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Purification and characterisation of alkaline cellulase produced by a novel isolate, Bacillus sphaericus JS1

  • Original Paper
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Journal of Industrial Microbiology and Biotechnology

Abstract

A novel strain of Bacillus sphaericus JS1 producing thermostable alkaline carboxymethyl cellulase (CMCase; endo-1,4-β-glucanase, E.C. 3.2.1.4) was isolated from soil using Horikoshi medium at pH 9.5. CMCase was purified 192-fold by (NH4)2SO4 precipitation, ion exchange and gel filtration chromatography, with an overall recovery of 23%. The CMCase is a multimeric protein with a molecular weight estimated by native-PAGE of 183 kDa. Using SDS-PAGE a single band is found at 42 kDa. This suggests presence of four homogeneous polypeptides, which would differentiate this enzyme from other known alkaline cellulases. The activity of the enzyme was significantly inhibited by bivalent cations (Fe3+ and Hg2+, 1.0 mM each) and activated by Co2+, K+ and Na+. The purified enzyme revealed the products of carboxymethyl cellulose (CMC) hydrolysis to be CM glucose, cellobiose and cellotriose. Thermostability, pH stability, good hydrolytic capability, and stability in the presence of detergents, surfactants, chelators and commercial proteases make this enzyme potentially useful in laundry detergents.

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References

  1. Au K-S, Chan K-Y (1987) Purification and properties of the endo-1,4-β-glucanase from Bacillus subtilis. J Gen Microbiol 133:2155–2162

    Google Scholar 

  2. Bajpai P (1999) Application of enzyme in pulp and paper industry. Biotechnol Progr 15:147–157

    Google Scholar 

  3. Beguin P (1983) Detection of cellulase activity in polysaccharide gels using Congo Red-stained agar replicas. Anal Biochem 131:333–336

    Google Scholar 

  4. Bhat MK (2000) Cellulases and related enzymes in biotechnology. Biotechnol Adv 18:355–383

    Google Scholar 

  5. Cavaco-Paulo A (1998) Mechanism of cellulase action in textile process. Carbohydr Polym 37:273–277

    Google Scholar 

  6. Christakopoulos P, Hatzinikolaou DG, Fountoukidis G, Kekos D, Claeyssens M, Macris BJ (1999) Purification and mode of action of an alkali-resistant endo-1,4-beta-glucanase from Bacillus pumilus. Arch Biochem Biophys 364:61–66

    Google Scholar 

  7. Dasilva R, Yim DK, Asquieri ER, Park YK (1993) Production of microbial alkaline cellulase and studies of their characteristics. Microbiol Rev 24:269–274

    Google Scholar 

  8. Endo K, Hakamada Y, Takizawa S, Kubota H (2001) A novel alkaline endoglucanase from an alkaliphilic Bacillus isolate: enzymatic properties, and nucleotide and deduced amino acid sequences. Appl Microbiol Biotechnol 57:109–116

    Google Scholar 

  9. Fukumori F, Kudo T, Horikoshi K (1985) Purification and properties of a cellulase from alkalophilic Bacillus sp. No. 1139. J Gen Microbiol 131:3339–3345

    Google Scholar 

  10. Hakamada Y, Koike K, Yoshimatsu T, Mori H, Kobayashi T, Ito S (1997) Thermostable alkaline cellulase from an alkalophilic isolate, Bacillus sp. KSM-S237. Extremophiles 1:151–156

    Google Scholar 

  11. Hakamada Y, Endo K, Takizawa S, Kobayashi T, Shirai T, Yamane T, Ito S (2002) Enzymatic properties, crystallisation, and deduced amino acid sequence of an alkaline endoglucanase from Bacillus circulans. Biochem Biophys Acta 1570:174–180

    Google Scholar 

  12. Horikoshi K (1997) Alkaliphiles—from an industrial point of view. FEMS Microbiol Rev 18:259–270

    Google Scholar 

  13. Horikoshi K (1999) Alkaliphiles: some applications of their products for biotechnology. Microbiol Mol Biol Rev 63:735–750

    Google Scholar 

  14. Horikoshi K, Nakao M, Kurono Y, Sashihara N (1984) Cellulase of an alkalophilic Bacillus strain isolated from soil. Can J Microbiol 30:774–779

    Google Scholar 

  15. Hoshino E, Ito S (1997) Application of alkaline cellulases that contribute to soil removal in detergents. In: van Ee JH, Misset O, Baas EJ (eds) Enzymes in detergency. Dekker, New York, pp 149–174

  16. Ito S (1997) Alkaline cellulase from alkaliphilic Bacillus: enzymatic properties, genetics, and application to detergents. Extremophiles 1:61–66

    Google Scholar 

  17. Ito S, Shikata S, Ozaki K, Kawai S, Okamoto K, Inoue S, Takei A (1989) Alkaline cellulase for laundry detergent: production by Bacillus sp. KSM-635 and enzymatic properties. Agric Biol Chem 53:1275–1281

    Google Scholar 

  18. Ito S, Shimooka Y, Takaiwa M, Ozaki K, Adachi S, Okamoto K (1991) Enhanced production of extracellular enzymes by mutant of Bacillus that have required resistance to vancomycin and ristocetin. Agric Biol Chem 55:2387–2391

    Google Scholar 

  19. Ito S, Kobayashi T, Ara K, Ozaki K, Kawai S, Hatada Y (1998) Alkaline detergent enzymes from alkaliphiles: enzymatic properties, genetics, and structures. Extremophiles 2:185–190

    Google Scholar 

  20. Kawai S, Okoshi H, Ozaki K, Shikata S, Ara K, Ito S (1988) Neutrophilic Bacillus strains KSM-522 that produces an alkaline carboxymethyl cellulase. Agric Biol Chem 52:1425–1431

    Google Scholar 

  21. Lenting HB, Warmoeskerken MM (2001) Mechanism of interaction between cellulase action and applied shear force, an hypothesis. J Biotechnol 89:217–226

    Google Scholar 

  22. Lowry OH, Rosenbrough W, Farr AL, Rondal RJ (1951) Protein measurement with the folin-phenol reagent. J Biol Chem 193:265–273

    Google Scholar 

  23. Lusterio DD, Suizo FG, Labunos NM, Valledor MN, Ureda S, Kawai S, Koike K, Shikata S, Yoshimatsu T, Ito S (1992) Alkali-resistant, alkaline endo-1,4-β-glucanase produced by Bacillus sp. PKM-5430. Biosci Biotechnol Biochem 56:1671–1672

    Google Scholar 

  24. Mawadza C, Hatti-Kaul R, Zvauya R, Mattiasson B (2000) Purification and characterisation of cellulases produced by Bacillus strain. J Biotechnol 83:177–187

    Google Scholar 

  25. Miettinenen-Oinonen A, Suominen P (2002) Enhanced production of Trichoderma reesei endoglucanases and use of new cellulase preparations in producing the stonewashed effect on denim fabrics. Appl Environ Microbiol 68:3956–3964

    Google Scholar 

  26. Murata M, Hoshino E, Yokosuka M, Suzuki A (1991) New detergent mechanism with use of novel alkaline cellulase. J Am Oil Chem Soc 68:553–557

    Google Scholar 

  27. Murata M, Hoshino E, Yokosuka M, Suzuki A (1993) New detergent mechanism using cellulase revealed by change in physicochemical properties of cellulose. J Am Oil Chem Soc 70:53–58

    Google Scholar 

  28. Okoshi H, Ozaki K, Oshino S, Kawai S, Ito S (1990) Purification and characterisation of multiple carboxymethyl cellulases from Bacillus sp. KSM-522. Agric Biol Chem 54:83–89

    Google Scholar 

  29. Ozaki K, Ito S (1991) Purification and properties of an acid endo β-1,4-glucanase from Bacillus sp. KSM-330. J Gen Microbiol 137:41–48

    Google Scholar 

  30. Poutanen K (1997) Enzymes: an important tool in the improvement of the quality of cereal food. Trends Food Sci Technol 8:300–306

    Google Scholar 

  31. Riisgaard S (1990) The enzyme industry and modern biotechnology. Genet Eng Biotechnol 10:11–14

    Google Scholar 

  32. Robson LM, Chambliss GH (1984) Characterisation of the cellulolytic activity of a Bacillus isolate. Appl Environ Microbiol 47:1039–1046

    Google Scholar 

  33. Saito K, Seko M, Masatsuji E (1994) Detergent comprising isolated cellulase from Bacillus ferm bp-3431 or a mutant strain thereof, surfactant, and builder. US Patent 5 314 637

  34. Screws GA, Pedersen G (1998) Process for the treatment of cellulosic fibres with cellulases. US Patent 5 707 858

    Google Scholar 

  35. Sharma P, Gupta JK, Vadehra DV, Dube DK (1990) Purification and properties of an endoglucanase from a Bacillus isolate PDV. Enzyme Microb Technol 12:132–137

    Google Scholar 

  36. Shikata S, Saeki K, Okoshi H, Yoshimatsu T, Ozaki K, Kawai S, Ito S (1990) Alkaline cellulase laundry detergents: production by alkalophilic strains of Bacillus and some properties of the crude enzymes. Agric Biol Chem 54:91–96

    Google Scholar 

  37. Shirai T, Ishida H, Noda J, Yamane T, Ozaki K, Hakamada Y, Ito S (2001) Crystal structure of alkaline cellulase K: insight into the alkaline adaptation of an industrial enzyme. J Mol Biol 310:1079–1087

    Google Scholar 

  38. Singh J, Batra N, Sobti RC (2001) A highly thermostable, alkaline CMCase produced by a newly isolated Bacillus sp. VG1. World J Microbiol Biotechnol 17: 761–765

    Google Scholar 

  39. Summer JB, Somers GF (1954) Dinitrosalicylic acid for glucose. In: Laboratory experiments in Biological Chemistry. Academic Press, New York, pp 34–39

  40. Suzuki A, Ito S, Okamoto K, Hoshino E, Yokosyka M, Murarta M (1990) Detergent composition for clothing: containing cellulase enzyme with non-degrading index of 500 or more. US Patent 4 978 470

    Google Scholar 

  41. Tian X, Wong X (1998) Purification and properties of alkaline cellulase from alkalophilic Bacillus N6–27. Wei Sheng Wu Xue Bao 38:310–312

    Google Scholar 

  42. Uhlig H (1998) Industrial enzymes and their applications. Wiley, New York

  43. Van Solingen P (1999) Alkaline cellulase and method of producing the same. US Patent 5 856 165

    Google Scholar 

  44. Wong KKY, Saddler JN (1993) Applications of hemicellulases in the food, feed and pulp, and pulp industries. In: Coughlan MP, Hazlewood GP (eds) Hemicellulose and hemicellulase. Portland Press, London, pp 127–143

  45. Yoshimatsu T, Ozaki K, Shikata S, Ohta Y, Koike K, Kawai S, Ito S (1990) Purification and characterisation of alkaline endo-1,4- β-glucanases from alkalophilic Bacillus sp. KSM-635. J Gen Microbiol 136:1973–1979

    Google Scholar 

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Acknowledgement

J.S. is thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi (India), for providing a research fellowship.

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Authors and Affiliations

  1. Department of Biotechnology, D.D.U. Gorakhpur University, Gorakhpur, 273-009 , Uttar Pradesh, India

    Jagtar Singh

  2. Department of Biotechnology, Lyallpur Khalsa College, Jallandhar, Punjab, India

    Navneet Batra

  3. Department of Biotechnology, Panjab University, 160 014, Chandigarh , India

    Ranbir Chander Sobti

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  1. Jagtar Singh
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  2. Navneet Batra
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  3. Ranbir Chander Sobti
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Correspondence to Ranbir Chander Sobti.

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Singh, J., Batra, N. & Sobti, R.C. Purification and characterisation of alkaline cellulase produced by a novel isolate, Bacillus sphaericus JS1. J IND MICROBIOL BIOTECHNOL 31, 51–56 (2004). https://doi.org/10.1007/s10295-004-0114-0

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  • DOI: https://doi.org/10.1007/s10295-004-0114-0

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