Applied Microbiology and Biotechnology

, Volume 89, Issue 3, pp 673–684 | Cite as

Salt-dependent thermo-reversible α-amylase: cloning and characterization of halophilic α-amylase from moderately halophilic bacterium, Kocuria varians

  • Rui Yamaguchi
  • Hiroko Tokunaga
  • Matsujiro Ishibashi
  • Tsutomu Arakawa
  • Masao Tokunaga
Biotechnologically Relevant Enzymes and Proteins

Abstract

A moderately halophilic bacterium, Kocuria varians, was found to produce active α-amylase (K. varians α-amylase (KVA)). We have observed at least six different forms of α-amylase secreted by this bacterium into the culture medium. Characterization of these KVA forms and cloning of the corresponding gene revealed that KVA comprises pre-pro-precursor form of α-amylase catalytic domain followed by the tandem repeats, which show high similarity to each other and to the starch binding domain (SBD) of other α-amylases. The observed six forms were most likely derived by various processing of the protein product. Recombinant KVA protein was successfully expressed in Escherichia coli as a fusion protein and was purified with affinity chromatography after cleavage from fusion partner. The highly acidic amino acid composition of KVA and the highly negative electrostatic potential surface map of the modeled structure strongly suggested its halophilic nature. Indeed, KVA showed distinct salt- and time-dependent thermal reversibility: when α-amylase was heat denatured at 85°C for 3 min in the presence of 2 M NaCl, the activity was recovered upon incubation on ice (50% recovery after 15 min incubation). Conversely, KVA denatured in 0.1 M NaCl was not refolded at all, even after prolonged incubation. KVA activity was inhibited by proteinaceous α-amylase inhibitor from Streptomyces nitrosporeus, which had been implicated to inhibit only animal α-amylases. KVA with putative SBD regions was found to digest raw starch.

Keywords

Halophilic Moderate halophile α-Amylase Reversibility α-Amylase inhibitor Acidic protein 

References

  1. Aghajari N, Feller G, Gerday C, Haser R (1998) Crystal structures of the psychrophilic α-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor. Protein Sci 7:564–572CrossRefGoogle Scholar
  2. Arakawa T, Tokunaga M (2004) Electrostatic and hydrophobic interactions play a major role in the stability and refolding of halophilic proteins. Protein Pept Lett 11:125–132CrossRefGoogle Scholar
  3. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL Workspace: a web-based environment for protein structure homology modeling. Bioinformatics 22:195–201CrossRefGoogle Scholar
  4. Coronado M-J, Vargas C, Mellado E, Tegos G, Drainas C, Nieto JJ, Ventosa A (2000) The alpha-amylase gene amyH of the moderate halophile Halomonas meridiana: cloning and molecular characterization. Microbiology 146:861–868Google Scholar
  5. Eisenberg H, Mevarech M, Zaccai G (1992) Biochemical, structural, and molecular genetic aspects of halophilism. Adv Protein Chem 43:1–62CrossRefGoogle Scholar
  6. Frillingos S, Linden A, Niehaus F, Vargas C, Nieto JJ, Ventosa A, Antranikian G, Drainas C (2000) Cloning and expression of alpha-amylase from the hyperthermophilic archaeon Pyrococcus woesei in the moderately halophilic bacterium Halomonas elongata. J Appl Microbiol 88:495–503CrossRefGoogle Scholar
  7. Fukushima T, Mizuki T, Echigo A, Inoue A, Usami R (2005) Organic solvent tolerance of halophilic alpha-amylase from a Haloarchaeon, Haloarcula sp. strain S-1. Extremophiles 9:85–89CrossRefGoogle Scholar
  8. Galinski EA (1995) Osmoadaptation in bacteria. Adv Microb Physiol 37:272–328Google Scholar
  9. Hutcheon GW, Vasisht N, Bolhuis A (2005) Characterisation of a highly stable alpha-amylase from the halophilic archaeon Haloarcula hispanica. Extremophiles 9:487–495CrossRefGoogle Scholar
  10. Ikezono T, Omori A, Ichinose S, Pawankar R, Watanabe A, Yagi T (2001) Identification of the protein product of the Coch gene (hereditary deafness gene) as the major component of bovine inner ear protein. Biochim Biophys Acta 1535:258–265Google Scholar
  11. Ishibashi M, Sakashita K, Tokunaga H, Arakawa T, Tokunaga M (2003) Activation of halophilic nucleoside diphosphate kinase by a non-ionic osmolyte, trimethylamine N-oxide. J Protein Chem 22:345–351CrossRefGoogle Scholar
  12. Kamekura M, Onishi H (1978) Flocculation and adsorption of enzymes during growth of a moderate halophile, Micrococcus varians var. halophilus. Can J Microbiol 24:703–709CrossRefGoogle Scholar
  13. Kawaguchi T, Nagae H, Murao S, Arai M (1992) Purification and some properties of a Haim-sensitive α-amylase from newly isolated Bacillus sp. No. 195. Biosci Biotechnol Biochem 56:1792–1796CrossRefGoogle Scholar
  14. Kiran KK, Chandra TS (2008) Production of surfactant and detergent-stable, halophilic, and alkalitolerant alpha-amylase by a moderately halophilic Bacillus sp. Strain TSCVKK. Appl Microbiol Biotechnol 77:1023–1031CrossRefGoogle Scholar
  15. Kobayashi T, Kamekura M, Kanlayakrit W, Onishi H (1986) Production, purification, and characterization of an amylase from the moderate halophile, Micrococcus varians subspecies halophilus. Microbios 46:165–177Google Scholar
  16. Kushner DJ (1985) The halobacteriaceae. Bacteria 8:171–214Google Scholar
  17. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  19. Madern D, Ebel C, Zaccai G (2000) Halophilic adaptation of enzymes. Extremophiles 4:91–98CrossRefGoogle Scholar
  20. Mevarech M, Frolow F, Gloss LM (2000) Halophilic enzymes: proteins with a grain of salt. Biophys Chem 86:155–164CrossRefGoogle Scholar
  21. Mijts BN, Patel BK (2002) Cloning, sequencing and expression of an alpha-amylase gene, amyA, from the thermophilic halophile Halothermothrix orenii and purification and biochemical characterization of the recombinant enzyme. Microbiology 148:2343–2349Google Scholar
  22. Oren A, Larimer F, Richardson P, Lapidus A, Csonka LN (2005) How to be moderately halophilic with broad salt tolerance: clues from the genome of Chromohalobacter salexigens. Extremophiles 9:275–279CrossRefGoogle Scholar
  23. Pérez-Pomares F, Bautista V, Ferrer J, Pire C, Marhuenda-Egea FC, Bonete MJ (2003) Alpha-amylase activity from the halophilic archaeon Haloferax mediterranei. Extremophiles 7:299–306CrossRefGoogle Scholar
  24. Rao JKM, Argos P (1981) Structure stability of halophilic proteins. Biochemistry 20:6536–6543CrossRefGoogle Scholar
  25. Rohban R, Amoozegar MA, Ventosa A (2009) Screening and isolation of halophilic bacteria producing extracellular hydrolyses from Howz Soltan Lake, Iran. J Ind Microbiol Biotechnol 36:333–340CrossRefGoogle Scholar
  26. Saito H, Miura K (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72:619–629CrossRefGoogle Scholar
  27. Sivakumar N, Li N, Tang JW, Patel BK, Swaminathan K (2006) Crystal structure of AmyA lacks acidic surface and provide insights into protein stability at poly-extreme condition. FEBS Lett 580:2646–2652CrossRefGoogle Scholar
  28. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85CrossRefGoogle Scholar
  29. Somogyi M (1952) Notes on sugar determination. J Biol Chem 195:19–23Google Scholar
  30. Srimathi S, Jayaraman G, Feller G, Danielsson B, Narayanan PR (2007) Intrinsic halotolerance of the psychrophilic alpha-amylase from Pseudoalteromonas haloplanktis. Extremophiles 11:505–515CrossRefGoogle Scholar
  31. Sumitani J, Kawaguchi T, Hattori N, Murao S, Arai M (1993) Molecular cloning and expression of proteinaceous α-amylase inhibitor gene from Streptomyces nitrosporeus in Escherichia coli. Biosci Biotechnol Biochem 57:1243–1248CrossRefGoogle Scholar
  32. Sumitani J, Tottori T, Kawaguchi T, Arai M (2000a) New type of starch-binding domain: the direct repeat motif in the C-terminal region of Bacillus sp. no 195 α-amylase contributes to starch binding and raw starch degrading. Biochem J 350:477–484CrossRefGoogle Scholar
  33. Sumitani J, Tsujimoto Y, Kawaguchi T, Arai M (2000b) Cloning and secretive expression of the gene encoding the proteinaceous α-amylase inhibitor Paim from Streptomyces corchorusii. J Biosci Bioeng 90:214–216Google Scholar
  34. Tokunaga H, Ishibashi M, Arakawa T, Tokunaga M (2004) Highly efficient renaturation of beta-lactamase isolated from moderately halophilic bacteria. FEBS Lett 558:7–12CrossRefGoogle Scholar
  35. Tokunaga H, Arakawa T, Fukada H, Tokunaga M (2006a) Opposing effects of NaCl on reversibility and thermal stability of halophilic beta-lactamase from a moderate halophile, Chromohalobacter sp. 560. Biophys Chem 119:316–320CrossRefGoogle Scholar
  36. Tokunaga H, Oda Y, Yonezawa Y, Arakawa T, Tokunaga M (2006b) Contribution of halophilic nucleoside diphosphate kinase sequence to the heat stability of chimeric molecule. Protein Pept Lett 13:525–530CrossRefGoogle Scholar
  37. Tokunaga H, Arakawa T, Tokunaga M (2008a) Engineering of halophilic enzymes: two acidic amino acid residues at the carboxy-terminal region confer halophilic characteristics to Halomonas and Pseudomonas nucleoside diphosphate kinases. Protein Sci 17:1603–1610CrossRefGoogle Scholar
  38. Tokunaga H, Ishibashi M, Arisaka F, Arai S, Kuroki R, Arakawa T, Tokunaga M (2008b) Residue 134 determines the dimer-tetramer assembly of nucleoside diphosphate kinase from moderately halophilic bacteria. FEBS Lett 582:1049–1054CrossRefGoogle Scholar
  39. Tokunaga H, Izutsu K, Arai S, Yonezawa Y, Kuroki R, Arakawa T, Tokunaga M (2010a) Dimer-tetramer assembly of nucleoside diphosphate kinase from moderately halophilic bacterium Chromohalobacter salexigens DSM3043:Both residues 134 and 136 are critical for the tetramer assembly. Enzyme Microb Technol 46:129–135CrossRefGoogle Scholar
  40. Tokunaga H, Saito S, Sakai K, Yamaguchi R, Katsuyama I, Arakawa T, Onozaki K, Arakawa T, Tokunaga M (2010b) Halophilic β-lactamase as a new solubility- and folding-enhancing tag protein: production of native human interleukin 1α and human neutrophil α-defensin. Appl Microbiol Biotechnol 86:649–658CrossRefGoogle Scholar
  41. Ventosa A, Nieto JJ, Oren A (1998) Biology of moderately halophilic aerobic bacteria. Microbiol Mol Biol Rev 62:504–544Google Scholar
  42. Wilkinson DL, Harrison RG (1991) Predicting the solubility of recombinant proteins in Escherichia coli. Bio Technol 9:443–448Google Scholar
  43. Yin XH, Gerbaud C, Francou FX, GuerineauM VMJ (1998) amlC, Another amylolytic gene maps close to the amlB locus in Streptomyces lividans TK24. Gene 215:171–180CrossRefGoogle Scholar
  44. Yonezawa Y, Izutsu K, Tokunaga H, Maeda H, Arakawa T, Tokunaga M (2007) Dimeric structure of nucleoside diphosphate kinase from moderately halophilic bacterium: contrast to the tetrameric Pseudomonas counterpart. FEMS Microbiol Lett 268:52–58CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Rui Yamaguchi
    • 1
  • Hiroko Tokunaga
    • 2
  • Matsujiro Ishibashi
    • 2
  • Tsutomu Arakawa
    • 3
  • Masao Tokunaga
    • 2
  1. 1.Biochemistry and Applied BiosciencesThe United Graduate School of Agricultural Sciences, Kagoshima UniversityKagoshimaJapan
  2. 2.Applied and Molecular Microbiology, Faculty of AgricultureKagoshima UniversityKagoshimaJapan
  3. 3.Alliance Protein LaboratoryThousand OaksUSA

Personalised recommendations