, Volume 22, Issue 2, pp 189–202 | Cite as

Optimization of the production of an extracellular and thermostable amylolytic enzyme by Thermus thermophilus HB8 and basic characterization

  • Mounia AkassouEmail author
  • Denis Groleau
Original Paper


The objective of this study was to determine the potential of Thermus thermophilus HB8 for accumulating a high level of extracellular, thermostable amylolytic enzyme. Initial production tests indicated clearly that only very low levels of amylolytic activity could be detected, solely from cells after extraction using the mild, non-ionic detergent Triton X-100. A sequential optimization strategy, based on statistical designs, was used to enhance greatly the production of extracellular amylolytic activity to achieve industrially attractive enzyme titers. Focus was placed on the optimal level of initial biomass concentration, culture medium composition and temperature for maximizing extracellular amylolytic enzyme accumulation. Empirical models were then developed describing the effects of the experimental parameters and their interactions on extracellular amylolytic enzyme production. Following such efforts, extracellular amylolytic enzyme accumulation was increased more than 70-fold, with enzyme titers in the 76 U/mL range. The crude extracellular enzyme was thereafter partially characterized. The optimal temperature and pH values were found to be 80 °C and 9.0, respectively. 100% of the initial enzyme activity could be recovered after incubation for 24 h at 80 °C, therefore, proving the very high thermostability of the enzyme preparation.


Thermus thermophilus HB8 Thermostable amylolytic enzyme Factorial design Central composite design Fermentation 



This research was financially supported by a Canada Research Chair grant (to D. Groleau) managed by the Natural Sciences and Engineering Council of Canada (NSERC). The authors wish to thank Iulian-Zoltan Bobescu, Pisey Neang, Geneviève Fugère and Mélodie Loriaux, postdoctoral fellows and students at the University of Sherbrooke for their technical and scientific support.


  1. Acer Ö, Matpan Bekler F, Pirinççioğlu H et al (2016) Purification and characterization of thermostable and detergent-stable α- amylase from Anoxybacillus sp. AH1. Food Technol Biotechnol 54:70–77. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Arikan B (2008) Highly thermostable, thermophilic, alkaline, SDS and chelator resistant amylase from a thermophilic Bacillus sp. isolate A3-15. Bioresour Technol 99:3071–3076. CrossRefPubMedGoogle Scholar
  3. Berger JL, Lee BH, Lacroix C (1995) Identification of new enzyme activities of several strains of Thermus species. Appl Microbiol Biotechnol 44:81–87. CrossRefPubMedGoogle Scholar
  4. Cava F, Hidalgo A, Berenguer J (2009) Thermus thermophilus as biological model. Extremophiles 13:213–231. CrossRefPubMedGoogle Scholar
  5. Crolla A, Kennedy KJ (2001) Optimization of citric acid production from Candida lipolytica Y-1095 using n-paraffin. J Biotechnol 89:27–40. CrossRefPubMedGoogle Scholar
  6. de Souza PM, de Magalhães P (2010) Application of microbial α-amylase in industry—a review. Brazilian J Microbiol 41:850–861CrossRefGoogle Scholar
  7. Doman-Pytka M, Bardowski J (2004) Pullulan degrading enzymes of bacterial origin. Crit Rev Microbiol 30:107–121. CrossRefPubMedGoogle Scholar
  8. Elleuche S, Schröder C, Sahm K, Antranikian G (2014) Extremozymes—biocatalysts with unique properties from extremophilic microorganisms. Curr Opin Biotechnol 29:116–123. CrossRefPubMedGoogle Scholar
  9. Farhat-Khemakhem A, Ali MB, Boukhris I et al (2013) Crucial role of Pro 257 in the thermostability of Bacillus phytases: biochemical and structural investigation. Int J Biol Macromol 54:9–15CrossRefPubMedGoogle Scholar
  10. Farris PL (2009) Economic growth and organisation of the US corn starch industry. In: BeMiller J, Whistler R (eds) Starch: chemistry and technology, 3rd edn. Academic Press, New York, pp 11–21CrossRefGoogle Scholar
  11. Fujiwara S (2002) Extremophiles. J Biosci Bioeng 94:518CrossRefPubMedGoogle Scholar
  12. Gomes I, Gomes J, Steiner W (2003) Highly thermostable amylase and pullulanase of the extreme thermophilic eubacterium Rhodothermus marinus: production and partial characterization. Bioresour Technol 90:207–214. CrossRefPubMedGoogle Scholar
  13. Gupta GN, Srivastava S, Khare SK, Prakash V (2014) Extremophiles: an overview of microorganism from extreme environment. Int J Agric Environ Biotechnol 7:371CrossRefGoogle Scholar
  14. Helenius A, Simons K (1975) Solubilization of membranes by detergents. BBA Rev Biomembr 415:29–79. Google Scholar
  15. Horomi K, Masatake M, Kanaya K, Matsumoto T (1975) The pH Jump Study of enzyme proteins I. Liquefying α-amylase from Bacillus subtilis. Biochem J 77:957–963CrossRefGoogle Scholar
  16. Kolawole AO, Ajele JO, Sirdeshmukh R (2011) Purification and characterization of alkaline-stable β-amylase in malted African finger millet (Eleusine coracana) seed. Process Biochem 46:2178–2186. CrossRefGoogle Scholar
  17. Kumar L, Awasthi G, Singh B (2011) Extremophiles: a novel source of industrially important enzymes. Biotechnology 10:121–135CrossRefGoogle Scholar
  18. Maalej H, Hmidet N, Ghorbel-Bellaaj O, Nasri M (2013) Purification and biochemical characterization of a detergent stable α-amylase from Pseudomonas stutzeri AS22. Biotechnol Bioprocess Eng 18:878–887CrossRefGoogle Scholar
  19. Miller PS, Blum PH (2010) Extremophile-inspired strategies for enzymatic biomass saccharification. Environ Technol 31:1005–1015. CrossRefPubMedGoogle Scholar
  20. Montgomery CD (2012) Design and analysis of experiments, 8th edn. Wiley, New YorkGoogle Scholar
  21. Morozkina EV, Slutskaya ES, Fedorova TV et al (2010) Extremophilic microorganisms: biochemical adaptation and biotechnological application (review). Appl Biochem Microbiol 46:1–14. CrossRefGoogle Scholar
  22. Nielsen JE, Borchert TV, Vriend G (2001) The determinants of α-amylase pH-activity profiles. Protein Eng 14:505–512. CrossRefPubMedGoogle Scholar
  23. Pantazaki AA, Papaneophytou CP, Lambropoulou DA (2011) Simultaneous polyhydroxyalkanoates and rhamnolipids production by Thermus thermophilus HB8. AMB Express 1:1–17. CrossRefGoogle Scholar
  24. Papaneophytou CP, Pantazaki AA, Kyriakidis DA (2009) An extracellular polyhydroxybutyrate depolymerase in Thermus thermophilus HB8. Appl Microbiol Biotechnol 83:659–668. CrossRefPubMedGoogle Scholar
  25. Plant AR, Morgan HW, Daniel RM (1986) A highly stable pullulanase from Thermus aquaticus YT-1. Enzyme Microb Technol 8:668–672. CrossRefGoogle Scholar
  26. Schiraldi C, De Rosa M (2002) The production of biocatalysts and biomolecules from extremophiles. Trends Biotechnol 20:515–521CrossRefPubMedGoogle Scholar
  27. Shaw J-F, Lin F-P, Chen S-C, Chen H-C (1995) Purification and properties of an extracellular α-amylase from Thermus sp. Bull Acad Sin 36:195–200Google Scholar
  28. Swarup A, Lu J, DeWoody KC, Antoniewicz MR (2014) Metabolic network reconstruction, growth characterization and 13C-metabolic flux analysis of the extremophile Thermus thermophilus HB8. Metab Eng 24:173. CrossRefPubMedGoogle Scholar
  29. Takami H, Horikoshi K (2000) Analysis of the genome of an alkaliphilic Bacillus strain from an industrial point of view. Extremophiles 4:99–108. CrossRefPubMedGoogle Scholar
  30. Tomiyasu K, Yato K, Yasuda M et al (2001) Cloning and nucleotide sequence of the pullulanase gene of Thermus thermophilus HB8 and production of the enzyme in Escherichia coli. Biosci Biotechnol Biochem 65:2090–2094CrossRefPubMedGoogle Scholar
  31. van den Burg B (2003) Extremophiles as a source for novel enzymes. Curr Opin Microbiol 6:213–218. CrossRefPubMedGoogle Scholar
  32. Varnavskaia OV, Illarionova NG, Selezneva AASG (1978) Effect of pH on the conformation properties and enzyme activity of α-amylase from Aspergillus terricola. Prikl Biokhim Mikrobiol 14:866–870PubMedGoogle Scholar
  33. Wu H, Yu X, Chen L, Wu G (2014) Cloning, overexpression and characterization of a thermostable pullulanase from Thermus thermophilus HB27. Protein Expr Purif 95:22–27. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Japan KK, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Department of Chemical Engineering and Biotechnological Engineering, Faculty of EngineeringUniversity of SherbrookeSherbrookeCanada

Personalised recommendations