduction Glucose oxidase (GOD; β-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) is an important dimeric enzyme that catalyzes the oxidation of β-D-glucose to D-glucono-1,5-lactone and hydrogen peroxide and finally to gluconic acid, using molecular oxygen as electron acceptor. It is widely used (i) in the removal of traces of oxygen or glucose from different foods, such as dried eggs, beer, wine, and fruit juices; (ii) as a source of hydrogen peroxide in food preservation; and (iii) in gluconic acid production (Kapat et al. 1998). Antimicrobial enzymes are ubiquitous in nature, playing a significant role in the defense Received 19 September 2005. Revision received 23 November 2005. Accepted 21 December 2005. Published on the NRC Research Press Web site at http://cjm.nrc.ca on 31 May 2006. H.N. Bhatti, M. Madeeha, M. Asgher,1 and N. Batool. Industrial Biotechnology Laboratory, Department of Chemistry, University of Agriculture, Faisalabad, Pakistan. 1

Corresponding author (e-mail: [email protected]).

Can. J. Microbiol. 52: 519–524 (2006)

mechanisms of living organisms against infections. GOD is the most promising enzyme that exerts its effect by generating reactive molecules in situ that can destroy vital proteins in the cell. Clinical applications of GOD in diagnostic tests are likely to be the most promising of its uses. The catalytic properties of GOD form the basis of assaying body fluids, such as blood and urine, for glucose (Schmid and Karube 1988). It has also been used as an ingredient of toothpaste. A new application for GOD is its use in biosensors (Petruccioli et al. 1999). In this respect, emerging preservation techniques, including immobilization, have received particular attention (Fugslang et al. 1995). Immobilized enzymes are very robust and stable and are capable of functioning in a wide range of conditions. However, the stability of the immobilized enzyme derivatives depends on enzyme concentration, suggesting that the dissociation of subunits could be playing a key role in the inactivation of multimeric enzymes (Hidalgo et al. 2003; Wilson et al. 2004). GOD also contains tightly bound flavin adenine dinucleotide (FAD), which may dissociate from the protein part at higher temperature treatments. Dissociation of

doi:10.1139/W05-158

© 2006 NRC Canada

520

subunits or FAD may become even more relevant when working at an industrial scale, as a loss of a small fraction of subunits per reaction cycle could promote a very rapid deactivation of the enzyme bio-reactor. Stabilization of the quaternary structure of multimeric enzymes may, therefore, have very profitable effects on their industrial performance (Wilson et al. 2004). Stabilization via multi-subunit covalent immobilization on highly activated supports like agarose–glutaraldehyde can improve functional and thermostabilization properties of multimeric enzymes (Balcao et al. 2001). Stabilization of the quaternary structures of multimeric enzymes has been achieved by multi-subunit immobilization of the enzyme followed by its further solid-phase chemical inter-subunit cross-linking with different bifunctional (Cao et al. 2000) and polyfunctional (dextran–aldehyde) reagents (Fernandez-Lafuente et al. 2001). These cross-linked enzyme aggregates stabilize the quaternary structure even better than multi-subunit covalent attachment in preexisting supports and have improved stability, selectivity, and specificity compared with that of soluble enzymes (Cao et al. 2001). Immobilization of GOD on microfiltration polyamide membrane (Vasileva and Godjevargova 2004), glyoxyl agarose and epoxy sepabeads (Betancor et al. 2006), and alkylamine controlled pore glass beads (Vojinovic et al. 2005) has been reported to enhance enzyme stability. Stabilization of GOD by glutaraldehyde cross-linking of adsorbed enzyme proteins on aminated supports (Lopez-Gallego et al. 2005) and entrapment of GOD in liposomes (Rodriguez-Nogales 2004) have gained considerable significance not only for separation purposes but also as means of carrying catalytically active substances. Multilayer nanofilms of photoreactive diazoresin with poly(styrene sulfonate) have also been used for stabilization of GOD for biosensor applications (Srivastava et al. 2005). The uncross-linked and cross-linked (diazoresincoated or poly(styrene sulfonate)-coated) spheres retained more than 50% of their initial activity after 4 weeks, which remained stable even after 24 weeks for the two and three bilayer films. It is generally accepted that the suitability of an enzyme in biosensors and for other practical purposes depends on the enzyme’s thermal stability and stability in various media (Eremin et al. 2001; Vasileva and Godjevargova 2005). Thermodynamic parameters are also important for inactivation studies of enzymes (Rashid and Siddiqui 1998; Iqbal et al. 2003). Immobilization of GOD with glass beads as support and with 4% silane has been reported to considerably enhance the thermostability of GOD at 70 °C (Sarath Babu et al. 2004), which may be attributed to the increase in the surface hydrophobicity of the support. The mycelial fungi Aspergillus niger, Penicillium amagasakiense, and Penicillium vitale serve as industrial producers of GOD. The carbon sources employed in the production of GOD from A. niger are mainly glucose and to a lesser extent sucrose. The use of cheaper carbon sources appears to be essential for the improvement of process economy (Hatzinikolaou and Macris 1995). To exploit new industrial potentials of GOD, it is necessary to investigate new microbial strains and to understand the structure–stability relationship of this important enzyme. In this manuscript, we describe the production and characterization of GOD from a locally

Can. J. Microbiol. Vol. 52, 2006

isolated strain of A. niger NFCCP as a part of the efforts being made to develop an indigenous technology for the production of this industrially important enzyme.

Materials and methods Chemicals All chemicals were purchased from the Sigma Chemical Co., St. Louis, Missouri, USA, unless otherwise mentioned. Organism and inoculum preparation A pure culture of A. niger NFCCP was obtained from the National Fungal Culture Collection of Pakistan (NFCCP), Department of Plant Pathology, University of Agriculture, Faisalabad. It was maintained on potato dextrose agar (PDA) slants at 4 °C. Inoculum was developed in 250 mL Erlenmeyer flasks with a working volume of 50 mL. The medium composition was as follows (g/L): (NH4)2HPO4, 0.4; KH2PO4, 0.2; MgSO4·7H2O, 0.2; peptone, 10.0; sucrose, 70.0. The pH was adjusted to 5.5 using 1 mol L–1 NaOH prior to sterilization (Hatzinikolaou and Macris 1995). The flasks were then incubated on a rotary shaker operating at 200 r/min at 30 °C for 48 h to get homogenous growth containing 106–107 spores/mL. Production of GOD Studies on GOD production were carried out in 250 mL Erlenmeyer flasks with 50 mL working volumes of the fermentation medium (Hatzinikolaou and Macris 1995). The medium contained the following (g/L): molasses, 4.0; (NH4)2HPO4, 0.4; KH2PO4, 0.2; MgSO4·7H2O, 0.2; peptone, 2.0; sucrose, 100.0; and CaCO3, 5.0. Sterilization was affected at 121 °C for 15 min and cooled to 30 °C. Experiments were carried out at an initial pH of 5.5 and 30 °C, unless CaCO3 was added in the growth medium (resulting in an initial pH of 6.5–6.8). The flasks were inoculated with 5 mL of inoculum and incubated in a rotary shaker operating at 150 r/min for 48–72 h for GOD production. At the end of the fermentation, the cell mass was centrifuged (10 000g, 20 min) and disrupted to release the GOD. Protein estimation Total proteins were estimated by the Bradford microassay (Bradford 1976) with bovine serum albumin as the standard. GOD assay GOD activity was determined with the help of a coupled o-dianisidine peroxidase reaction as described earlier (Gouda et al. 2003). Appropriately diluted GOD solution (200 µL) was added to the dianisidine buffer mixture (pH 6.0) containing glucose and peroxidase after proper mixing. The increase in absorbance at 460 nm was monitored for 5 min at 25 °C with a spectrophotometer. One unit of GOD activity was defined as the amount of enzyme required to oxidize 1 micromole of glucose per minute under the above assay conditions. Isolation and purification of glucose oxidase At the end of fermentation, the mycelia were separated from the culture liquid by filtration, suspended in 0.1 mol/L citrate–phosphate buffer (pH 4.0), and finally disrupted in a commercial blender, as described earlier (Hatzinikolaou and Macris 1995). The crude extract (200 mL) was subjected to © 2006 NRC Canada

Bhatti et al.

521

Table 1. Purification of Aspergillus niger NFCCP glucose oxidase. Treatment

Total activity (U)

Total protein (mg)

Crude 85% (NH4)2SO4 precipitation Anion-exchange chromatography Gel filtration

2374 1589 1197 675

132 74 22 5

Fig. 1. Effect of pH on the activity of Aspergillus niger NFCCP glucose oxidase.

85% saturation with ammonium sulfate saturation at 0 °C. It was kept at 4 °C overnight. After 24 h, the resulting precipitate was collected by centrifugation at 10 000g for 30 min. The precipitate was dispersed in 5 mmol L–1 acetate buffer (pH 5.5) and dialyzed extensively against the same buffer to remove the salts. The dialyzed sample was applied to a DEAE– cellulose column (2.4 cm × 26.0 cm) equilibrated with 5 mmol L–1 acetate buffer (pH 5.5). Fractions of 2 mL were collected. The active fractions were pooled and dialyzed against distilled water. The sample from the DEAE–cellulose column was then applied to a Sephadex G-100 column (2.4 cm × 26.0 cm) previously equilibrated with 5 mmol L–1 acetate buffer (pH 5.5) and eluted with the same buffer, as described by Sukhacheva et al. (2004). The eluates containing GOD activity were pooled and used for kinetic and thermodynamic characterization. Effect of pH The effect of pH on GOD activity was determined by assaying the enzyme as mentioned before, the only difference being that the activity was determined at different pH values ranging from 4 to 8 in various buffer solutions, as described earlier (Siddiqui et al. 1997). Effect of temperature and activation energy GOD was assayed at different temperatures ranging from 20 to 70 °C at pH 5.5, as described before. Activation energy was determined from the Arrhenius plot as described earlier (Siddiqui et al. 1997; Rashid and Siddiqui 1998). Effect of substrate GOD from A. niger NFCCP was assayed in the reaction mixtures containing variable amounts of glucose (4.0%–25%,

Specific activity (U mg–1) 17.98 21.47 54.41 135.00

Purification factor

% recovery

1.00 1.19 3.03 7.50

100.00 66.93 50.42 28.43

m/v) at pH 5.5. The data were plotted according to Lineweaver–Burk to determine the values of kinetic constants (Vmax and Km). Kinetics of thermal denaturation Kinetic and thermodynamic parameters for irreversible thermal denaturation of GOD were determined by incubating the enzyme in 50 mmol L–1 MES (morpholineethanesulphonic) monohydrate buffer (pH 5.5) at a particular temperature. Aliquots were withdrawn at different times, cooled on ice for 3 h (Violet and Meunier 1989), and assayed for enzyme activity at 25 °C, as described above. This procedure was repeated at five different temperatures ranging from 45 to 60 °C. The data were fitted to first-order plots (Fig. 1) and analyzed as described earlier (Munch and Tritsch 1990; Montes et al. 1995). The thermodynamic parameters for thermostability were calculated by rearranging the Eyring’s absolute rate equation derived from the transition state theory (Eyring and Stearn 1939) as described by Siddiqui et al. (1999). [1]

kd = (kbT/h) e(–H*/RT) e(S*/R)

where h is Planck’s constant (6.63 × 10–34 Js), kb is Boltzmann’s constant (R/NA; 1.38 × 10–23 J K–1), R is the gas constant (8.314 J K–1 mol–1), NA is Avogadro’s No. (6.02 × 1023 mol–1), and T is the absolute temperature. [2]

H* (enthalpy of activation) = Ea – RT

where Ea is the activation energy. [3]

G* (free energy of activation) = –RT ln[kdh/(kbT)]

[4]

S* (entropy of inactivation) = (H* – G*)/T

Results Enzyme production The intracellular GOD production continued to increase up to 3 days (72 h), followed by a decrease (data not shown). Disintegration of the mycelia after 3 days of submerged fermentation resulted in a GOD activity of 10.85 U mL–1 in the mycelial extract. Purification of GOD The specific activity of crude extract was 17.98 U mg–1 (Table 1). The complete precipitation of GOD was observed with 85% ammonium sulfate treatment. Purification of the enzyme on an anion-exchange column was 3.03-fold with 50.42% recovery. On a gel filtration column the enzyme was © 2006 NRC Canada

522 Fig. 2. Arrhenius plot for the determination of activation energy of Aspergillus niger NFCCP glucose oxidase for the oxidation of D-glucose.

Fig. 3. Irreversible thermal inactivation of Aspergillus niger NFCCP glucose oxidase. 䉱, 45 °C; 䉭, 48 °C; 䊉, 52 °C; 䊊, 56 °C; and ⵧ, 60 °C.

Can. J. Microbiol. Vol. 52, 2006 Fig. 4. Arrhenius plots for the determination of energy of activation for irreversible thermal inactivation of Aspergillus niger NFCCP glucose oxidase.

Thermal denaturation studies The GOD from A. niger NFCCP was thermally stable at 45 °C with a half-life of 173 min. However, at 60 °C it was less stable and displayed a half life of 30 min under similar conditions (Fig. 3). The enzyme had H* values of 99.79 and 99.66 kJ mol–1 at 45 and 60 °C, respectively (Fig. 4). The G* value for GOD was 103.47 kJ mol–1 at 45 °C, showing a decreasing trend with an increase in temperature. A ∆G* value of 103.63 kJ mol–1 was observed at 60 °C. When S* was calculated at each temperature, negative values were found (Table 2). GOD from A. niger NFCCP showed an S* value of –11.92 J mol–1 K–1 at 60 °C.

Discussion

purified 7.5-fold to a specific activity of 135 U mg–1 with a yield of 28.43%. Effect of pH The optimum pH was determined by incubating the enzyme in a wide pH range at 25 °C. The enzyme exhibited high activity in a pH range spanning 5.0–6.0 (Fig. 1), with the optimum pH at 5.5. The activity was found to decrease above and below pH 5.5. Effect of temperature and activation energy The activation energy (Ea) and optimum temperature of the GOD were found to be 15.46 kJ mol–1 and 40 °C, respectively. It is obvious from the Arrhenius plot (Fig. 2) that the enzyme had a single conformation up to the transition temperature. Effect of substrate Purified GOD having a protein content of 4.7 × 10–3 mg mL–1 was used for the kinetic and thermodynamic characterization. The Km and Vmax values as determined from Lineweaver– Burk plots were 25 mmol L–1 and 435 U mg–1, respectively.

GOD activities as high as 10.85 U mL–1 were produced, comparing favorably with those reported for other GODproducing microorganisms. Hatzinikolaou and Macris (1995) reported a maximum activity (5.7 U mL–1) by A. niger BTL after 6 days. A purification procedure applied to A. niger GOD resulted in 7.5-fold purification with a 28.43% recovery. The optimum pH for GOD from A. niger NFCCP was found to be 5.5. The change in pH affects the ionization of essential active-site amino acid residues, which are involved in substrate binding and catalysis, i.e., in the breakdown of substrate into products. Some ionizable residues may be located on the periphery of the active site, commonly known as nonessential residues. The ionization of these residues may cause distortion of active site cleft and, hence, indirectly affect the enzyme activity. The results obtained are comparable to those of Weibel and Bright (1971) who reported that GOD worked in the pH range of 4.0–7.0, with a similar optimum pH of 5.5. A pH optimum of 5.6 has previously been reported (Swoboda and Massey 1965) for GOD from A. niger. GOD from A. niger NFCCP was found to be better than that from Penicillium pinophilum, which was unstable in the pH range of 2.0–4.0 and above 7.0 (Rando et al. 1997). GOD of P. pinophilum could also be rapidly deactivated at 40 °C by increasing the pH to 7.0 or decreasing it to 4.0. GOD from A. niger NFCCP had optimum activity at 40 °C. GOD is a dimeric enzyme (Rando et al. 1997), and © 2006 NRC Canada

Bhatti et al.

523 Table 2. Kinetics and thermodynamics of irreversible thermal denaturation of Aspergillus niger NFCCP glucose oxidase. Temperature (K)

kd (min–1)

t½ (min)

H* (kJ mol–1)

G* (kJ mol–1)

S* (J mol–1 K–1)

318 321 325 329 333

0.004 0.007 0.012 0.018 0.023

173 99 58 39 30

99.79 99.76 99.73 99.70 99.66

103.47 102.98 102.84 103.03 103.63

–11.57 –10.03 –9.57 –10.12 –11.92

Note: kd, (first-order rate constant of denaturation) as determined from Fig. 4. t1/2 (half-life) = 0.693/kd. H* (enthalpy of denaturation) = Ea – RT. Ea (102.43 kJ mol–1; activation energy of denaturation) as calculated from Fig. 4. G* (free energy of activation) = –RT In[kdh/(kbT)]. S* (entropy of inactivation) = (H* – G*)/T.

the enzyme concentration might affect enzyme stability, suggesting that the dissociation of subunits could be playing a key role in its inactivation as observed for other multimaric enzymes (Hidalgo et al. 2003; Wilson et al. 2004). The enzyme showed a low Ea at 40 °C. This Ea value makes this GOD superior to those from various other sources (Ye et al. 1988; Eremin et al. 2001). Referring to properties of biotechnological relevance, this GOD exhibited a high affinity for D-glucose (Km = 25 mmol L–1). This value is less than the reported value (Km = 33 mmol L–1) for GOD from A. niger (Swoboda and Massey 1965. This high substrate affinity and specificity, in addition to its stability at 40 °C, makes the A. niger NFCCP GOD a suitable biocatalyst for industrial applications, such as in food technology and in biosensors. Purified GOD showed appreciably stable kinetic and thermodynamic characteristics between 45 and 60 °C. Thermal denaturation occurs in two steps as shown below: N ø U ÷ D where N is the native enzyme, U is the unfolded enzyme, which could be reversibly refolded upon cooling, and D is the denatured enzyme formed after prolonged exposure to heat and, therefore, cannot be recovered on cooling. Thermostability is the ability of enzymes to resist thermal unfolding in the absence of substrates, while thermophilicity is the capability of enzymes to work at elevated temperatures in the presence of substrate (Georis et al. 2000; Iqbal et al. 2003; Sarath Babu et al. 2004). The thermal denaturation of multimeric enzymes is accompanied by the disruption of noncovalent linkages, including hydrophobic interactions, leading to subunit dissociation with a concomitant increase in the enthalpy of activation (Rodriguez-Nogales 2004; Srivastava et al. 2005). The opening up of the enzyme structure is accompanied by an increase in the disorder, randomness, or entropy of inactivation (Vieille and Zeikus 1996). On the other hand, the transition state of α-amylase from Bacillus licheniformis was found to be more ordered, as revealed by its negative ∆S* at a high temperature of 80 °C (Violet and Meunier 1989; Declerck et al. 2003). The negative entropy of inactivation (∆S*) observed for GOD suggested that there was negligible disorderness, like that of β-glucosidase from Aspergillus wentii (Kvesitadze et al. 1990). The values of ∆H* and ∆S* decreased with an increase in the temperature, indicating that the conformation of the enzyme was altered. Moreover, a high value for free energy of thermal denaturation (∆G*) at 60 °C indicated that

the GOD exhibited a resistance against thermal unfolding at higher temperatures. Eremin et al. (2001) reported a value of 123.1 J mol–1 K–1 and 25.2 kcal mol–1 for ∆S* and ∆G*, respectively, for A. niger GOD, indicating that the enzyme has more unfolding configuration and that it is unstable at 60 °C. GOD is a glycoprotein that contains tightly bound FAD, which is not cleaved from the protein part at even higher temperature treatments (Swoboda and Massey 1965). GOD isolated from A. niger NFCCP strain was thermally stable and could be used for analytical and other industrial applications.

References Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254. Balcao, V.M., Mateo, C., Fernandez-Lafuente, R., Malcata, F.X., and Guisan, J.M. 2001. Structural and functional stabilization of L-asparaginase via multisubunit immobilization onto highly activated supports. Biotechnol. Prog. 17: 537–542. Betancor, L., Lopez-Gallego, F., Hidalgo, A., Alonso-Morales, N., Dellamora-Ortiz, G., Guisan, J.M., and Fernandez-Lafuente, R. 2006. Preparation of a very stable immobilized biocatalyst of glucose oxidase from Aspergillus niger. J. Biotechnol. 121(2): 284–289. Cao, L., Van Rantwijk, F., and Sheldon, R. 2000. Cross-linked enzyme aggregates: a simple and effective method for the immobilization of penicillin acylase. Org. Lett. 2: 1361–1364. Cao, L., Van Langen, F., Van Rantwijk, F., and Sheldon, R. 2001. Cross-linked aggregates of penicillin acylase: robust catalysts for the synthesis of β-lactam antibiotics. J. Mol. Catal. B Enzym. 11: 665–670. Declerck, N., Machius, M., Joyet, P., Weigand, G., Hubere, R., and Gaillardin, C. 2003. Hyperthermostabilization of Bacillus licheniformis α-amylase and modulation of its stability over a 50 °C temperature range. Protein Eng. 16: 287–293. Eremin, A.N., Metelitsa, D.I., Shishko, Z.F., Mikhailova, R.V., Yasenko, M.I., and Lobanok, A.G. 2001. Thermal stability of glucose oxidase from Penicillium adametzii. Appl. Biochem. Microbiol. 37: 578–586. Eyring, H., and Stearn, A.E. 1939. The application of the theory of absolute reaction rates to protein. Chem. Rev. 24: 253–270. Fernandez-Lafuente, R., Hernandez-Justiz, O., Mateo, C., Terreni, M., Fernandez-Lorente, G., Moreno, M.A., Alonso, J., Garcia-Lopez, J.L., and Guisan, J.M. 2001. Biotransformations catalyzed by multimeric enzymes: stabilization of tetrameric ampicillin acylase permits the optimization of ampicillin synthesis under dissociation conditions. Biomacromolecules, 2: 95–104. © 2006 NRC Canada

524 Fugslang, C.C., Johanson, C., Christgau, S., and Alder-Nissen, J. 1995. Antimicrobial enzymes: applications and future trends in the food industry. Trends Food Sci. Technol. 6: 390–396. Georis, J., Esteves, F.L., Brasseur, J.L., Bougnet, V., Devreese, B., Giannotta, F., Granier, B., and Frere, J.M. 2000. An additional aromatic interaction improves the thermostability and thermophilicity of a mesophilic family 11 xylanase: structural basis and molecular study. Protein Sci. 9: 466–475. Gouda, M.D., Singh, S.A., Appu Rao, A.G., Thakur, M.S., and Karanth, N.G. 2003. Thermal inactivation of glucose oxidase: mechanism and stabilization using additives. J. Biol. Chem. 278: 24324–24333. Hatzinikolaou, D.G., and Macris, B.J. 1995. Factors regulating production of glucose oxidase by Aspergillus niger. Enzyme Microb. Technol. 17: 530–534. Hidalgo, A., Betancor, L., Lopez-Gallego, F., Moreno, R., Berenguer, J., Fernández-Lafuente, R., and Guisan, J.M. 2003. Design of an immobilized preparation of catalase from Thermus thermophilus to be used in a wide range of conditions. Structural stabilization of a multimeric enzyme. Enzyme Microb. Technol. 33: 278–285. Iqbal, Z., Rashid, M.H., Jabar, A., Malana, M.A., Khalid, A.M., and Rajoka, M.I. 2003. Kinetics of enhanced thermostability of an extracellular glucoamylase from Arachniotus sp. Biotechnol. Lett. 25: 1667–1670. Kapat, A., Jang, J.K., Park, Y.H., Hong, S.Y., and Choi, H.K. 1998. Effects of agitation and aeration on the production of extracellular glucose oxidase from a recombinant Saccharomyces cereviiae. Bioprocess Eng. 18: 247–351. Kvesitadze, G.I., Svanbidez, R.S., Tsuprun, V.L., Nizharadze, D.N., and Buachidze, T.S.H. 1990. Quaternary structure and properties of enzyme, β-glucosidase, isolated from a thermophilic culture of Aspergillus wentii. Bioorg. Khim. 16: 881–888. López-Gallego, F., Betancor, L., Mateo, C., Hidalgo, A., AlonsoMorales, N., Dellamora-Ortiz, G., Guisan, J.M., and FernándezLafuente, R. 2005. Enzyme stabilization by glutaraldehyde crosslinking of adsorbed proteins on aminated supports. J. Biotechnol. 119: 70–75. Montes, F.J., Battanar, E., Catalan, J., and Galan, M.A. 1995. Kinetics and heat inactivation mechanism of D-amino oxidase. Process Biochem. 30: 217–224. Munch, O., and Tritsch, D. 1990. Irreversible thermoinactivation of glycoamylase from Aspergillus niger and thermostabilization by chemical modification of carboxyl groups. Biochim. Biophys. Acta, 1041: 111–116. Petruccioli, M., Federici, F., Bucke, C., and Keshavarz, T. 1999. Enhancement of glucose oxidase production by Penicillium variabile P16. Enzyme Microb. Technol. 24: 397–401. Rando, D., Kohring, G.W., and Giffhorn, F. 1997. Production, purification and characterization of glucose oxidase from a newly isolated strain of Penicillium pinophilum. Appl. Microbiol. Biotechnol. 48: 34–40. Rashid, M.H., and Siddiqui, K.S. 1998. Thermodynamic and kinetic study of stability of the native and chemically modified β-glucosidase from Aspergillus niger. Process Biochem. 33: 109–115.

Can. J. Microbiol. Vol. 52, 2006 Rodriguez-Nogales, J.M. 2004. Kinetic behaviour and stability of glucose oxidase entrapped in liposomes. J. Chem. Technol. Biotechnol. 79: 72–78. Sarath Babu, V.R., Kumar, M.A., Karanth, N.G., and Thakur, M.S. 2004. Stabilization of immobilized glucose oxidase against thermal inactivation by silanization for biosensor applications. Biosens. Bioelectron. 19: 1337–1341. Schmid, R.D., and Karube, I. 1988. Biosensors and bioelectronics. In Biotechnology. Edited by H.J. Rehm and G. Reeds. Vol. 6b. Verlage Chemie GmbH, Weinheim, Germany. pp. 317–365. Siddiqui, K.S., Azhar, M.J., Rashid, M.H., and Rajoka, M.I. 1997. Stability and identification of active site residues of carboxymethylcellulase from Aspergillus niger and Cellulomonas biazotea. Folia Microbiol. 42: 312–318. Siddiqui, K.S., Shamsi, A.M., Anwar, M.A., Rashid, M.H., and Rajoka, M.I. 1999. Partial and complete alteration of surface charges of carboxymethylcellulase by chemical modification: thermostabilization in water-miscible organic solvent. Enzyme Microb. Technol. 24: 599–608. Srivastava, R., Brown, J.Q., Zhu, H., and McShane, M.J. 2005. Stabilization of glucose oxidase in alginate microspheres with photoreactive diazoresin nanofilm coatings. Biotechnol. Bioeng. 91: 24–131. Sukhacheva, M.V., Davydova, M.E., and Netrusov, A.I. 2004. Production of Penicillium funiculosum 433 glucose oxidase and its properties. Appl. Biochem. Microbiol. 40: 32–36. Swoboda, B.E.P., and Massey, V. 1965. Purification and properties of the glucose oxidase from Aspergillus niger. J. Biol. Chem. 240: 2209–2215. Vasileva, N., and Godjevargova, T. 2004. Study on the behaviour of glucose oxidase immobilized on microfiltration polyamide membrane. J. Membr. Sci. 239: 157–161. Vasileva, N., and Godjevargova, T. 2005. Study of the effect of some organic solvents on the activity and stability of glucose oxidase. Mater. Sci. Eng., C, 25: 17–21. Vieille, C., and Zeikus, J.G. 1996. Thermozymes: identifying molecular determinants of protein structural and functional stability. Trends Biotechnol. 14: 183–190. Violet, M., and Meunier, J.C. 1989. Kinetic study of irreversible thermal denaturation of Bacillus licheniformis α-amylase. Biochem. J. 263: 665–670. Vojinovic, V., Calado, C.R., Silva, A.I., Mateus, M., Cabral, J.M.S., and Fonseca, L.P. 2005. Micro-analytical GO/HRP bioreactor for glucose determination and bioprocess monitoring. Biosens. Bioelectron. 20: 1955–1961. Weibel, M., and Bright, H. 1971. The glucose oxidase mechanism: interpretation of the pH dependence. J. Biol. Chem. 246: 273– 278. Wilson, L., Betancor, L., Fernandez-Lorente, G., Fuentes, M., Hidalgo, A., Guisan, J.M., Pessela, B.C.C., and FernandezLafuente, R. 2004. Cross-linked aggregates of multimeric enzymes: a simple and efficient methodology to stabilize their quaternary structure. Biomacromolecules, 5: 814–817. Ye, W., Combes, D., and Monsan, P. 1988. Influence of additives on the thermostability of glucose oxidase. Enzyme Microb. Technol. 10: 498–502.

© 2006 NRC Canada