Antonie van Leeuwenhoek

, Volume 81, Issue 1–4, pp 73–83 | Cite as

Novel thermoactive glucoamylases from the thermoacidophilic Archaea Thermoplasma acidophilum, Picrophilus torridus and Picrophilus oshimae

  • Ehab Serour
  • Garabed AntranikianEmail author


The thermoacidophilic Archaea Thermoplasma acidophilum (optimal growth at 60 °C and pH 1–2), Picrophilus torridus and Picrophilus oshimae (optimal growth at 60 °C and pH 0.7) were able to utilize starch as sole carbon source. During growth these microorganisms secreted heat and acid-stable glucoamylases into the culture fluid. Applying SDS gel electrophoresis activity bands were detected with appearent molecular mass (Mw) of 141.0, 95.0 kDa for T. acidophilum, 133.0, 90.0 kDa for P. torridus and 140.0, 85.0 kDa for P. oshimae. The purified enzymes were incubated with various polymeric substrates such as starch, pullulan, panose and isomaltose. The product pattern, analyzed by HPLC, showed that in all cases glucose was formed as the sole product of hydrolysis. The purified glucoamylases were optimally active at pH 2.0 and 90 °C and have an isoelectric points (pI) between 4.5 and 4.8. Enzymatic activity was detected even at pH 1.0 and 100 °C. The glucoamylases were thermostable at elevated temperature with a half-life of 24 h at 90 °C for both P. torridus and T. acidophilum, and 20 h at 90 °C for P. oshimae. The enzyme system of T. acidophilum has a lower K m value for soluble starch (1.06 mg/ml) than the enzymes from P. oshimae and P. torridus (4.35 mg/ml and 2.5 mg/ml), respectively. Enzyme activity was not affected by Na+, Mg++, Ca++, Ni++, Zn++, Fe++, EDTA and DTT.

Archaea Glucoamylase starch thermoacidophilic Thermoplasma Picrophilus 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Alazard D & Baldensperger JF (1982) Amylolytic enzymes from Aspergillus hennebergi (A. niger group): purification and characterization of amylases from solid liquid cultures. Carbohydr. Res. 107: 231–241.CrossRefGoogle Scholar
  2. Antranikian G (1992) Microbial degradation of starch. In Windelmann G (Ed), Microbial Degradation of Natural Products, (pp 27–56) VCH Weinheim.Google Scholar
  3. Bailey JM (1988) A note on the use of dinitrosalicylic acid for determining the products of enzymatic reactions. Appl. Microbiol. Biotechnol. 29: 494–496.CrossRefGoogle Scholar
  4. Barros C, Rawlings E & Woods R (1984) Mixotrophic growth of a Thiobacillus ferrooxidans strain. Appl. Environ. Microbiol. 47: 593–595.PubMedGoogle Scholar
  5. Bender H (1981) A bacterial glucoamylase degrading cyclodextrins. Partial purification and properties of the enzyme from Flavobacterium species. Eur. J. Biochem. 115: 287–329.PubMedCrossRefGoogle Scholar
  6. Brandani de Silva W & Peralta WM Purification and characterization of a thermostable glucoamylase from Aspergillus fumigatus. Can. J. Microbiol. 44: 493-497.Google Scholar
  7. Brock TD & Gustafson J (1976) Ferric iron reduction by sulfur-and iron-oxidizing bacteria. Appl. Environ. Microbiol. 32: 567–571.PubMedGoogle Scholar
  8. Brown SH & Kelly RM (1993) Characterization of amylolytic enzymes, having both ά-1,4 and ά-1,6 hydrolyltic activity, from the thermophilic archaea Pyrococcus furiosus and Thermococcus litoralis. Appl. Environ. Microbiol. 59: 2614–2621.PubMedGoogle Scholar
  9. Campos L & Felix CR (1995) Purification and characterization of a glucoamylase from Humicola grisea. Appl. Environ. Microbiol. 61: 2436–2438.PubMedGoogle Scholar
  10. De Mot R & Verachtert H (1987) Purification and characterization of extracellular a-amylase and glucoamylase from the yeast Candida antarctica CBS6678. Eur. J. Biochem. 164: 643–654.PubMedCrossRefGoogle Scholar
  11. Fusek M, Lin XL & Tang J (1990) Enzymatic properties of thermopsin. J. Biol. Chem. 265: 1496–1501.PubMedGoogle Scholar
  12. Golovacheva S & Karavaiko G (1978) A new genus of thermophilic spore-forming bacteria, Sulfobacillus. Microbiology 47: 658–664.Google Scholar
  13. Guay R & Silver M (1975) Thiobacillus acidophilus sp. nov.: isolation and some physiological characteristics. Can. J. Microbiol. 21: 281–288.PubMedCrossRefGoogle Scholar
  14. Hallberg KB & Lindström EB (1994) Characterization of Thiobacillus caldus sp. nov., a moderately thermophilic acidophilic. Microbiology 140: 3451–3456.PubMedGoogle Scholar
  15. Heukeshoven J & Dernick R (1985) Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electophoresis 7: 103–112.CrossRefGoogle Scholar
  16. Huber G & Stetter KO (1990) Thiobacillus cuprinus sp. nov., a novel facultatively organotrophic metal-mobilizing bacterium. Appl. Environ. Microbiol. 56: 315–322.PubMedGoogle Scholar
  17. Hyun H & Zeikus J (1985) General biochemical characterization of thermostable pullulanase and glucoamylase from Clostridium thermohydrosulfuricum. Appl. Environ. Microbiol. 49: 1168–1173.PubMedGoogle Scholar
  18. James A & Lee B (1996) Characterization of glucoamylase from Lactobacillus amylovorus ATCC 33621. Biotech. Lett. 18: 1401–1406.CrossRefGoogle Scholar
  19. James A & Berger J-L, Lee B (1997) Purification of glucoamylase from Lactobacillus amylovorus ATCC 33621. Curr. Microbiol. 34: 186–191.PubMedCrossRefGoogle Scholar
  20. Jansz R, Pieris N, JeyaRaj E & De Silva N (1977) Cultivation, isolation, purification and some properties of the enzyme glucoamylase from Aspergillus niger. J. Natl. Sci. Coun. Sri Lanka 5: 59–74.Google Scholar
  21. Katkocin M, Word S & Yang S (1985) Thermostable glucoamylase and method for its production. US Patent [4,536,477].Google Scholar
  22. Kelkar S & Deshpande M (1993) Purification and charaterization of a pullulan-hydrolyzing glucoamylase from Sclerotium rolfsii. Starch 45: 361–368.Google Scholar
  23. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680–685.CrossRefGoogle Scholar
  24. Li D-C, Yang Y-J, Peng Y-L & Shen C-Y (1998) Purification and characterization of extracellular glucoamylase from the thermophilic Thermomyces lanuginosus. Mycol. Rev. 102: 568–572.CrossRefGoogle Scholar
  25. Lin XL & Tang J (1990) Purification, characterization, and gene cloning of thermopsin, a thermostable acid protease from Sulfolobus acidocaldarius. J. Biol. Chem. 265: 1490–1495.PubMedGoogle Scholar
  26. Lineweaver H & Burk D (1934) The determination for the enzyme dissociation constants. J. Am. Chem. Soc. 56: 658–666.CrossRefGoogle Scholar
  27. Lowry OH, Rosenbrough NJ, Farr AL & Randall JR (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265–275.PubMedGoogle Scholar
  28. Matzke J, Schwermann B & Bakker E (1997) Acidostable and acidophilic proteins. The example of the ά-amylase from Alicyclobacillus acidocaldarius. Comp. Biochem. Physiol. 118A: 475–479.CrossRefGoogle Scholar
  29. McCleary BV & Anderson MA (1980) Hydrolysis of ά-D-glucan and ά-D-gluco-oligosaccharides by Cladosporium resinae glucoamylase. Carbohydr. Res. 86: 77–96.PubMedCrossRefGoogle Scholar
  30. Norris R, Clark D, Owen J & Waterhouse S (1996) Characterization of Sulfobacillus acidophilus sp. nov. and other moderately thermophilic mineral-sulphide-oxidizing bacteria. Microbiology 142: 775–783.PubMedCrossRefGoogle Scholar
  31. Ordoñez RG, Morlon-Guyot J, Gasparlan S & Guyot JP (1998) Occurrence of a thermoacidophilic cell-bound exo-pectinase in Alicyclobacillus acidocaldarius. Folia Microbiol. 43: 657–660.Google Scholar
  32. Oren Aharon (1983) A thermophilic amyloglucosidase from Halobacterium sodomense, a halophilic bacterium from the Dead sea. Curr. Microbiol. 8: 225–230.CrossRefGoogle Scholar
  33. Rao VB, Sastri NVS & Rao PVS (1981) Purification and characterization of a thermostable glucoamylase from the thermophilic fungus Thermomyces lanuginosa. Biochem. J. 193: 379–387.Google Scholar
  34. Rutloff H, Friese R, Kupke G & Täufel A (1969) Differentiation and characteristics of glucoamylase isoenzymes from Endomycopsis bispora. Z. Allg. Mikrobiol 9: 39–47.Google Scholar
  35. Schleper C, Pühler G, Holz I, Janekovic D, Santarius U, Klenk HP & Zillig W (1995) Picrophilus gen. nov. fam. nov.: a novel aerobic heterotrophic, thermoacidophilic genus and family comprising archaea capable of growth around pH 0. J. Bacteriol. 177: 7050–7059.PubMedGoogle Scholar
  36. Schleper C, Pühler G, Klenk HP & Zillig W (1996) Picrophilus oshimae and Picrophilus torridus fam, nov., gen. nov., sp. nov., two species of hyperacidophilic, thermophilic, heterotophic, aerobic archaea.J. Syst. Bacteriol. 46: 814–816.CrossRefGoogle Scholar
  37. Schwermann B, Pfau K, Liliensiek B, Schleyer M, Fischer T & Bakker E (1994) Purification, properties and structural aspects of a thermoacidophilic ά-amylase from Alicyclobacillus acidocaldarius ATCC 27009. Eur. J. Biochem. 226: 981–991.PubMedCrossRefGoogle Scholar
  38. Smith PF, Langworthy TA & Smith MR (1975) Polypeptide nature of growth requirement in Yeast extract for Thermoplasma acidophilum. J. Bacteriol. 124: 884–892.PubMedGoogle Scholar
  39. Specka U, Mayer F & Antranikian G (1991) Purification and properties of a thermoactive glucoamylase from Clostridium thermosaccharolyticum. Appl. Environ. Microbiol. 57: 2317–2323.PubMedGoogle Scholar
  40. Tucker M, Grohman K & Himmel M (1984) Isolation and characterization of a glucoamylase from Saccharomyces diastaticus. Biotechnol. Bioeng. Symp. 14: 279–293.Google Scholar
  41. Vandersall AS, Cameron RG, Nairn III CJ, Yelenosky G & Wodzinski RJ (1995) Identification, charaterization, and partial purification of glucoamylase from Aspergillus niger (syn A. ficuum) NRRL 3135. Preparat. Biochem. 25: 29–55.Google Scholar
  42. Yamasaki Y, Tsuboi A & Suzuki Y (1977) Two forms of glucoamylase from Mucor rouxianus. II. Properties of the two glucoamylases. Agric. Biol. Chem. 41: 2139–2148.Google Scholar
  43. Yamashita I, Hatano T & Fukui S (1984) Subunit structure of glucoamylase of Saccharomyces diastaticus. Agric. Biol. Chem. 48: 1611–1616.Google Scholar
  44. Yamashita I, Suzuki K & Fukui S (1985) Nucleotide sequence of the extracellular glucoamylase gene STA 1 in the yeast Saccharomyces diastaticus. J. Bacteriol. 161: 567–573.PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  1. 1.Institute of Technical MicrobiologyTechnical University Hamburg-HarburgHamburgGermany

Personalised recommendations