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Purification and biochemical characterization of a transglucosilating β-glucosidase of Stachybotrys strain

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Abstract

The filamentous fungus Stachybotrys sp has been shown to possess a rich β-glucosidase system composed of five β-glucosidases. One of them was already purified to homogeneity and characterized. In this work, a second β-glucosidase was purified and characterized. The filamentous fungal A19 strain was fed-batch cultivated on cellulose, and its extracellular cellulases (mainly β-glucosidases) were analyzed. The purified enzyme is a monomeric protein of 78 kDa molecular weight and exhibits optimal activity at pH 6.0 and at 50°C. The kinetic parameters, K m and V max, on para-nitro-phenyl-β-d-glucopyranosid (p-NPG) as a substrate were, respectively, 1.846 ± 0.11 mM and 211 ± 0.08 μmol min−1 ml−1. One interesting feature of this enzyme is its high stability in a wide range of pH from 4 to 10. Besides its aryl β-glucosidase activity towards salicin, methylumbellypheryl-β-d-glucoside (MU-Glc), and p-NPG, it showed a true β-glucosidase activity because it splits cellobiose into two glucose monomers. This enzyme has the capacity to synthesize short oligosaccharides from cellobiose as the substrate concentration reaches 30% with a recovery of 40%. We give evidences for the involvement of a transglucosylation to synthesize cellotetraose by a sequential addition of glucose to cellotriose.

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References

  1. Ajisaka K, Nishida H, Fujimoto H (1987) The synthesis of oligosaccharides by the reversed hydrolysis reaction of . β. -glucosidase at high substrate concentration and at high temperature. Biotechnol Lett 9:243–248

  2. Amouri B, Gargouri A (2006) Characterization of a novel . β. -glucosidase from a Stachybotrys strain. Biochem Eng J 32:191–197

  3. Bhat MK, Bhat S (1997) Cellulose degrading enzymes and their potential industrial applications. Biotechnol Adv 15:583–620

  4. Bradford MM (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

  5. Bronnenmeyer K, Staudenbauer WL (1988) Purification and properties of an extracellular b-glucosidase from the cellulolytic thermophile Clostridium stercorarium. Appl Microbiol Biotechnol 28:380–386

  6. Cai YJ, Buswell JA, Chang ST (1998) β. -Glucosidase components of the cellulolytic system of the edible straw mushroom, Volvariella volvacea. Enzyme Microb Technol 22:122–129

  7. Cao W, Crawford DL (1993) Purification and some properties of . β. -glucosidase from the ectomycorrhizae Pisolithus tinctorius strain SMF. Can J Microbiol 39:125–129

  8. Christakopoulos P, Goodenoogh PW, Kebos D, Macris BG, Clayssens M, Bhat MK (1994) Purification and characterization of an extracellular β-glucosidase with transglycoaylation and exo-glucosidase activities from Fusarium oxysporum. Eur J Biochem 224:379–385

  9. Deshpande V, Eriksson KE, Pettersson B (1978) Production, purification and partial characterization of 1,4-b-glucosidase enzymes from Sporotrichum pulverulentum. Eur J Biochem 90:191–119

  10. Gong CS, Ladisch MR, Tsao GT (1977) Cellobiase from Trichoderma viride: purification, properties, kinetics, and mechanism. Biotechnol Bioeng 19:959–981

  11. Hansson T, Ablercreutz P (2002) Enzymatic synthesis of hexyl glycosides from lactose at low water activity and high temperature using hyperthermostables . β. -glucosidases. Biocatal Biotrans 220:167–178

  12. Hidalgo M, Steiner J, Eyzaguirre J (1992) β. -Glucosidase from Penicillium purpurogenum: purification and properties. Biotechnol Appl Biochem 15:185–191

  13. Kengen SWM, Luesink EJ, Stams AJM, Zehnder AJB (1993) Purification and characterization of an extremely thermostable b-glucosidase from the hyperthermophilic Pyrococcus furiosus. Eur J Biochem 213:305–312

  14. Laemmli UK, Favre M (1973) Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol 80:575–592

  15. Mandels M, Weber J (1969) The production of the cellulases. Adv Chem Ser 95:391

  16. Mandels M, Parrish FW, Reese T (1962) Sophorose as in inducer of cellulase in Trichoderma reesei. J Bacteriol. 83:400–408

  17. Matsumura S, Yohikawa S, Kawada K, Uchibori T (1991) Surface activities, biodegradability and antimicrobial properties of n-alkylglucosides, mannosides and galactosides. J Am Oil Chem Soc 67:966–1001

  18. Saha BC, Bothast RJ (1996) Production, purification and characterization of highly glucose tolerant novel . β. -glucosidase from Candida peltata. Appl Environ Microbiol 62:3165–3170

  19. Smaali MI, Gargouri M, Limmam F, Fattouch S, Maugard T, Legoy MD, Marzouki N (2003) Production, purification and biochemical characterization of two . β. -Glucosidases from Sclerotinia sclerotiorum. Appl Biochem Biotechnol 111:29–39

  20. Smaali MI, Gargouri M, Limmam F, Maugard T, Legoy MD, Marzouki N (2004) A . β. -glucosidase from Sclerotinia sclerotiorum: biochemical characterization and use in oligosaccharides synthesis. Appl Biochem Biotechnol 112:63–78

  21. Zanoelo FF, Polizeli Mde L, Terenzi HF, Jorge JA (2004) Beta-glucosidase activity from the thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose. FEMS Microbiol Lett 240:137–143

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Acknowledgment

This work is dedicated to the memory of our colleagues Said Hamdi and Nejib Trigui. We deeply thank Najla Masmoudi and Hédi Aouissaoui for their expert help in chromatographic analysis. Hafedh Belghith is thanked for helpful discussion and advises. Mosbeh Dardouri is thanked for his technical help.

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Correspondence to Ali Gargouri.

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Saibi, W., Amouri, B. & Gargouri, A. Purification and biochemical characterization of a transglucosilating β-glucosidase of Stachybotrys strain. Appl Microbiol Biotechnol 77, 293–300 (2007). https://doi.org/10.1007/s00253-007-1141-3

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Keywords

  • Stachybotrys
  • Cellulases
  • β-glucosidase
  • Purification
  • Transglucosylation