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Cell Bound and Extracellular Glucose Oxidases from Aspergillus niger BTL: Evidence for a Secondary Glycosylation Mechanism

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Abstract

Two glucose oxidase (GOX) isoforms where purified to electrophoretic homogeneity from the mycelium extract (GOXI) and the extracellular medium (GOXII) of Aspergillus niger BTL cultures. Both enzymes were found to be homodimers with nonreduced molecular masses of 148 and 159 kDa and pI values of 3.7 and 3.6 for GOXI and GOXII, respectively. The substrate specificity and the kinetic characteristics of the two GOX forms, as expressed through their apparent K m values on glucose, as well as pH and T activity optima, were almost identical. The only structural difference between the two enzymes was in their degrees of glycosylation, which were determined equal to 14.1 and 20.8% (w/w) of their molecular masses for GOXI and GOXII, respectively. The above difference in the carbohydrate content between the two enzymes seems to influence their pH and thermal stabilities. GOXII proved to be more stable than GOXI at pH values 2.5, 3.0, 8.0, and 9.0. Half-lives of GOXI at pH 3.0 and 8.0 were 8.9 and 17.5 h, respectively, whereas the corresponding values for GOXII were 13.5 and 28.1 h. As far as the thermal stability is concerned, GOXII was also more thermostable than GOXI as judged by the deactivation constants determined at various temperatures. More specifically, the half-lives of GOXI and GOXII, at 45°C, were 12 and 49 h, respectively. These results suggest A. niger BTL probably possesses a secondary glycosylation mechanism that increases the stability of the excreted GOX.

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References

  1. Malherbe, D., du Toit, M., Cordero Otero, R., van Rensburg P., & Pretorius I. (2003). Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential applications in wine production. Applied Microbiology and Biotechnology, 61, 502–511.

    CAS  Google Scholar 

  2. Kapat, A., Jung, J. K., & Park, Y. H. (1998). Improvement of extracellular recombinant glucose oxidase production in fed-batch culture of Saccharomyces cerevisiae: Effect of different feeding strategies. Biotechnology Letters, 20, 319–323.

    Article  CAS  Google Scholar 

  3. Castillo, J., Gaspar, S., Sakharov, I., & Csoregi, E. (2003). Bienzyme biosensors for glucose, ethanol and putrescine built on oxidase and sweet potato peroxidase. Biosensors and Bioelectronics, 18, 705–714.

    Article  CAS  Google Scholar 

  4. Rasiah, I. A., Sutton, K. H., Low, F. L., Lin, H. M., & Gerrard, J. A. (2005). Crosslinking of wheat dough proteins by glucose oxidase and the resulting effects on bread and croissants. Food Chemistry, 89, 325–332.

    Article  CAS  Google Scholar 

  5. Szynol, A., de Soet, J. J., Sieben-van Tuyl, E., Bos, J. W., & Frenken, L. G. (2004). Bactericidal effects of a fusion protein of Llama heavy-chain antibodies coupled to glucose oxidase on oral bacteria. Antimicrobial Agents and Chemotherapy, 48, 3390–3395.

    Article  CAS  Google Scholar 

  6. Kelley, R. L., & Reddy, C. A. (1986). Purification and characterization of glucose oxidase from ligninolytic cultures of Phanerochaete chrysosporium. Journal of Bacteriology, 166, 269–274.

    CAS  Google Scholar 

  7. Shin, K. S., Youn, H. D., Han, Y. H., Kang, S. O., & Hah, Y. C. (1993). Purification and characterisation of D-glucose oxidase from white-rot fungus Pleurotus ostreatus. European Journal of Biochemistry, 215, 747–752.

    Article  CAS  Google Scholar 

  8. Liu, S., Oeljeklaus, S., Gerhardt, B., & Tudzynski, B. (1998). Purification and characterization of glucose oxidase of Botrytis cinerea. Physiological and Molecular Plant Pathology, 53, 123–132.

    Article  CAS  Google Scholar 

  9. Kalisz, H. M., Hendle, J., & Schmid, R. D. (1997). Structural and biochemical properties of glycosylated and deglycosylated glucose oxidase from Penicillium amagasakiense. Applied Microbiology and Biotechnology, 47, 502–507.

    Article  CAS  Google Scholar 

  10. Eriksson, K. O., Kourteva, I., Yao, K. Q., Liao, J. L., Kilar, F., Hjerten, S., et al. (1987). Application of high-performance chromatographic and electrophoretic methods to the purification and characterization of glucose oxidase and catalase from Penicillium chrysogenum. Journal of Chromatography, 397, 239–249.

    Article  CAS  Google Scholar 

  11. Rando, D., Kohring, G. W., & Giffhorn, F. (1997). Production, purification and characterization of glucose oxidase from a newly isolated strain of Penicillium pinophilum. Applied Microbiology and Biotechnology, 48, 34–40.

    Article  CAS  Google Scholar 

  12. Semashko, T. V., Mikhailova, R. V., & Lobanok, A. G. (2004). Growth characteristics and glucose oxidase production in mutant Penicillium funiculosum strains. Microbiology, 73, 286–291.

    Article  CAS  Google Scholar 

  13. Pazur, J. (1966). Glucose oxidase from Aspergillus niger. Methods in Enzymology, 9, 82–87.

    Article  CAS  Google Scholar 

  14. Zetelaki, K., & Vas, K. (1968). The role of aeration and agitation in the production of glucose oxidase in submerged culture. Biotechnology and Bioengineering, 10, 45–59.

    Article  CAS  Google Scholar 

  15. Zetelaki, K. (1970). The role of aeration and agitation in the production of glucose oxidase in submerged culture II. Biotechnology and Bioengineering, 12, 379–397.

    Article  CAS  Google Scholar 

  16. van Dijken, J. P. & Veenhuis, M. (1980). Cytochemical localization of glucose oxidase in peroxisomes of Aspergillus niger. European Journal of Applied Microbiology and Biotechnology, 9, 275–283.

    Article  Google Scholar 

  17. Rogalski, J., Fiedurek, J., Szczordrak, J., Kapusta, K., & Leonowicz, A. (1988). Optimization of glucose oxidase synthesis in submerged cultures of Aspergillus niger G-13 mutant. Enzyme and Microbial Technology, 10, 508–511.

    Article  CAS  Google Scholar 

  18. Fiedurek, J., Rogalski, J., Ilczuk, Z., & Leonowicz, A. (1986). Screening and mutagenesis of moulds for the improvement of glucose oxidase production. Enzyme and Microbial Technology, 8, 734–736.

    Article  CAS  Google Scholar 

  19. Traeger, M., Qazi, G. N., Onken, U., & Chopra, C. L. (1991). Contribution of endocellular glucose oxidase and exocellular glucose oxidase to glutonic acid production at increased dissolved oxygen concentrations. Journal of Chemical Technology and Biotechnology, 50, 1–11.

    CAS  Google Scholar 

  20. Hatzinikolaou, D. G., & Macris, B. J. (1995). Factors regulating production of glucose oxidase by Aspergillus niger. Enzyme and Microbial Technology, 17, 530–534.

    Article  CAS  Google Scholar 

  21. Mischak, H., Kubicek, C. P., & Rohr, M. (1985). Formation and location of glucose oxidase in citric acid producing mycelia of Aspergillus niger. Applied Microbiology and Biotechnology, 21, 27–31.

    Article  CAS  Google Scholar 

  22. Fiedurek, J., & Gromada, A. (2000). Production of catalase and glucose oxidase by Aspergillus niger using unconventional oxygenation of culture. Journal of Applied Microbiology, 89, 85–89.

    Article  CAS  Google Scholar 

  23. Pluschkell, S., Hellmuth, K., & Rinas, U. (1996). Kinetics of glucose oxidase excretion by recombinant Aspergillus niger. Biotechnology and Bioengineering, 51, 215–220.

    Article  Google Scholar 

  24. Rothberg, A., Weegar, J., von Schalien, R., Fagervik, K., Rydstrom, M., & Lind, K. (1999). Optimization of an Aspergillus niger glucose oxidase production process. Bioprocess Engineering, 21, 307–312.

    CAS  Google Scholar 

  25. Witteveen, C. F. B., Veenhuis, M., & Visser, J. (1992). Localization of glucose oxidase and catalase activities in Aspergillus niger. Applied and Environmental Microbiology, 58, 1190–1194.

    CAS  Google Scholar 

  26. Pazur, J., Kleppe, K., & Cepure, A. (1965). A glycoprotein structure for glucose oxidase from Aspergillus niger. Archives of Biochemistry and Biophysics, 111, 351–357.

    Article  CAS  Google Scholar 

  27. Tsuge, H., Natsuaki, O., & Ohashi, K. (1975). Purification, properties, and molecular features of glucose oxidase from Aspergillus niger. Journal of Biochemistry, 78, 835–843.

    CAS  Google Scholar 

  28. Nakamura, S., Hayashi, S., & Koga, K. (1976). Effect of periodate oxidation on the structure and properties of glucose oxidase. Biochimica et Biophysica Acta, 445, 294–308.

    CAS  Google Scholar 

  29. Kalisz, H., Hecht, H., Schomburg, D., & Schmid, R. (1991). Effects of carbohydrate depletion on the structure, stability and activity of glucose oxidase from Aspergillus niger. Biochimica et Biophysica Acta, 1080, 138–142.

    CAS  Google Scholar 

  30. Takegawa, K., Fujiwara, K., Iwahara, S., Yamamoto, K., & Tochikura, T. (1989). Effect of deglycosylation of N-linked sugar chains on glucose oxidase from Aspergillus niger. Biochemistry and Cell Biology, 67, 460–464.

    Article  CAS  Google Scholar 

  31. Ahmad, A., Akhtar, M. S., & Bhakuni, V. (2001). Monovalent cation-induced conformational change in glucose oxidase leading to stabilization of the enzyme. Biochemistry, 40, 1945–1955.

    Article  CAS  Google Scholar 

  32. Hecht, H. J., Kalisz, H. M., Hendle, J., Schmid, R. D., & Schomburg, D. (1993). Crystal structure of glucose oxidase from Aspergillus niger refined at 2·3 Å resolution. Journal of Molecular Biology, 229, 153–172.

    Article  CAS  Google Scholar 

  33. Akhtar, M. S., & Bhakuni, V. (2003). Alkaline treatment has contrasting effects on the structure of deglycosylated and glycosylated forms of glucose oxidase. Archives of Biochemistry and Biophysics, 413, 221–228.

    Article  CAS  Google Scholar 

  34. Hayashi, S., & Nakamura, S. (1981). Multiple forms of glucose oxidase with different carbohydrate compositions. Biochimica et Biophysica Acta, 657, 40–51.

    CAS  Google Scholar 

  35. Hatzinikolaou, D. G., Hansen, O. C., Macris, B. J., Tingey, A., Kekos, D., Goodenough, P., et al. (1996). A new glucose oxidase from Aspergillus niger: Characterization and regulation studies of enzyme and gene. Applied Microbiology and Biotechnology, 46, 371–381.

    CAS  Google Scholar 

  36. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193, 265–275.

    CAS  Google Scholar 

  37. Bradford, M. (1976). A rapid and sensitive method for the quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.

    Article  CAS  Google Scholar 

  38. Dubois, M., Gilles, K., Hamilton, J., Rebers, P., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Biochemistry, 28, 350–356.

    CAS  Google Scholar 

  39. Sukhacheva, M. V., Davydova, M. E., & Netrusov, A. I. (2004). Production of Penicillium funiculosum 433 glucose oxidase and its properties. Applied Biochemistry and Microbiology, 40, 32–36.

    CAS  Google Scholar 

  40. Swoboda, B. E. P. & Massey, V. (1965). Purification and properties of the glucose oxidase from Aspergillus niger. Journal of Biological Chemistry, 240, 2209–2215.

    CAS  Google Scholar 

  41. Kim, K. K., Fravel, D. R., & Papavizas, G. C. (1990). Production, purification and properties of glucose oxidase from the bioconrtol fungus Talaromyces flavus. Canadian Journal of Microbiology, 36, 199–205.

    Article  CAS  Google Scholar 

  42. Underkofler, L. (1958). Properties and applications of the fungal enzyme glucose oxidase. International symposium in enzyme chemistry. Tokyo and Kyoto, Japan. New York: Academic Press.

    Google Scholar 

  43. Garzillo, A. M. V., Dipaolo, S., Fenice, M., Petruccioli, M., Buonocore, V., & Federici, F. (1995). Production, purification and characterization of glucose oxidase from Penicillium variabile P16. Biotechnology and Applied Biochemistry, 22, 169–178.

    CAS  Google Scholar 

  44. O’Malley, J. J., & Ulmer, R. W. (1973). Thermal stability of glucose oxidase and its admixtures with synthetic polymers. Biotechnology and Bioengineering, 15, 917–925.

    Article  CAS  Google Scholar 

  45. Witt, S., Singh, M., & Kalisz, H. M. (1998). Structural and kinetic properties of nonglycosylated recombinant Penicillium amagasakiense glucose oxidase expressed in Escherichia coli. Applied and Environmental Microbiology, 64, 1405–1411.

    CAS  Google Scholar 

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Acknowledgment

This work has been partially supported by the Research Committee of the National and Kapodistrian University of Athens, “Kapodistrias” Research Program

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Authors and Affiliations

  1. Laboratory of Microbiology, Department of Botany, School of Biology, National and Kapodistrian University of Athens, Panepistimioupoli Zografou, 15781, Athens, Greece

    Dimitris G. Hatzinikolaou

  2. Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Polytechnioupoli Zografou, 15780, Athens, Greece

    Diomi Mamma, Paul Christakopoulos & Dimitris Kekos

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  1. Dimitris G. Hatzinikolaou
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Correspondence to Dimitris Kekos.

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Hatzinikolaou, D.G., Mamma, D., Christakopoulos, P. et al. Cell Bound and Extracellular Glucose Oxidases from Aspergillus niger BTL: Evidence for a Secondary Glycosylation Mechanism. Appl Biochem Biotechnol 142, 29–43 (2007). https://doi.org/10.1007/s12010-007-0006-7

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