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Purification and Properties of Pullulanase from Bacillus halodurans

Author Affiliations

  • 1 Department of Biochemistry, Center for Post Graduate Studies, Jain University, Jayanagar 3rd Block, Bangalore-560011, INDIA

Int. Res. J. Biological Sci., Volume 2, Issue (3), Pages 35-43, March,10 (2013)


A pullulanase-producing bacterium was isolated from soil and identified as Bacillus halodurans based on the microscopic examination and biochemical tests. Maximum pullulanase production occurred in the presence of soluble pullulan at 1.5% concentration and in the presence of 0.5% peptone at pH 10.0 and 37°C. The purified alkaline pullulanase had a molecular mass of 37 ± 1 kDa, an optimum pH in the alkaline region (10.0) and optimum temperature of 50°C. The pullulanase activity was inhibited by Zn2+ and Cu2+ ions. Mg2+, Mn2+ and Fe2+ slightly inhibited the enzyme whereas Ca2+ had a stimulating effect on the enzyme activity. Ethylenediaminetetraacetate (EDTA), dithiothreitol (DTT), phenylmethylsulphonylfluoride (PMSF), tocylchloride methylketone (TLCK) and sodium azide (NaI) did not obviously inhibit the enzyme whereas N-ethylmalamide (NEM) and iodoacetic acid (IAA) had moderately inhibited the enzyme. N-bromosuccinimide (NBS) inhibited completely the enzyme activity suggesting that tryptophan is important for enzyme activity. The isolated enzyme was thermotolerant and alkolophilic which can thus be used in starch processing, detergent industry and other biotechnological applications.


  1. Abdullah M. and French D., Reverse action of pullulanase, Nature, 200, 210 (1966)
  2. Hyun H.H. and Zeikus J.G., General biochemical characterization of thermostable pullulanase and glucoamylase from Clostridium thermohydrosulfurium, Appl. Environ. Microbial., 49, 1168-1173 (1985)
  3. Teague W.M. and Brumm P.J., Commercial enzymes for starch hydrolysis products. In: Starch hydrolysis products: Worldwide technology production and applications, Schenck F.W., Hebeda R.E. Eds., VCH Publishers, New York, 277-291 (1992)
  4. Uhlig H., Industrial enzymes and their applications, John Wiley & Sons, Inc., New York, 435 (1998)
  5. Nair S.U., Singhal R.S. and Kamat M.Y., Induction of pullulanase production in Bacillus cereus FDTA-13, Bioresour. Technol., 98, 856–859 (2007)
  6. Van D. and Willem R., Dishwashing detergents containing enzyme capable of breaking -1,6-glucoside linkage, Brit. UK Patent Appl. GB2, 228, 945 (1990)
  7. Jensen B.D. and Norman B.E., Bacillus acidopullyticuspullulanase: applications and regulatory aspects for use in food industry, Proc. Biochem., 397-400 (1984)
  8. Olsen H.S., Goddard P. and Nordisk A.S., Enzymes at work: a concise guide to industrial enzymes and their uses. In Novo Nordisk A/S, 58 (2000)
  9. Marotta M., Martino A., De Rosa A., Farina E., Carteni M. and De Rosa M., Degradation of dental plaque glucans and prevention of glucan formation using commercial enzymes, Proc. Biochem., 38(1), 101-108 (2002)
  10. Sneath P.H.A., Mair N.S., Sharpe M.E. and Holt J.G., Bergey’s Manual of Systematic Bacteriology, Vol. 2 Baltimore,Williams and Wilkins, (1986)
  11. Kim C.H., Nashiru O. and Ko J.H., Purification and biochemical characterization of pullulanase type I from Thermus aldophilus GK-24, FEMS Microbiol. Lett. 138, 147-152 (1996)
  12. Miller G.L., Use of dinitrosalicylic agent for determination of reducing sugars, Anal. Chem., 31, 426-428 (1959)
  13. Lowry O.H., Rosebrough N.J., Farr A.L. and Randall R.J., Quantitation of protein using Folin Ciocalteu reagent, J. Biol. Chem., 193, 265 (1951)
  14. Obi S.K.C. and Odibo F.J.C., Partial purification and characterization of a thermostable actinomycete -Amylase, Appl. Environ. Microbiol, 47(3), 571-575 (1984)
  15. Laemmli U.K., Cleavage of structural proteins during the assembly of the head of becteriophage T4, Nature, 227, 680-685 (1970)
  16. Balkan B. and Ertan F., Production and properties of amylase from Penicillium chrysogenum and its application in starch hydrolysis, Prep. Biochem. Biotechnol. 35, 169-178 (2005)
  17. Lineweaver H. and Burk D., The determination of enzyme dissociation constants, J. Am. Chem. Soc., 56, 658-666 (1934)
  18. Swamy M.V. and Seenayya G., Thermostable pullulanase and -amylase activity from Clostridium thermosulfurogenes SV9 - optimization of culture conditions for enzyme production, Proc. Biochem., 31, 157-62 (1966)
  19. Ara K., Sneki K.., Igarashi K. and Ito S., Alkaline pullulanase manufacture with Bacillus, Eur. Pat. App. EP. 415, 397 (1991)
  20. Castro G.R., Baigori M.D., Mendez B.S. and Sineriz F., Effect of pH and temperature on the continuous production of amylolytic enzymes by Bacillus amyloliquefaciens, J. Chem. Technol. Biotechnol., 58(3), 277-280 (1993)
  21. Ara K., Igarashi K., Saeki K., Kawai S. and Ito S., Purification and some properties of an alkaline pullulanase from alkalophilic Bacillus sp. KSM-1876, Biosc. Biotechnol. Biochem., 56, 62-65 (1992)
  22. Ling H.S., Ling T.C., Mohamad R. and Ariff A.B., Characterization of pullulanase type II from Bacillus cereusH1.5, Am. J. Biochem. Biotechnol., 5(4), 170-179 (2009)
  23. Kim C.H., Choi H.I. and Lee D.S., Purification and biochemical properties of an alkaline pullulanase from alkalophilic Bacillus sp. S-1, Biosci. Biotechnol. Biochem., 57(10), 1632-1637 (1993)
  24. Yamanobe T. and Takasaki T., Production of maltose from starch of various origins by -amylase and pullulanase of Bacillus cereus var.mycoides, Biseibutsu Kenkyu Hakoku, 52, 17-23 (1979)
  25. Zareian S., Khajeh K., Ranjbar B., Dabirmanesh B., Ghollasi M. and Mollania N., Purification and characterization of a novel amylopullulanase that converts pullulan to glucose, maltose, and maltotriose and starch to glucose and maltose, Enzyme Microbial. Technol., 46, 57–63 (2010)
  26. Ara K., Saeki K., Igarashi K., Takaiwa M., Uemura T., Hagihara H., Kawai S. and Ito S. Purification and characterization of an alkaline amylopullulanase with both alpha-1,4 and alpha-1,6 hydrolytic activity from alkalophilic Bacillus sp. KSM-1378, Biochim. Biophys. Acta., 1243(3), 315-324 (1995)
  27. Singh R.S., Saini G.K. and Kennedy J.F., Maltotriose syrup preparation from pullulan using pullulanase, Carbohydr. Polym., 80, 401-407 (2010)
  28. Gesu G., Jun Y.S. and Zhang S., Studies on pullulanase from Aerobacter aerogenes I. Purification and some properties, Wei Sheng WU Ilsueh Pao, 20(4), 415-420 (1980)
  29. Markosyan L.S. and Balayan A.M., Extracellular pullulanase of thermophilic strain of Bacillus sp., Priki. Biokhim. Microbiol., 26(3), 313-320 (1990)
  30. Shen G.J., Srivastava K.C., Saha B.C. and Zeikus J.G., Physiological and enzymatic characterization of a novel pullulan-degrading thermophilic Bacillus strain 3183, Appl. Microbiol. Biotechnol., 33(3), 340-344 (1990)