Annals of Microbiology

, Volume 60, Issue 3, pp 557–563 | Cite as

Isolation of a novel cold-adapted amylase-producing bacterium and study of its enzyme production conditions

  • Ming Sheng Lu
  • Yaowei Fang
  • Huangzhong Li
  • Hongfei Liu
  • Shujun Wang
Original Article


A novel cold-adapted amylase-producing bacterium was isolated from seawater collected from Gaogong island of Jiangsu Province, China. The isolate was identified based on its phenotypes, biochemical test, and 16 S rRNA gene sequence as Pseudoalteromonas arctica and named GS230. The optimal activity temperature of isolate GS230 amylase was 30°C, and the activity decreased dramatically at temperatures above 40°C. Compared with the amylase from mesophiles and thermophiles, the cold-adapted amylase showed higher enzyme activity at low temperature and a greater degree of heat sensitivity at temperatures higher than 40°C. Ca2+ showed a significant effect on maintaining the activity of the enzyme. The α-amylase could have important applications in the food industry. A number of factors affecting the production of the extracellular amylase were investigated. Soluble starch and beef extract were the most promising carbon and nitrogen sources, respectively. When the strain was cultured at 20°C, pH 8.0 for 24 h, the amylase activity peaked at 780.4 U/ml.


Screening Identification Cold-adapted amylase Pseudoalteromonas arctica Production 



This work was financially supported by National Natural Science Foundation of China (40746030), Natural Science Foundation of the Science and Technology Department of Jiangshu (BE200830094), Natural Science Foundation of the Science and Technology Department of Lianyungang City (CG0714), Natural Science Foundation of the Jiangsu Higher Education Institutions of China (08KJB550001).


  1. Aghajari N, Feller G, Gerday C, Haser R (1998a) Structures of the psychrophilic Alteromonas haloplanctis α-amylase give insights into cold adaptation at a molecular level. Structure 6:1503–1516CrossRefPubMedGoogle Scholar
  2. Aghajari N, Feller G, Gerday C, Haser R (1998b) Crystal structures of the psychrophilic a-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor. Protein Sci 7:564–572CrossRefPubMedGoogle Scholar
  3. Amico SD, Sohier JS, Feller G (2006) Kinetics and energetics of ligand binding determined by microcalorimetry: insights into active site mobility in a psychrophilic alpha-amylase. J Mol Biol 358:1296–1304CrossRefPubMedGoogle Scholar
  4. Ballschmiter M, Futterer O, Liebl W (2006) Identification and characterization of a novel intracellular alkaline alpha-amylase from the hyperthermophilic bacterium Thermotoga maritima MSB8. Appl Environ Microbiol 72:2206–2211CrossRefPubMedGoogle Scholar
  5. Chessa JP, Feller G, Gerday C (1999) Purification and characterization of the heat-labile a-amylase secreted by the psychrophilic bacterium TAC 240B. Can J Microbiol 45:452–457CrossRefPubMedGoogle Scholar
  6. Chung YC, Kobayashi T, Kanai H, Akiba T, Kudo T (1995) Purification and properties of extracellular amylase from the hyperthermophilic archaeon Thermococcus profundus DT5432. Appl Environ Microbiol 61:1502–1506PubMedGoogle Scholar
  7. Cusano AM, Parrilli E, Duilio A, Sannia G, Marino G, Tutino ML (2006) Secretion of psychrophilic a-amylase deletion mutants in Pseudoalteromonas haloplanktis TAC125. FEMS Microbiol Lett 258:67–71CrossRefPubMedGoogle Scholar
  8. Declerck N, Machius M, Joyet P, Wiegand G, Huber R, Gaillardin C (2003) Hyperthermostabilization of Bacillus licheniformis alpha-amylase and modulation of its stability over a 50 degrees C temperature range. Protein Eng 16:287–293CrossRefPubMedGoogle Scholar
  9. Feller G, Lonhienne T, Deroanne C, Libioulle CJ, Beeumen V, Gerday C (1992) Purification, characterization, and nucleotide sequence of the thermolabile α-amylase from the Antarctic psychrotroph Alteromonas haloplanctis A23. J Biol Chem 267:5217–5221PubMedGoogle Scholar
  10. Feller G, Payan F, Theys F, Qian M, Haser R, Gerday C (1994) Stability and structural analysis of α-amylase from the Antarctic psychrophile Alteromonas haloplanctis A23. Eur J Biochem 222:441–447CrossRefPubMedGoogle Scholar
  11. Gerday C, Aittaleb M, Bentahir M, Chessa JP, Claverie P, Collins T, Amico SD, Dumont J, Garsoux G, Georlette D, Hoyoux A, Lonhienne T, Meuwis MA, Feller G (2000) Cold-adapted enzymes: from fundamentals to biotechnology. Trends Biotechnol 18:103–107CrossRefPubMedGoogle Scholar
  12. Groudieva T, Kambourova M, Yusef H, Royter M, Grote R, Trinks H, Antranikian G (2004) Diversity and cold-active hydrolytic enzymes of culturable bacteria associated with Arctic sea ice, Spitzbergen. Extremophiles 8:475–488CrossRefPubMedGoogle Scholar
  13. Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B (2003) Microbial α-amylases: a biotechnological perspective. Process Biochem 381:599–1616Google Scholar
  14. Hagihara H, Igarashi K, Hayashi Y, Endo K, Ikawa-Kitayama K, Ozaki K, Kawai S (2001) Novel alpha-amylase that is highly resistant to chelating reagents and chemical oxidants from the alkaliphilic Bacillus isolate KSM-K38. Appl Environ Microbiol 67:1744–1750CrossRefPubMedGoogle Scholar
  15. Hmidet N, Maalej H, Haddar A, Nasri M (2010) A novel α-amylase from Bacillus mojavensis A21: purification and biochemical characterization. Appl Biochem Biotechnol. doi: 10.1007/s12010-009-8902-7 Google Scholar
  16. Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST (1994) Bergey’s manual of determinative bacteriology, 9th edn. Williams & Wilkins, BaltimoreGoogle Scholar
  17. Jinwei Z, Runying Z (2008) Purification and characterization of a cold-adapted α-amylase produced by Nocardiopsis sp. 7326 isolated from Prydz Bay, Antarctic. Mar Biotechnol 10:75–82CrossRefGoogle Scholar
  18. Lee S, Mouri Y, Minoda M, Oneda H, Inouye K (2006a) Comparison of the wild-type α-amylase and its variant enzymes of Bacillus amyloliquefaciens in their activity and thermal stability, and insights into engineering the thermal stability of Bacillus α-amylase. J Biochem 139:1007–1101CrossRefPubMedGoogle Scholar
  19. Lee S, Oneda H, Minoda M, Tanaka A, Inouye K (2006b) Comparison of starch hydrolysis activity and thermal stability of two Bacillus licheniformis α-amylase and insights into engineering α-amylase variants active at acidic conditions. J Biochem 139:997–1005CrossRefPubMedGoogle Scholar
  20. Macgregor EA, Svensson B (1989) A super-secondary structure predicted to be common to several α-1, 4-D-glucan-cleaving enzymes. Biochem J 259:145–152PubMedGoogle Scholar
  21. Marshall CJ (1997) Cold-adapted enzymes. Trends Biotechnol 15:359–364CrossRefPubMedGoogle Scholar
  22. Pandey A, Nigam P, Soccol CR, Soccol VT, Singh D, Mohan R (2000) Advances in microbial amylases. Biotechnol Appl Biochem 31:135–152CrossRefPubMedGoogle Scholar
  23. Siddiqui KS, Poljak A, Guilhaus M, Francisci DD, Curmi PM, Feller G, Amico SD, Gerday C, Uversky VN, Cavicchioli R (2006) Role of lysine versus arginine in enzyme cold-adaptation: modifying lysine to homo-arginine stabilizes the cold-adapted alpha-amylase from Pseudoalteramonas haloplanktis. Proteins 64:486–501CrossRefPubMedGoogle Scholar
  24. Srimathi S, Jayaraman G, Feller G, Danielsson B, Narayanan PR (2007) Intrinsic halotolerance of the psychrophilic a-amylase from Pseudoalteromonas haloplanktis. Extremophiles 11:505–515CrossRefPubMedGoogle Scholar
  25. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence weighing, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680CrossRefPubMedGoogle Scholar
  26. Vallee BL, Stein EA, Sumerwell WN, Fischer EH (1959) Metal content of α-amylase of various origins. J Biol Chem 234:2901–2905PubMedGoogle Scholar

Copyright information

© Springer-Verlag and the University of Milan 2010

Authors and Affiliations

  • Ming Sheng Lu
    • 1
  • Yaowei Fang
    • 1
  • Huangzhong Li
    • 2
  • Hongfei Liu
    • 1
    • 2
  • Shujun Wang
    • 1
  1. 1.School of Marine Science and TechnologyHuaihai Institute of TechnologyLianyungangChina
  2. 2.School of BioengineeringJiangnan UniversityWuxiChina

Personalised recommendations