Molecular cloning and expression of an extracellular α-amylase gene from an Antarctic deep sea psychrotolerant Pseudomonas stutzeri strain 7193

Original Paper


Psychrotolerant Pseudomonas stutzeri strain 7193 capable of producing an extracellular α-amylase was isolated from deep sea sediments of Prydz Bay, Antarctic. The 59678-Da protein (AmyP) was encoded by 1665-bp gene (amyP). The deduced amino acid sequence was identified with four regions, which are conserved in amylolytic enzymes and form a catalytic domain, and was predicted to be maltotetraose forming extracellular amylase by using the I-TASSER online server. Purification of AmyP amylases from both the recombinant of Escherichia coli Top 10 F′ and strain 7193 was conducted. Biochemical characterization revealed that the optimal amylase activity was observed at pH 9.0 and temperature 40°C. The enzymes were unstable at temperatures above 30°C, and only retain half of their highest activity after incubation at 60°C for 5 min. Thin-layer chromatography analysis of the products of the amylolytic reaction showed the presence of maltotetraose, maltotriose, maltose and glucose in the starch hydrolysate.


Deep sea sediment Extracellular α-amylase Sequencing and expression Characterization 



This work was supported by National High Technology Research and Development Program (863) 2007AA091407 and China Ocean Mineral Resources R&D Association (COMRA) Program (DYXM-115-02-2-04).


  1. Beaudry AA, Joyce GF (1992) Directed evolution of an RNA enzyme. Science 257:635–641CrossRefGoogle Scholar
  2. Bornscheuer UT, Pohl M (2001) Improved biocatalysts by directed evolution and rational protein design. Curr Opin Chem Biol 5:137–143CrossRefGoogle Scholar
  3. Cladera AM, Bennasar A, Barcelo M, Lalucat J, Garcia-Valdes E (2004) Comparative genetic diversity of Pseudomonas stutzeri genomovars, clonal structure, and phylogeny of the species. J Bacteriol 186:5239–5248CrossRefGoogle Scholar
  4. Dalby PA (2003) Optimising enzyme function by directed evolution. Curr Opin Struct Biol 13:500–505CrossRefGoogle Scholar
  5. Damager I, Numao S, Chen H, Brayer GD, Withers SG (2004) Synthesis and characterisation of novel chromogenic substrates for human pancreatic alpha-amylase. Carbohydr Res 339:1727–1737CrossRefGoogle Scholar
  6. Debashish G, Malay S, Barindra S, Joydeep M (2005) Marine enzymes. Adv Biochem Eng 96:189–218Google Scholar
  7. Dey G, Palit S, Banerjee R, Maiti BR (2002) Purification and characterization of maltooligosaccharide-forming amylase from Bacillus circulans GRS 313. J Ind Microbiol Biotechnol 28:193–200CrossRefGoogle Scholar
  8. Feller G, Gerday C (2003) Psychrophilic enzymes: hot topics in cold adaptation. Nat Rev Microbiol 1:200–208CrossRefGoogle Scholar
  9. Feller G, Lonhienne T, Deroanne C, Libioulle C, Van Beeumen J, Gerday C (1992) Purification, characterization, and nucleotide sequence of the thermolabile alpha-amylase from the Antarctic psychrotroph Alteromonas haloplanctis A23. J Biol Chem 267:5217–5221Google Scholar
  10. Fogarty WM, Bourke AC, Kelly CT, Doyle EM (1994) A constitutive maltotetraose-producing amylase from Pseudomonas sp. IMD 353. Appl Microbiol Biotechnol 42:198–203Google Scholar
  11. Fujii R, Nakagawa Y, Hiratake J, Sogabe A, Sakata K (2005) Directed evolution of Pseudomonas aeruginosa lipase for improved amide-hydrolyzing activity. Protein Eng Des Sel 18:93–101CrossRefGoogle Scholar
  12. Fujita M, Torigoe K, Nakada T, Tsusaki K, Kubota M, Sakai S, Tsujisaka Y (1989) Cloning and nucleotide sequence of the gene (amyP) for maltotetraose-forming amylase from Pseudomonas stutzeri MO-19. J Bacteriol 171:1333–1339Google Scholar
  13. Gerday C, Aittaleb M, Bentahir M, Chessa JP, Claverie P, Collins T, D’Amico S, Dumont J, Garsoux G, Georlette D, Hoyoux A, Lonhienne T, Meuwis MA, Feller G (2000) Cold-adapted enzymes: from fundamentals to biotechnology. Trends Biotechnol 18:103–107CrossRefGoogle Scholar
  14. Guzman-Maldonado H, Paredes-Lopez O (1995) Amylolytic enzymes and products derived from starch: a review. Crit Rev Food Sci Nutr 35:373–403CrossRefGoogle Scholar
  15. Hasegawa K, Kubota M, Matsuura Y (1999) Roles of catalytic residues in alpha-amylases as evidenced by the structures of the product-complexed mutants of a maltotetraose-forming amylase. Protein Eng 12:819–824CrossRefGoogle Scholar
  16. Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280(Pt 2):309–316Google Scholar
  17. Henrissat B, Bairoch A (1993) New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 293(Pt 3):781–788Google Scholar
  18. Kim TU, Gu BG, Jeong JY, Byun SM, Shin YC (1995) Purification and characterization of a maltotetraose-forming alkaline (alpha)-amylase from an alkalophilic Bacillus strain, GM8901. Appl Environ Microbiol 61:3105–3112Google Scholar
  19. Kobayashi H, Takaki Y, Kobata K, Takami H, Inoue A (1998) Characterization of alpha-maltotetraohydrolase produced by Pseudomonas sp. MS300 isolated from the deepest site of the mariana trench. Extremophiles 2:401–407CrossRefGoogle Scholar
  20. Kuriki T, Imanaka T (1999) The concept of the alpha-amylase family: structural similarity and common catalytic mechanism. J Biosci Bioeng 87:557–565CrossRefGoogle Scholar
  21. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  22. MacGregor EA, Janecek S, Svensson B (2001) Relationship of sequence and structure to specificity in the alpha-amylase family of enzymes. Biochim Biophys Acta 1546:1–20CrossRefGoogle Scholar
  23. Matsuura Y, Kusunoki M, Harada W, Kakudo M (1984) Structure and possible catalytic residues of Taka-amylase A. J Biochem 95:697–702Google Scholar
  24. Nagarajan DR, Rajagopalan G, Krishnan C (2006) Purification and characterization of a maltooligosaccharide-forming alpha-amylase from a new Bacillus subtilis KCC103. Appl Microbiol Biotechnol 73:591–597CrossRefGoogle Scholar
  25. Nakajima R, Imanaka T, Aiba S (1986) Comparison of amino acid sequences of eleven different α-amylases. Appl Microbiol Biotechnol 23:355–360CrossRefGoogle Scholar
  26. Ohdan K, Kuriki T, Kaneko H, Shimada J, Takada T, Fujimoto Z, Mizuno H, Okada S (1999) Characteristics of two forms of alpha-amylases and structural implication. Appl Environ Microbiol 65:4652–4658Google Scholar
  27. Palleroni N (1984) Bergey’s manual of systematic bacteriology. In: Krieg NR, Holt JG (eds) Genus I Pseudomonas, 1st edn. Williams and Wilkins, Baltimore/London, pp 141–199Google Scholar
  28. Roy A, Kucukural A, Zhang Y (2010) I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5:725–738CrossRefGoogle Scholar
  29. Sakano Y, Kashiwagi Y, Kobayashi T (1982) Purification and properties of an exo-oa-amylase from Pseudomonas stutzeri. Agric Biol Chem Tokyo 46:639–646Google Scholar
  30. Siddiqui KS, Cavicchioli R (2006) Cold-adapted enzymes. Annu Rev Biochem 75:403–433CrossRefGoogle Scholar
  31. Siezen RJ, Wilson G (2009) Genomics of deep-sea and sub-seafloor microbes. Microbial Biotechnol 2:157–163CrossRefGoogle Scholar
  32. Sikorski J, Lalucat J, Wackernagel W (2005) Genomovars 11 to 18 of Pseudomonas stutzeri, identified among isolates from soil and marine sediment. Int J Syst Evol Microbiol 55:1767–1770CrossRefGoogle Scholar
  33. Simonato F, Campanaro S, Lauro FM, Vezzi A, D’Angelo M, Vitulo N, Valle G, Bartlett DH (2006) Piezophilic adaptation: a genomic point of view. J Biotechnol 126:11–25CrossRefGoogle Scholar
  34. van der Maarel MJ, van der Veen B, Uitdehaag JC, Leemhuis H, Dijkhuizen L (2002) Properties and applications of starch-converting enzymes of the alpha-amylase family. J Biotechnol 94:137–155CrossRefGoogle Scholar
  35. Woo GJ, McCord JD (1991) Maltotetraose production using Pseudomonas stutzeri exo-alpha-amylase in a membrane recycle bioreactor. J Food Sci 56:1019–1023, 1033CrossRefGoogle Scholar
  36. Xu M, Wang P, Wang F, Xiao X (2005) Microbial diversity at a deep-sea station of the Pacific nodule province. Biodivers Conserv 14:3363–3380CrossRefGoogle Scholar
  37. Yan Y, Yang J, Dou Y, Chen M, Ping S, Peng J, Lu W, Zhang W, Yao Z, Li H, Liu W, He S, Geng L, Zhang X, Yang F, Yu H, Zhan Y, Li D, Lin Z, Wang Y, Elmerich C, Lin M, Jin Q (2008) Nitrogen fixation island and rhizosphere competence traits in the genome of root-associated Pseudomonas stutzeri A1501. Proc Natl Acad Sci USA 105:7564–7569CrossRefGoogle Scholar
  38. Yu Y, Li H, Zeng Y, Chen B (2009) Extracellular enzymes of cold-adapted bacteria from Arctic sea ice, Canada Basin. Polar Biol 32:1539–1547CrossRefGoogle Scholar
  39. Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9:40CrossRefGoogle Scholar
  40. Zhang Y (2009) I-TASSER: fully automated protein structure prediction in CASP8. Proteins 77(S9):100–113CrossRefGoogle Scholar
  41. Zhang JW, Zeng RY (2006) Cloning, expression and characterization of the cold active lipase (Lip3) from metagenomic DNA of an Antarctic deep sea sediment. Prog Biochem Biophys 33:1207–1214Google Scholar
  42. Zhang JW, Zeng RY (2007) Psychrotolerant amylolytic bacteria from deep sea sediment of Prydz Bay, Antarctic: diversity and characterization of amylases. World J Microbiol Biotechnol 23:1551–1557CrossRefGoogle Scholar
  43. Zhang JW, Zeng RY (2008a) Molecular cloning and expression of a cold-adapted lipase gene from an Antarctic deep sea psychrotolerant bacterium Pseudomonas sp. 7323. Mar Biotechnol 10:612–621CrossRefGoogle Scholar
  44. Zhang JW, Zeng RY (2008b) Purification and characterization of a cold-adapted alpha-amylase produced by Nocardiopsis sp. 7326 isolated from Prydz Bay, Antarctic. Mar Biotechnol 10:75–82CrossRefGoogle Scholar
  45. Zhang JW, Lin S, Zeng RY (2007) Cloning, expression, and characterization of a cold-adapted lipase gene from an Antarctic deep-sea psychrotolerant bacterium, Psychrobacter sp. 7195. J Microbiol Biotechnol 17:604–610Google Scholar
  46. Zhou JH, Baba T, Takano T, Kobayashi S, Arai Y (1989) Nucleotide sequence of the maltotetraohydrolase gene from Pseudomonas saccharophila. FEBS Lett 255:37–41CrossRefGoogle Scholar
  47. Zhu J, Che F, Yan Z, Liang G, Zhang S (1997) Studies on multiple forms of maltotetraose-forming amylase from Alcaligenes sp. Chin J Biotechnol 13:25–30Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  1. 1.Key Laboratory of Marine Biogenetic ResourcesState Oceanic AdministrationXiamenPeople’s Republic of China
  2. 2.Third Institute of Oceanography, State Oceanic AdministrationXiamenPeople’s Republic of China
  3. 3.Dove Marine Laboratory, School of Marine Science and TechnologyNewcastle University Upon TyneNewcastle Upon TyneUK

Personalised recommendations