Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Purification and Characterization of an Extracellular $\beta$-Glucosidase from Monascus purpureus

  • Daroit, Daniel J. (Laboratorio de Bioquimica e Microbiologia Aplicada, Departamento de Cienda de Alimentos (ICTA), Universidade Federal do Rio Grande do Sui) ;
  • Simonetti, Aline (Laboratorio de Bioquimica e Microbiologia Aplicada, Departamento de Cienda de Alimentos (ICTA), Universidade Federal do Rio Grande do Sui) ;
  • Hertz, Plinho F. (Laboratorio de Bioquimica e Microbiologia Aplicada, Departamento de Cienda de Alimentos (ICTA), Universidade Federal do Rio Grande do Sui) ;
  • Brandelli, Adriano (Laboratorio de Bioquimica e Microbiologia Aplicada, Departamento de Cienda de Alimentos (ICTA), Universidade Federal do Rio Grande do Sui)
  • Published : 2008.05.31
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Abstract

An extracellular $\beta$-glucosidase produced by Monascus purpureus NRRL1992 in submerged cultivation was purified by acetone precipitation, gel filtration, and hydrophobic interaction chromatography, resulting in a purification factor of 92-fold. A $2^2$ central-composite design (CCD) was performed to find the best temperature and pH conditions for enzyme activity. Maximum activity was observed in a wide range of temperature and pH values, with optimal conditions set at $50^{\circ}C$ and pH 5.5. The $\beta$-glucosidase showed moderate thermostability, was inhibited by $HgCl_2$, $K_2Cr_O_4$, and $K_2Cr_2O_7$, whereas other reagents including $\beta$-mercaptoethanol, SDS, and EDTA showed no effect. Activity was slightly stimulated by low concentrations of ethanol and methanol. Hydrolysis of p-nitrophenyl-$\beta$-D-glucopyranoside (pNPG), cellobiose, salicin, n-octyl-$\beta$-D-glucopyranoside, and maltose indicates that the $\beta$-glucosidase has broad substrate specificity. Apparently, glucosyl residues were removed from the nonreducing end of p-nitrophenyl-$\beta$-D-cellobiose. $\beta$-Glucosidase affinity and hydrolytic efficiency were higher for pNPG, followed by maltose and cellobiose. Glucose and cellobiose competitively inhibited pNPG hydrolysis.

Keywords

References

  1. Badhan, A. K., B. S. Chadha, J. Kaur, H. S. Saini, and M. K. Bhat. 2007. Production of multiple xylanolytic and cellulolytic enzymes by thermophilic fungus Myceliophthora sp. IMI 387099. Bioresour. Technol. 98: 504-510 https://doi.org/10.1016/j.biortech.2006.02.009
  2. Belancic, A., Z. Gunata, M. J. Vallier, and E. Agosin. 2003. $\beta$- Glucosidase from the native yeast Debaryomyces vanrijiae: Purification, characterization, and its effect on monoterpene content of a Muscat grape juice. J. Agric. Food Chem. 51: 1453-1459 https://doi.org/10.1021/jf025777l
  3. Bhatia, Y., S. Mishra, and V. S. Bisaria. 2002. Microbial $\beta$- glucosidases: Cloning, properties and applications. Crit. Rev. Biotechnol. 22: 375-407 https://doi.org/10.1080/07388550290789568
  4. Box, G. E. P., W. G. Hunter, and J. S. Hunter. 1978. Statistics for Experiments. Wiley, New York
  5. Cervantes, C., J. Campos-Garcia, S. Devars, F. Gutierrez- Corona, H. Loza-Tavera, J. C. Torres-Guzman, and R. Moreno- Sanchez. 2001. Interactions of chromium with microorganisms and plants. FEMS Microbiol. Rev. 25: 335-347 https://doi.org/10.1111/j.1574-6976.2001.tb00581.x
  6. Chen, H., M. Hayn, and H. Esterbauer. 1992. Purification and characterization of two extracellular $\beta$-glucosidases from Trichoderma reesei. Biochim. Biophys. Acta 1121: 54-60 https://doi.org/10.1016/0167-4838(92)90336-C
  7. Christakopoulos, P., P. H. Goodenough, D. Kekos, B. J. Macris, M. Claeyssens, and M. K. Bhat. 1994. Purification and characterisation of an extracellular $\beta$-glucosidase with transglycosylation and exo-glucosidase activities from Fusarium oxysporum. Eur. J. Biochem. 224: 379-385 https://doi.org/10.1111/j.1432-1033.1994.00379.x
  8. Daroit, D. J., S. T. Silveira, P. F. Hertz, and A. Brandelli. 2007. Production of extracellular $\beta$-glucosidase by Monascus purpureus on different growth substrates. Process Biochem. 42: 904-908 https://doi.org/10.1016/j.procbio.2007.01.012
  9. Dufosse, L., P. Galaup, A. Yaron, S. M. Arad, P. Blanc, K. M. C. Murthy, and G. A. Ravishankar. 2005. Microorganisms and microalgae as sources of pigments for food use: A scientific oddity or an industrial reality? Trends Food Sci. Technol. 16: 389-406 https://doi.org/10.1016/j.tifs.2005.02.006
  10. Eveleigh, D. E., J. D. Bok, H. El-Dorry, S. El-Gogary, K. Elliston, A. Goyal, C. Waldron, R. Wright, and Y. M. Wu. 1995. Cellulase lessons revealed through the microbe's perspective. Appl. Biochem. Biotechnol. 51/52: 169-177 https://doi.org/10.1007/BF02933422
  11. Gallifuoco, A., F. Alfani, M. Cantarella, G. Spagna, and P. G. Pifferi. 1999. Immobilized $\beta$-glucosidase for the winemaking industry: Study of biocatalyst operational stability in laboratoryscale continuous reactors. Process Biochem. 35: 179-185 https://doi.org/10.1016/S0032-9592(99)00049-7
  12. Gueguen, Y., P. Chemardin, A. Arnaud, and P. Galzy. 1995. Purification and characterization of an intracellular $\beta$-glucosidase from Botrytis cinerea. Enzyme Microb. Technol. 17: 900-906 https://doi.org/10.1016/0141-0229(94)00143-F
  13. Hang, Y. D. and E. E. Woodams. 1994. Apple pomace: A potential substrate for production of $\beta$-glucosidase by Aspergillus foetidus. Food Sci. Technol. 27: 587-589
  14. Jager, S., A. Brumbauer, E. Feher, K. Reczey, and L. Kiss. 2001. Production and characterization of $\beta$-glucosidases from diferent Aspergillus strains. World J. Microbiol. Biotechnol. 17: 455-461 https://doi.org/10.1023/A:1011948405581
  15. Jeoh, T., J. O. Baker, M. K. Ali, M. E. Himmel, and W. S. Adney. 2005. $\beta$-D-Glucosidase reaction kinetics from isothermal titration microcalorimetry. Anal. Biochem. 347: 244-253 https://doi.org/10.1016/j.ab.2005.09.031
  16. Kalil, S. J., F. Maugeri, and M. I. Rodrigues. 2000. Response surface analysis and simulation as a tool for bioprocess design and optimization. Process Biochem. 35: 539-550 https://doi.org/10.1016/S0032-9592(99)00101-6
  17. Kawai, R., K. Igarashi, M. Kitaoka, T. Ishii, and M. Samejima. 2004. Kinetics of substrate transglycosylation by glycoside hydrolase family 3 glucan $(1\rightarrow3)$-$\beta$-glucosidase from the whiterot fungus Phanerochaete chrysosporium. Carbohydr. Res. 339: 2851-2857 https://doi.org/10.1016/j.carres.2004.09.019
  18. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 https://doi.org/10.1038/227680a0
  19. Liang, T. W., J. J. Lin, Y. H. Yen, C. L. Wang, and S. L. Wang. 2006. Purification and characterization of a protease extracellularly produced by Monascus purpureus CCRC31499 in a shrimp and crab shell powder medium. Enzyme Microb. Technol. 38: 74-80 https://doi.org/10.1016/j.enzmictec.2005.04.023
  20. Lo, A. C., J. R. Barbier, and G. E. Willick. 1990. Kinetics and specificities of two closely related $\beta$-glucosidases secreted by Schizophyllum commune. Eur. J. Biochem. 192: 175-181 https://doi.org/10.1111/j.1432-1033.1990.tb19211.x
  21. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275
  22. Lucas, R., A. Robles, G. A. De Cienfuegos, and A. Galvez. 2000. $\beta$-Glucosidase from Chalara paradoxa CH32: Purification and properties. J. Agric. Food Chem. 48: 3698-3703 https://doi.org/10.1021/jf0002591
  23. Lusterio, D. D., F. G. Suizo, N. M. Labunos, M. N. Valledor, S. Ueda, S. Kawai, K. Koike, S. Shikata, T. Yoshimatsu, and S. Ito. 1992. Alkali resistant, alkaline endo-1,4-$\beta$-glucanase produced by Bacillus sp. PKM-5430. Biosci. Biotechnol. Biochem. 56: 1671-1672 https://doi.org/10.1271/bbb.56.1671
  24. Mateo, J. J. and R. Di Stefano. 1997. Description of the $\beta$- glucosidase activity of wine yeasts. Food Microbiol. 14: 583-591 https://doi.org/10.1006/fmic.1997.0122
  25. Palmieri, R. and G. Spagna. 2007. $\beta$-Glucosidase in cellular and acellular form for winemaking application. Enzyme Microb. Technol. 40: 382-389 https://doi.org/10.1016/j.enzmictec.2006.07.007
  26. Park, J. K., L. X. Wang, H. V. Patel, and S. Roseman. 2002. Molecular cloning and characterization of a unique $\beta$-glucosidase from Vibrio cholerae. J. Biol. Chem. 277: 29555-29560 https://doi.org/10.1074/jbc.M202978200
  27. Perez-Pons, J. A., X. Rebordosa, and E. Querol. 1995. Properties of a novel glucose-enhanced $\beta$-glucosidase purified from Streptomyces sp. (ATCC 11238). Biochim. Biophys. Acta 1251: 145-153 https://doi.org/10.1016/0167-4838(95)00074-5
  28. Queiroz, J. A., C. T. Tomaz, and J. M. S. Cabral. 2001. Hydrophobic interaction chromatography of proteins. J. Biotechnol. 87: 143-159 https://doi.org/10.1016/S0168-1656(01)00237-1
  29. Riccio, P., R. Rossano, M. Vinella, P. Domizio, F. Zito, F. Sansevrino, A. D'Elia, and I. Rossi. 1999. Extraction and immobilization in one step of two $\beta$-glucosidases released from a yeast strain of Debaryomyces hansenii. Enzyme Microb. Technol. 24: 123-129 https://doi.org/10.1016/S0141-0229(98)00066-0
  30. Riou, C., J. M. Salmon, M. J. Vallier, Z. Gunata, and P. Barre. 1998. Purification, characterization, and substrate specificity of a novel highly glucose-tolerant $\beta$-glucosidase from Aspergillus oryzae. Appl. Environ. Microbiol. 64: 3607-3614
  31. Sanyal, A., R. K. Kundu, S. Dube, and D. K. Dube. 1988. Extracellular cellulolytic enzyme system of Aspergillus japonicus: 2. Purification and characterization of an inducible extracellular $\beta$-glucosidase. Enzyme Microb. Technol. 10: 91-99 https://doi.org/10.1016/0141-0229(88)90004-X
  32. Sarry, J. E. and Z. Gunata. 2004. Plant and microbial glycoside hydrolases: Volatile release from glycosidic aroma precursors. Food Chem. 87: 509-521 https://doi.org/10.1016/j.foodchem.2004.01.003
  33. Scopes, R. K. 1994. Protein Purification: Principles and Practice, 3rd Ed. Springer-Verlag, New York
  34. Shrikhande, A. J. 2000. Wine by-products with health benefits. Food Res. Int. 33: 469-474 https://doi.org/10.1016/S0963-9969(00)00071-5
  35. Singh, A. and K. Hayashi. 1998. Microbial cellulases: Protein architecture, molecular properties, and biosynthesis. Adv. Appl. Microbiol. 41: 1-44
  36. Sun, Y. and J. Chen. 2002. Hydrolysis of lignocellulosic materials for ethanol production: A review. Bioresour. Technol. 83: 1-11 https://doi.org/10.1016/S0960-8524(01)00212-7
  37. Wallecha, A. and S. Mishra. 2003. Purification and characterization of two $\beta$-glucosidases from a thermo-tolerant yeast Pichia etchellsii. Biochim. Biophys. Acta 1649: 74-84 https://doi.org/10.1016/S1570-9639(03)00163-8
  38. Wang, S. L., Y. H. Yen, W. J. Tsiao, W. T. Chang, and C. L. Wang. 2002. Production of antimicrobial compounds by Monascus purpureus CCRC31499 using shrimp and crab shell powder as a carbon source. Enzyme Microb. Technol. 31: 337-344 https://doi.org/10.1016/S0141-0229(02)00135-7
  39. Woodward, J. and A. Wiseman. 1982. Fungal and other $\beta$-Dglucosidases - their properties and applications. Enzyme Microb. Technol. 4: 73-79 https://doi.org/10.1016/0141-0229(82)90084-9
  40. Yan, T. R., Y. H. Lin, and C. L. Lin. 1998. Purification and characterization of an extracellular $\beta$-glucosidase II with high hydrolysis and transglycosylation activities from Aspergillus niger. J. Agric. Food Chem. 46: 431-437 https://doi.org/10.1021/jf9702499
  41. Yun, S. I., C. S. Jeong, D. K. Chung, and H. S. Choi. 2001. Purification and some properties of a $\beta$-glucosidase from Trichoderma harzianum type C-4. Biosci. Biotechnol. Biochem. 65: 2028-2032 https://doi.org/10.1271/bbb.65.2028
  42. Zanoelo, F. F., M. L. T. M. Polizeli, H. F. Terenzi, and J. A. Jorge. 2004. $\beta$-Glucosidase activity from the thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose. FEMS Microbiol. Lett. 240: 137-143 https://doi.org/10.1016/j.femsle.2004.09.021