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. 1987 May 1;243(3):779–787. doi: 10.1042/bj2430779

Purification and properties of a stable beta-glucosidase from an extremely thermophilic anaerobic bacterium.

M L Patchett 1, R M Daniel 1, H W Morgan 1
PMCID: PMC1147925  PMID: 3117033

Abstract

A beta-glucosidase (EC 3.2.1.21) was purified to homogeneity from cell-free extracts of an extremely thermophilic anaerobic bacterium. The enzyme has an Mr of 43,000 as determined by molecular-exclusion chromatography, has a pI of 4.55 and shows optimum activity at pH 6.2. The enzyme is active against a wide range of aryl beta-glycosides and beta-linked disaccharides, with beta-galactosidase activity only slightly less than beta-glucosidase activity, and significant beta-xylosidase activity. Lineweaver-Burk plots for p-nitrophenyl beta-glucoside, o-nitrophenyl beta-glucoside and cellobiose substrates are biphasic concave-downwards. Inhibition of the beta-glucosidase by substrates and glucose is negligible. Thermal inactivation follows first-order kinetics, with t1/2 (65 degrees C) 45 h, t1/2 (75 degrees C) 47 min and t1/2 (85 degrees C) 1.4 min and a deactivation energy of 380 kJ/mol at pH 6.2. At pH 7.0, which is the optimum pH for thermostability, t1/2 (75 degrees C) is 130 min. At 75 degrees C, at pH 6.2, the thermostability is enhanced about 8-fold by 10% (w/v) glycerol, about 6-fold by 0.2 M-cellobiose and about 3-fold by 5 mM-dithiothreitol and 5 mM-2-mercaptoethanol.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aït N., Creuzet N., Cattanéo J. Characterization and purification of thermostable beta-glucosidase from Clostridium thermocellum. Biochem Biophys Res Commun. 1979 Sep 27;90(2):537–546. doi: 10.1016/0006-291x(79)91269-5. [DOI] [PubMed] [Google Scholar]
  2. Beldman G., Searle-Van Leeuwen M. F., Rombouts F. M., Voragen F. G. The cellulase of Trichoderma viride. Purification, characterization and comparison of all detectable endoglucanases, exoglucanases and beta-glucosidases. Eur J Biochem. 1985 Jan 15;146(2):301–308. doi: 10.1111/j.1432-1033.1985.tb08653.x. [DOI] [PubMed] [Google Scholar]
  3. Bissett F., Sternberg D. Immobilization of Aspergillus beta-glucosidase on chitosan. Appl Environ Microbiol. 1978 Apr;35(4):750–755. doi: 10.1128/aem.35.4.750-755.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Bucht B., Eriksson K. E. Extracellular enzyme system utilized by the rot fungus Stereum sanguinolentum for the breakdown of cellulose. IV. Separation of cellobiase and aryl beta-glucosidase activities. Arch Biochem Biophys. 1969 Feb;129(2):416–420. doi: 10.1016/0003-9861(69)90197-0. [DOI] [PubMed] [Google Scholar]
  6. Forsberg C. W., Groleau D. Stability of the endo-beta-1,4-glucanase and beta-1,4-glucosidase from Bacteroides succinogenes. Can J Microbiol. 1982 Jan;28(1):144–148. doi: 10.1139/m82-017. [DOI] [PubMed] [Google Scholar]
  7. Frieden C. Slow transitions and hysteretic behavior in enzymes. Annu Rev Biochem. 1979;48:471–489. doi: 10.1146/annurev.bi.48.070179.002351. [DOI] [PubMed] [Google Scholar]
  8. Garibaldi A., Gibbins L. N. Partial purification and properties of a beta-glucosidase from Erwinia herbicola Y46. Can J Microbiol. 1975 Apr;21(4):513–520. doi: 10.1139/m75-073. [DOI] [PubMed] [Google Scholar]
  9. Gekko K., Timasheff S. N. Thermodynamic and kinetic examination of protein stabilization by glycerol. Biochemistry. 1981 Aug 4;20(16):4677–4686. doi: 10.1021/bi00519a024. [DOI] [PubMed] [Google Scholar]
  10. Groleau D., Forsberg C. W. Cellulolytic activity of the rumen bacterium Bacteroides succinogenes. Can J Microbiol. 1981 May;27(5):517–530. doi: 10.1139/m81-077. [DOI] [PubMed] [Google Scholar]
  11. Han Y. W., Srinivasan V. R. Purification and characterization of beta-glucosidase of Alcaligenes faecalis. J Bacteriol. 1969 Dec;100(3):1355–1363. doi: 10.1128/jb.100.3.1355-1363.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hirayama T., Nagayama H., Matsuda K. Studies on cellulases of a phytopathogenic fungus, Pyricularia oryzae Cavara. IV. Kinetic studies on beta-glucosidases. J Biochem. 1980 Apr;87(4):1203–1208. [PubMed] [Google Scholar]
  13. Inglin M., Feinberg B. A., Loewenberg J. R. Partial purification and characterization of a new intracellular beta-glucosidase of Trichoderma reesei. Biochem J. 1980 Feb 1;185(2):515–519. doi: 10.1042/bj1850515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jermyn M. A. Fungal cellulases. XV. Acceptor specificity of the aryl beta-glucosidase of Stachybotrys atra. Aust J Biol Sci. 1966 Oct;19(5):903–917. doi: 10.1071/bi9660903. [DOI] [PubMed] [Google Scholar]
  15. Jorgensen B. B., Jorgensen O. B. Inhibition of barley malt alpha-glucosidase by Tris(hydroxymethyl)aminomethane and erythritol. Biochim Biophys Acta. 1967 Sep 12;146(1):167–172. doi: 10.1016/0005-2744(67)90083-6. [DOI] [PubMed] [Google Scholar]
  16. Lusis A. J., Becker R. R. The beta-glucosidase system of the thermophilic fungus Chaetomium thermophile var. coprophile n. var. Biochim Biophys Acta. 1973 Nov 2;329(1):5–16. doi: 10.1016/0304-4165(73)90003-2. [DOI] [PubMed] [Google Scholar]
  17. MAHADEVAN P. R., EBERHART B. THE BETA-GLUCOSIDASE SYSTEM OF NEUROSPORA CRASSA. II. PURIFICATION AND CHARACTERIZATION OF ARYL BETA-GLUCOSIDASE. Arch Biochem Biophys. 1964 Oct;108:22–29. doi: 10.1016/0003-9861(64)90350-9. [DOI] [PubMed] [Google Scholar]
  18. Macris B. J. Production and Characterization of Cellulase and beta-Glucosidase from a Mutant of Alternaria alternata. Appl Environ Microbiol. 1984 Mar;47(3):560–565. doi: 10.1128/aem.47.3.560-565.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Merril C. R., Goldman D., Sedman S. A., Ebert M. H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
  20. Meyer H. P., Canevascini G. Separation and Some Properties of Two Intracellular beta-Glucosidases of Sporotrichum (Chrysosporium) thermophile. Appl Environ Microbiol. 1981 Apr;41(4):924–931. doi: 10.1128/aem.41.4.924-931.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Moldoveanu N., Kluepfel D. Comparison of beta-Glucosidase Activities in Different Streptomyces Strains. Appl Environ Microbiol. 1983 Jul;46(1):17–21. doi: 10.1128/aem.46.1.17-21.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nari J., Noat G., Ricard J. pH-induced co-operative effects in hysteretic enzymes. 2. pH-induced co-operative effects in a cell-wall beta-glucosyltransferase. Eur J Biochem. 1984 Dec 3;145(2):319–322. doi: 10.1111/j.1432-1033.1984.tb08555.x. [DOI] [PubMed] [Google Scholar]
  23. Ohmiya K., Shirai M., Kurachi Y., Shimizu S. Isolation and properties of beta-glucosidase from Ruminococcus albus. J Bacteriol. 1985 Jan;161(1):432–434. doi: 10.1128/jb.161.1.432-434.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pitt-Rivers R., Impiombato F. S. The binding of sodium dodecyl sulphate to various proteins. Biochem J. 1968 Oct;109(5):825–830. doi: 10.1042/bj1090825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Reese E. T., Mandels M. Stability of the cellulase of Trichoderma reesei under use conditions. Biotechnol Bioeng. 1980 Feb;22(2):323–335. doi: 10.1002/bit.260220207. [DOI] [PubMed] [Google Scholar]
  26. Rodionova N. A., Rumiantseva G. N., Tiunova N. A., Martinovich L. I., Bakhtadze L. N. beta-Gliukozidazy iz griba Geotrichum candidum. Biokhimiia. 1977 Jan;42(1):43–50. [PubMed] [Google Scholar]
  27. Saddler J. N., Khan A. W. Cellulolytic enzyme system of Acetivibrio cellulolyticus. Can J Microbiol. 1981 Mar;27(3):288–294. doi: 10.1139/m81-045. [DOI] [PubMed] [Google Scholar]
  28. Sano K., Amemura A., Harada T. Purification and properties of a beta-1,6-clucosidase from Flavobacterium. Biochim Biophys Acta. 1975 Feb 19;377(2):410–420. doi: 10.1016/0005-2744(75)90321-6. [DOI] [PubMed] [Google Scholar]
  29. Schliemann W. Beta-D-Glucosidasen (EC 3.2.1.21) der Mikroorganismen. Eine tabellarische Ubersicht. Pharmazie. 1983 May;38(5):287–307. [PubMed] [Google Scholar]
  30. Shepherd M. G., Tong C. C., Cole A. L. Substrate specificity and mode of action of the cellulases from the thermophilic fungus Thermoascus aurantiacus. Biochem J. 1981 Jan 1;193(1):67–74. doi: 10.1042/bj1930067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Shewale J. G. Beta-Glucosidase: its role in cellulase synthesis and hydrolysis of cellulose. Int J Biochem. 1982;14(6):435–443. doi: 10.1016/0020-711x(82)90109-4. [DOI] [PubMed] [Google Scholar]
  32. Spears G., Sneyd J. G., Loten E. G. A method for deriving kinetic constants for two enzymes acting on the same substrate. Biochem J. 1971 Dec;125(4):1149–1151. doi: 10.1042/bj1251149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sternberg D., Vijayakumar P., Reese E. T. beta-Glucosidase: microbial production and effect on enzymatic hydrolysis of cellulose. Can J Microbiol. 1977 Feb;23(2):139–147. doi: 10.1139/m77-020. [DOI] [PubMed] [Google Scholar]
  34. Stoppok W., Rapp P., Wagner F. Formation, Location, and Regulation of Endo-1,4-beta-Glucanases and beta-Glucosidases from Cellulomonas uda. Appl Environ Microbiol. 1982 Jul;44(1):44–53. doi: 10.1128/aem.44.1.44-53.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tong C. C., Cole A. L., Shepherd M. G. Purification and properties of the cellulases from the thermophilic fungus Thermoascus aurantiacus. Biochem J. 1980 Oct 1;191(1):83–94. doi: 10.1042/bj1910083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Umezurike G. M. The beta-glucosidase from Botryodiplodia theobromae Pat. Kinetics of enzyme-catalysed hydrolysis of o-nitrophenyl beta-D-glucopyranoside in dioxan/water. Biochem J. 1978 Nov 1;175(2):455–459. doi: 10.1042/bj1750455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Umezurike G. M. The beta-glucosidase from Botryodiplodia theobromae. Mechanism of enzyme action. Biochem J. 1981 Oct 1;199(1):203–209. doi: 10.1042/bj1990203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wood T. M. The cellulase of Fusarium solani. Purification and specificity of the -(1-4)-glucanase and the -D-glucosidase components. Biochem J. 1971 Feb;121(3):353–362. doi: 10.1042/bj1210353. [DOI] [PMC free article] [PubMed] [Google Scholar]

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