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Purification and characterization of a cellulase (CMCase) from a newly isolated thermophilic aerobic bacterium Caldibacillus cellulovorans gen. nov., sp. nov

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Abstract

One thermostable endoglucanase (CMCase) was purified to homogeneity from the culture supernatant of a new isolated thermophilic bacterium Caldibacillus cellulovorans. The molecular weight of the enzyme was 85.1 kDa as determined by SDS Polyacrylamide gel electrophoresis (PAGE) and 174 kDa by size-exclusion chromatography. The isoelectric point of the enzyme was at pH 4.12. The temperature for maximum activity was 80 °C, with half-lives of 32 min at 80 °C, and 2 min at 85 °C, and 83% activity remaining after 3 h at 70 °C. Thermostability of the enzyme was increased twofold by the addition of bovine serum albumin. Maximal activity was observed between pH 6.5 and 7.0. The enzyme activity was significantly inhibited by Zn2+, Hg2+, and p-chloromercuribenzenesulphonic acid. The enzyme showed high activity on carboxymethylcellulose (CMC) with much lower activity on Avicel; a low level of activity was also found against xylan. Cellobiose was the major product of hydrolysis of amorphous cellulose and CMC. Viscometric analysis indicated that the enzyme hydrolysed CMC in an exo-acting fashion. Cellotriose and cellobiose were not degraded and at least four contiguous glucosyl residues were necessary for degradation by the enzyme. The K m and V max of the enzyme for CMC were 3.4 mg ml−1 and 44.7 μmol min−1 (mg protein)−1, respectively.

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References

  • American Type Culture Collection. 1992 Catalogue of Bacteria and Bacteriophages. Rockville, Maryland: ATCC, p. 460.

    Google Scholar 

  • Au, K.S. & Chan, K.Y. 1987 Purification and properties of the endo-1,4-β-glucanase from Bacillus subtilis. Journal of General Microbiology 133, 2155–2162.

    Google Scholar 

  • Bauer, M.W., Driskill, L.E., Callen, W., Snead, M.A., Mathur, E.J. & Kelly, R.M. 1999 An endoglucanase, EglA, from the hyperthermophilic archaeon Pyrococcus furiosus hydrolyzes β-1,4 bonds in mixed-linkage (1-3), (1-4)-β-D-glucans and cellulose. Journal of Bacteriology 181, 284–290.

    Google Scholar 

  • Bronnenmeier, K. & Staudenbauer, W.L. 1988 Resolution of Clostridium stercorarium cellulase by fast protein liquid chromatography (FPLC). Applied Microbiology and Biotechnology 27, 432–436.

    Google Scholar 

  • Bronnenmeier, K. & Staudenbauer, W.L. 1990 Cellulose hydrolysis by a highly thermostable endo-1,4-β-glucanase (Avicelase I) from Clostridium stercorarium. Enzyme and Microbial Technology 12, 431–436.

    Google Scholar 

  • Calza, R.E., Irwin, D.C. & Wilson, D.B. 1985 Purification and characterization of two β-1,4-endoglucanases from Thermomonospora fusca. Biochemistry 24, 7797–7804.

    Google Scholar 

  • Chan, K.Y. & Au, C.K. 1987 Studies on cellulase production by a Bacillus subtilis. Antonie van Leeuwenhoek Journal of Microbiology 53, 125–136.

    Google Scholar 

  • Coughlan, M.P. 1988 Staining techniques for the detection of the individual components of cellulolytic enzyme systems. Methods in Enzymology 160, 135–144.

    Google Scholar 

  • Coughlan, M.P. & Mayer, F. 1992 The cellulose-decomposing bacteria and their enzyme systems. In The Prokaryotes vol. 1, eds. Balows, A., Truper, H.G., Dworkin, M., Harder, W. & Schleifer, K.H. pp. 460–516. New York: Springer-Verlag. ISBN 0-38797258-7.

    Google Scholar 

  • Creuzet, N. & Frixon, C. 1983 Purification and characterization of an endoglucanase from a newly isolated thermophilic anaerobic bacterium. Biochimie 65, 149–156.

    Google Scholar 

  • Fauth, U.M., Romaniec, P.M., Kobayashi, T. & Demain, A. 1991 Purification and characterization of endoglucanase Ss from Clostridium thermocellum. Biochemical Journal 279, 67–73.

    Google Scholar 

  • Fukumori, F., Kudo, T. & Horikoshi, K. 1985 Purification and properties of a cellulase from alkalophilic Bacillus sp. No. 1139. Journal of General Microbiology 131, 3339–3345.

    Google Scholar 

  • Gal, L., Gaudin, C., Belaich, A., Pages, S., Tardif, C. & Belaich, J.P. 1997 CelG from Clostridium cellulolyticum: a multidomain endoglucanase acting efficiently on crystalline cellulose. Journal of Bacteriology 179, 6595–6601.

    Google Scholar 

  • Han, S.J., Yoo, Y.J. & Kang, H.S. 1995 Characterization of a bifunctional cellulase and its structural gene. Journal of Biological Chemistry 270, 26012–26019.

    Google Scholar 

  • Hazlewood, G.P., Romaniec, M.P.M., Davidson, K., Grepinet, O., Beguin, P., Millet, J., Raynaud, O. & Aubert, J.P.A. 1988 catalogue of Clostridium thermocellum endoglucanase, β-glucosidase and xylanase genes cloned in E. coli. FEMS Microbiology Letters 51, 231–236.

    Google Scholar 

  • Horikoshi, K., Nakao, M., Kurono, Y. & Sashihara, N. 1984 Cellulase of an alkalophilic Bacillus strain isolated from soil. Canadian Journal of Microbiology 30, 774–779.

    Google Scholar 

  • Hreggvidsson, G.O., Kaiste, E., Holst, O., Eggertsson, G., Palsdottir, A. & Kristjansson, J.K. 1996 An extremely thermostable cellulase from the thermophilic eubacteria Rhodothermus marinus. Applied and Environmental Microbiology 62, 3047–3049.

    Google Scholar 

  • Huang, X.P. 1999 Characterization of a novel aerobic cellulolytic thermophilic bacterium and its cellulases. PhD thesis, University of Waikato, New Zealand.

    Google Scholar 

  • Huang, L.C., Forsberg, W. & Thomas, D.Y. 1988 Purification and characterization of a chloride-stimulated cellobiosidase from Bacteroides succinogenes S85. Journal of Bacteriology 170, 2923–2932.

    Google Scholar 

  • Jorgensen, P.L. & Hansen, C.K. 1990 Multiple endo-β-1,4-glucanase-encoding genes from Bacillus lautus PL236 and characterization of the celB gene. Gene 93, 55–60.

    Google Scholar 

  • Kim, C.H. 1995 Characterization and substrate specificity of an endo-β-1,4-D-glucanase I (avicelase I) from an extracellular multienzyme complex of Bacillus circulans. Applied and Environmental Microbiology 61, 959–965.

    Google Scholar 

  • Lamed, R., Setter, E., Kenig, R. & Bayer, E.A. 1983 The cellulosome-A discrete cell surface organelle of Clostridium thermocellum which exhibits separate antigenic cellulose-binding and various cellulolytic activities. Biotechnology and Bioengineering Symposium 13, 163–181.

    Google Scholar 

  • Lever, M. 1973 Colorimetric and fluorometric carbohydrate determination with p-hydroxybenzoic acid hydrazide. Biochemical Medicine 7, 274–281.

    Google Scholar 

  • Lin, S.B. & Stutzenberger, F.J. 1995 Purification and characterization of the major β-1,4-endoglucanase from Thermomonospora curvata. Journal of Applied Microbiology 79, 447–453.

    Google Scholar 

  • Mohagheghi, A., Grohmann, K., Himmel, M., Leighton, L. & Updegraff, D.M. 1986 Isolation and characterization of Acidothermus cellulolyticus gen. nov., sp. nov., a new genus of thermophilic, acidophilic, cellulolytic bacteria. International Journal of Systematic Bacteriology 36, 435–443.

    Google Scholar 

  • Nakamura, K. & Kitamura, K. 1988 Cellulase of Cellulomonas uda. Methods in Enzymology 160, 211–216.

    Google Scholar 

  • Neal, T.L. 1987 A thermophilic CMCase from an extreme thermophile cloned into E. coli. Master of Science thesis, University of Waikato, New Zealand.

    Google Scholar 

  • Ng, T.K. & Zeikus, J.G. 1981 Purification and characterization of an endoglucanase (1,4-β-D-glucan glucanohydrolase) from Clostridium thermocellum. Biochemical Journal 199, 341–350.

    Google Scholar 

  • Ohmiya, K. & Schimizu, T. 1988 Cellobiosidase from Ruminococcus albus. Methods in Enzymology 160, 391–398.

    Google Scholar 

  • Peek, K., Daniel, R.M., Monk, C., Parker, L. & Coolbear, T. 1992 Purification and characterization of a thermostable proteinase isolated from Thermus sp. strain Rt41 A. European Journal of Biochemistry 207, 1035–1044.

    Google Scholar 

  • Priest, F. 1977 Extracellular enzyme synthesis in the genus Bacillus. Bacteriological Reviews 41, 711–753.

    Google Scholar 

  • Robson, L.M. & Chamblis, G.H. 1984 Characterization of the cellulolytic activity of a Bacillus isolate. Applied and Environmental Microbiology 47, 1039–1046.

    Google Scholar 

  • Romaniec, M.P.M., Fauth, U., Kobayashi, T., Huskisson, N., Barker, P.J. & Demain, A.L. 1992 Purification and characterization of a new endoglucanase from Clostridium thermocellum. Biochemical Journal 283, 69–73.

    Google Scholar 

  • Ruttersmith, J.V. 1991 A thermostable cellulase cloned from Caldocellum saccharolyticum. M.Phil. thesis, University of Waikato, New Zealand.

    Google Scholar 

  • Ruttersmith, L.D. & Daniel, R.M. 1993 Thermostable β-glucosidase and β-xylosidase from Thermotoga sp. strain FjSS3-B.1. Biochimica et Biophysica Acta 1156, 167–172.

    Google Scholar 

  • Sami, A.J., Akhtar, M.W., Malik, N.N. & Naz, B.A. 1988 Production of free and substrate-bound cellulases of Cellulomonas flavigena. Enzyme and Microbial Technology 10, 626–631.

    Google Scholar 

  • Schofield, L.R. & Daniel, R.M. 1993 Purification and properties of a β-1,4-xylanase from a cellulolytic extreme thermophile expressed in E. coli. The International Journal of Biochemistry 25, 609–617.

    Google Scholar 

  • Stutzenberger, F.J. 1991 Cellulose degradation by thermophilic aerobic bacteria. In Biosynthesis and Biodegradation of Cellulose eds. Haigler, C.H. & Weimer, P.J., pp. 445–489. New York: Marcel Dekker, Inc. ISBN 0-82478387-5.

    Google Scholar 

  • Tomme, P., Gilkes, N.R., Kwan, E.Y. & Warren, R.A.J. 1996 Characterization of CenC, an enzyme from Cellulomonas fimi with both endo-and exoglucanase activities. Journal of Bacteriology 178, 4216–4223.

    Google Scholar 

  • Tucker, M.P., Mohagheghi, A., Grohmann, K. & Himmel, M.E. 1989 Ultra-thermostable cellulases from Acidothermus cellulolyticus: comparison of temperature optima with previously reported cellulases. Biotechnology 7, 817–820.

    Google Scholar 

  • Vlasenko, E.Y., Ryan, A.I., Shoemaker, C.F. & Shoemaker, S.P. 1998 The use of capillary viscometry, reducing end-group analysis, and size exclusion chromatography combined with multi-angle laser light scattering to characterize endo-1,4-β-D glucanases on carboxymethylcellulose: a comparative evalution of the three methods. Enzyme and Microbial Technology 23, 350–359.

    Google Scholar 

  • Wang, W.K., Kruus, K. & Wu, J.H.D. 1994 Cloning and expression of the Clostridium thermocellum CelS gene in Escherichia coli. Applied Microbiology and Biotechnology 42, 346–352.

    Google Scholar 

  • Wood, T.M. & Bhat, K.M. 1988 Methods for measuring cellulase activities. Methods in Enzymology 160, 87–112.

    Google Scholar 

  • Wood, T.M. 1989 Mechanisms of cellulose degradation by enzymes from aerobic and anaerobic fungi. In Enzyme Systems for Lignocellulose Degradation, ed. Coughlan, M.P. pp. 17–35. London: Elsevier Applied Science. ISBN 1-85166411-4.

    Google Scholar 

  • Yablonsky, M.D., Bartley, T. & Eveleigh, D.E. 1988 Characterization and cloning of the cellulase complex of Microbispora bispora. In Biochemistry and Genetics of Cellulose Degradation, eds. Aubert, J.P., Beguin, P. & Millet, J. pp. 249–264. New York: Academic Press. ISBN 0-12-067575-7.

    Google Scholar 

  • Yoshimatsu, T., Ozaki, K., Shikata, S., Ohta, Y.I., Koike, K., Kawai, S. & Ito, S. 1990 Purification and characterization of alkaline endo-1,4-β-glucanases from alkalophilic Bacillus sp. KSM-635. Journal of General Microbiology 136, 1973–1979.

    Google Scholar 

  • Zverlov, V.V., Velikodvorskaya, G.V., Schwarz, W.H.J., Bronnenmeier K., Kellermann J. & Staudenbauer, W.L. 1998 Multidomain structure and cellulosomal localization of the C. thermocellum cellobiohydrolase CbhA. Journal of Bacteriology 180, 3091–3099.

    Google Scholar 

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  1. Xiao Ping Huang

    Present address: Biochemistry Department, College of Chemistry and Biology, Yantai University, Yantai, Shandong, China

Authors and Affiliations

  1. Thermophile Research Unit, The University of Waikato, Hamilton, New Zealand

    Xiao Ping Huang & Colin Monk

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Huang, X.P., Monk, C. Purification and characterization of a cellulase (CMCase) from a newly isolated thermophilic aerobic bacterium Caldibacillus cellulovorans gen. nov., sp. nov. World Journal of Microbiology and Biotechnology 20, 85–92 (2004). https://doi.org/10.1023/B:WIBI.0000013316.12730.e7

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  • DOI: https://doi.org/10.1023/B:WIBI.0000013316.12730.e7

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