Applied Microbiology and Biotechnology

, Volume 86, Issue 1, pp 131–141 | Cite as

A novel α-amylase from the cyanobacterium Nostoc sp. PCC 7119

  • Francisco M. Reyes-Sosa
  • Fernando P. Molina-Heredia
  • Miguel A. De la Rosa
Biotechnologically Relevant Enzymes and Proteins


Little information is yet available on the α-amylases of cyanobacteria. Here, the presence of an α-amylase in the cyanobacterium Nostoc sp. PCC 7119 is first demonstrated. A gene (amy1) encoding a cytoplasmic α-amylase (Amy1) protein has been identified, cloned, and overexpressed in Escherichia coli cells. The recombinant protein is a 56.7-kDa monomer, which has been purified to electrophoretic homogeneity by affinity chromatography. The substrate specificity and end product analyses confirm that it is a calcium-dependent α-amylase enzyme, which exhibits its maximum activity at 31°C and at pH between 6.5 and 7.5. The Amy1 protein breaks down mainly starch, is also able to cleave glycogen and dextrin, and exhibits no activity against xylan or pullulan. So the enzyme cannot efficiently attack the maltodextrins with degrees of polymerization below that of maltooctaose. Maltotriose, maltose, and maltotetraose are the major products of the enzymatic reaction with starch as substrate. The enzyme shows a very high turnover number against soluble potato starch (3,420 ± 270 s−1), as compared with other α-amylases reported in the literature. The high catalytic efficiency and relatively low optimum temperature of the Nostoc Amy1 protein make this previously unexplored group of cyanobacterial enzymes of great interest for further physiological studies and industrial applications.


Affinity chromatography α-amylase Cyanobacteria Endoglycosyl hydrolase Heterologous expression Nostoc Starch 



This work was supported by the Andalusian Government (CVI-387). We are grateful to Drs. Manuel Hervás, Marika Lindahl, and José Antonio Navarro for critically reading the manuscript. The technical support of Pilar Alcántara is gratefully acknowledged.


  1. Adolph KW, Haselkorn R (1971) Isolation and characterization of a virus infecting the blue-green alga Nostoc muscorum. Virology 46:200–208CrossRefGoogle Scholar
  2. Bernfeld P (1955) Amylases, α and β. Method Enzymol 1:149–158CrossRefGoogle Scholar
  3. Bolen DW, Santoro MM (1988) Unfolding free energy changes determined by the linear extrapolation method. 2. Incorporation of delta G degrees N-U values in a thermodynamic cycle. Biochemistry 27:8069–8074CrossRefGoogle Scholar
  4. Bordoli L, Kiefer F, Arnold K, Benkert P, Battey J, Schwede T (2009) Protein structure homology modeling using SWISS-MODEL workspace. Nat Protoc 4:1–13CrossRefGoogle Scholar
  5. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  6. Casadaban MJ, Cohen SN (1980) Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol 138:179–207CrossRefGoogle Scholar
  7. Curatti L, Porchia AC, Herrera-Estrella L, Salerno GL (2000) A prokaryotic sucrose synthase gene (susA) isolated from a filamentous nitrogen-fixing cyanobacterium encodes a protein similar to those of plants. Planta 211:729–735CrossRefGoogle Scholar
  8. Davies GJ, Gloster TM, Henrissat B (2005) Recent structural insights into the expanding world of carbohydrate-active enzymes. Curr Opin Struct Biol 15:637–645CrossRefGoogle Scholar
  9. De Mot R, Verachtert H (1987) Purification and characterization of extracellular alpha-amylase and glucoamylase from the yeast Candida antarctica CBS 6678. Eur J Biochem 164:643–654CrossRefGoogle Scholar
  10. Dey S, Agarwal SO (1999) Characterization of a thermostable alpha-amylase from a thermophilic Streptomyces megasporus strain SD12. Indian J Biochem Biophys 36:150–157Google Scholar
  11. Emanuelsson O, Brunak S, von Heijne G, Nielsen H (2007) Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2:953–971CrossRefGoogle Scholar
  12. Georlette D, Blaise V, Collins T, D'Amico S, Gratia E, Hoyoux A, Marx JC, Sonan G, Feller G, Gerday C (2004) Some like it cold: biocatalysis at low temperatures. FEMS Microbiol Rev 28:25–42CrossRefGoogle Scholar
  13. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95–98Google Scholar
  14. Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280(Pt 2):309–316Google Scholar
  15. 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
  16. Henrissat B, Bairoch A (1996) Updating the sequence-based classification of glycosyl hydrolases. Biochem J 316(Pt 2):695–696Google Scholar
  17. Janeček S (1997) alpha-Amylase family: molecular biology and evolution. Prog Biophys Mol Biol 67:67–97CrossRefGoogle Scholar
  18. Janeček S, Leveque E, Belarbi A, Haye B (1999) Close evolutionary relatedness of alpha-amylases from Archaea and plants. J Mol Evol 48:421–642CrossRefGoogle Scholar
  19. Kagawa M, Fujimoto Z, Momma M, Takase K, Mizuno H (2003) Crystal structure of Bacillus subtilis alpha-amylase in complex with acarbose. J Bacteriol 185:6981–6984CrossRefGoogle Scholar
  20. Kandra L (2003) Alpha-amylases of medical and industrial importance. J Mol Struc-Teochem 666–7:487–498CrossRefGoogle Scholar
  21. Kaneko T, Nakamura Y, Wolk CP, Kuritz T, Sasamoto S, Watanabe A, Iriguchi M, Ishikawa A, Kawashima K, Kimura T, Kishida Y, Kohara M, Matsumoto M, Matsuno A, Muraki A, Nakazaki N, Shimpo S, Sugimoto M, Takazawa M, Yamada M, Yasuda M, Tabata S (2001) Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120. DNA Res 8:205–213 227-253CrossRefGoogle Scholar
  22. Kurland CG, Dong H (1996) Bacterial growth inhibition by overproduction of protein. Mol Microbiol 21:1–4CrossRefGoogle Scholar
  23. MacGregor EA, Janeček S, Svensson B (2001) Relationship of sequence and structure to specificity in the alpha-amylase family of enzymes. Biochim Biophys Acta 1546:1–20Google Scholar
  24. Machius M, Wiegand G, Huber R (1995) Crystal structure of calcium-depleted Bacillus licheniformis alpha-amylase at 2.2 A resolution. J Mol Biol 246:545–559CrossRefGoogle Scholar
  25. Machovic M, Janeček S (2006) Starch-binding domains in the post-genome era. Cell Mol Life Sci 63:2710–2724CrossRefGoogle Scholar
  26. McCleary B, McNally M, Monaghan D, Mugford D (2002) Measurement of α-amylase activity in white wheat flour, milled malt, and microbial enzyme preparations, using the Ceralpha assay: collaborative study. J AOAC International 85:1096–1102Google Scholar
  27. Nishihara K, Kanemori M, Kitagawa M, Yanagi H, Yura T (1998) Chaperone coexpression plasmids: differential and synergistic roles of DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an allergen of Japanese cedar pollen, Cryj2, in Escherichia coli. Appl Environ Microbiol 64:1694–1699Google Scholar
  28. Ochman H, Lawrence JG, Groisman EA (2000) Lateral gene transfer and the nature of bacterial innovation. Nature 405:299–304CrossRefGoogle Scholar
  29. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612CrossRefGoogle Scholar
  30. Pidcock E, Moore GR (2001) Structural characteristics of protein binding sites for calcium and lanthanide ions. J Biol Inorg Chem 6:479–489CrossRefGoogle Scholar
  31. Pujadas G, Palau J (2001) Evolution of alpha-amylases: architectural features and key residues in the stabilization of the (beta/alpha)(8) scaffold. Mol Biol Evol 18:38–54Google Scholar
  32. Robertson EF, Dannelly HK, Malloy PJ, Reeves HC (1987) Rapid isoelectric focusing in a vertical polyacrylamide minigel system. Anal Biochem 167:290–294CrossRefGoogle Scholar
  33. Santoro MM, Bolen DW (1988) Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. Biochemistry 27:8063–8068CrossRefGoogle Scholar
  34. Sato N (2002) Comparative analysis of the genomes of cyanobacteria and plants. Genome Inform 13:173–182Google Scholar
  35. Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 31:3381–3385CrossRefGoogle Scholar
  36. Sinha A, Yadav S, Ahmad R, Ahmad F (2000) A possible origin of differences between calorimetric and equilibrium estimates of stability parameters of proteins. Biochem J 345(Pt 3):711–717CrossRefGoogle Scholar
  37. Somers WAC, Koenen PHM, Rozie HJ, Visser J, Rombouts FM, van't Riet K (1995) Isolation of -amylase on crosslinked starch. Enzyme Microb Tech 17:56–62CrossRefGoogle Scholar
  38. Stam MR, Danchin EG, Rancurel C, Coutinho PM, Henrissat B (2006) Dividing the large glycoside hydrolase family 13 into subfamilies: towards improved functional annotations of alpha-amylase-related proteins. Protein Eng Des Sel 19:555–562CrossRefGoogle Scholar
  39. Strobl S, Maskos K, Betz M, Wiegand G, Huber R, Gomis-Ruth FX, Glockshuber R (1998) Crystal structure of yellow meal worm alpha-amylase at 1.64 A resolution. J Mol Biol 278:617–628CrossRefGoogle Scholar
  40. Suvd D, Fujimoto Z, Takase K, Matsumura M, Mizuno H (2001) Crystal structure of Bacillus stearothermophilus alpha-amylase: possible factors determining the thermostability. J Biochem 129:461–468Google Scholar
  41. Svensson B (1994) Protein engineering in the alpha-amylase family: catalytic mechanism, substrate specificity, and stability. Plant Mol Biol 25:141–157CrossRefGoogle Scholar
  42. Takase K (1993) Effect of mutation of an amino acid residue near the catalytic site on the activity of Bacillus stearothermophilus alpha-amylase. Eur J Biochem 211:899–902CrossRefGoogle Scholar
  43. Teale FW (1960) The ultraviolet fluorescence of proteins in neutral solution. Biochem J 76:381–388Google Scholar
  44. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680CrossRefGoogle Scholar
  45. Vihinen M, Mäntsälä P (1989) Microbial amylolytic enzymes. Crit Rev Biochem Mol Biol 24:329–418CrossRefGoogle Scholar
  46. Weber M, Foglietti M-J, Percheron F (1976) Purification d´α-amylases par chromatographie d´affinité sur amidon réticulé. Biochimie 58:1299–1302CrossRefGoogle Scholar
  47. Wing-Ming C, Hsueh-Mei C, Hso-Freng Y, Jei-Fu S, Tan-Chi H (1994) The aerobic nitrogen-fixing Synechococcus RF-1 containing uncommon polyglucan granules and multiple forms of α-amylase. Curr Microbiol 29:201–205CrossRefGoogle Scholar
  48. Yang SJ, Lee HS, Park CS, Kim YR, Moon TW, Park KH (2004) Enzymatic analysis of an amylolytic enzyme from the hyperthermophilic archaeon Pyrococcus furiosus reveals its novel catalytic properties as both an alpha-amylase and a cyclodextrin-hydrolyzing enzyme. Appl Environ Microbiol 70:5988–5995CrossRefGoogle Scholar
  49. Yoon S-H, Robyt JF (2005) Activation and stabilization of 10 starch-degrading enzymes by Triton X-100, polyethylene glycols, and polyvinyl alcohols. Enzyme Microb Tech 37:556–562CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Francisco M. Reyes-Sosa
    • 1
  • Fernando P. Molina-Heredia
    • 1
  • Miguel A. De la Rosa
    • 1
  1. 1.Instituto de Bioquímica Vegetal y FotosíntesisUniversidad de Sevilla & CSIC, Centro de Investigaciones Científicas Isla de la CartujaSevillaSpain

Personalised recommendations