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The Protein Journal

, Volume 29, Issue 5, pp 355–364 | Cite as

Gene Sequence, Bioinformatics and Enzymatic Characterization of α-Amylase from Saccharomycopsis fibuligera KZ

  • Eva HostinováEmail author
  • Štefan Janeček
  • Juraj Gašperík
Article

Abstract

A fragment coding for a putative extracellular α-amylase, from the genomic library of the yeast Saccharomycopsis fibuligera KZ, has been subcloned into yeast expression vector pVT100L and sequenced. The nucleotide sequence revealed an ORF of 1,485 bp coding for a 494 amino acid residues long protein with 99% identity to the α-amylase Sfamy from S. fibuligera HUT 7212. The S. fibuligera KZ α-amylase (Sfamy KZ) belongs to typical extracellular fungal α-amylases classified in the glycoside hydrolase family 13, subfamily 1, as supported also by clustering observed in the evolutionary tree. Sfamy KZ, in addition to the essential GH13 α-amylase three-domain arrangement (catalytic TIM barrel plus domains B and C), does not contain any distinct starch-binding domain. Sfamy KZ was expressed as a recombinant protein in Saccharomyces cerevisiae and purified to electrophoretic homogeneity. The enzyme had a molecular mass 53 kDa and contained about 2.5% of carbohydrate. The enzyme exhibited pH and temperature optima in the range of 5–6 and 40–50 °C, respectively. Stable adsorption of the enzyme to starch granules was not detected but a low degradation of raw starch in a concentration-dependent manner was observed.

Keywords

Saccharomycopsis fibuligera Yeast α-amylase Protein bioinformatics Raw starch digestion 

Abbreviations

Sfamy

α-Amylase from Saccharomycopsis fibuligera HUT 7212

Sfamy KZ

α-amylase from S. fibuligera KZ

ALP1

Gene coding for α-amylase from S. fibuligera HUT 7212

LKA1

α-Amylase 1 from Lipomyces kononenkoae

LKA2

α-Amylase 2 from Lipomyces kononenkoae

CBM

Carbohydrate-binding module

SBD

Starch-binding domain

GH

Glycoside hydrolase

AMY1

Barley α-amylase 1 (low pI isozyme)

AMY2

Barley α-amylase 2 (high pI isozyme)

CAZy

Carbohydrate-active enzymes

Notes

Acknowledgments

This work was supported in part by the grant No. 2/0114/08 from the Slovak grant agency VEGA.

References

  1. 1.
    Apweiler R, Martin MJ, O’Donovan C, Magrane M, …, Zhang J (2010) Nucleic Acids Res 38 (Database issue): D142–D148Google Scholar
  2. 2.
    Baeva LF, Kozlov DG, Brevnova EE, Benevolenskij SV (1996) Prikl Biochim Mikrobiol 32:311–314Google Scholar
  3. 3.
    Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE (2000) Nucleic Acids Res 28:235–242CrossRefGoogle Scholar
  4. 4.
    Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) Nucleic Acids Res 37 (Database issue): D233–D238Google Scholar
  5. 5.
    Chi Z, Chi Z, Liu G, Wang F, Ju L, Zhang T (2009) Biotechnol Advan 27:423–431CrossRefGoogle Scholar
  6. 6.
    Christiansen C, Abou Hachem M, Janecek S, Viksø-Nielsen A, Blennow A, Svensson B (2009) FEBS J 276:5006–5029CrossRefGoogle Scholar
  7. 7.
    Da Lage JL, Feller G, Janecek S (2004) Cell Mol Life Sci 61:97–109CrossRefGoogle Scholar
  8. 8.
    Dubois M, Gilles GA, Hamilton JK, Rebers PA, Smith F (1956) Anal Chem 28:350–356CrossRefGoogle Scholar
  9. 9.
    Eksteen JM, Steyn AJ, van Rensburg P, Cordero Otero RR, Pretorius IS (2003) Yeast 20:69–78CrossRefGoogle Scholar
  10. 10.
    Eksteen JM, Van Rensburg P, Cordero Otero RR, Pretorius IS (2003) Biotechnol Bioeng 84:639–646CrossRefGoogle Scholar
  11. 11.
    Felsenstein J (1985) Evolution 39:783–791CrossRefGoogle Scholar
  12. 12.
    Galdino AS, Ulhoa CJ, Moraes L, Prates MV, Bloch C, Torres F (2008) FEMS Microbiol Lett 280:189–194CrossRefGoogle Scholar
  13. 13.
    Gašperík J, Hostinová E (1990) Biologia 45:1013–1019Google Scholar
  14. 14.
    Gašperík J, Hostinová E (1993) Curr Microbiol 27:11–14CrossRefGoogle Scholar
  15. 15.
    Gašperík J, Kováč Ľ, Mináriková O (1991) Int J Biochem 23:21–25CrossRefGoogle Scholar
  16. 16.
    Gietz RD, Woods RA (2002) Methods Enzymol 350:87–96CrossRefGoogle Scholar
  17. 17.
    Gupta R, Gigras P, Mohapatra H, Goswam VK, Chauhan B (2003) Process Biochem 38:1599–1616CrossRefGoogle Scholar
  18. 18.
    Hasan K, Ismaya WT, Kardi I, Andiyana Y, Kusumawidjaya S, Ishmayana S, Subroto T, Soemitro S (2008) Biologia 63:1044–1050CrossRefGoogle Scholar
  19. 19.
    Hostinová E, Balanová J, Zelinka J (1990) Biologia 45:301–306Google Scholar
  20. 20.
    Hostinová E, Solovicová A, Dvorský R, Gašperík J (2003) Arch Biochem Biophys 411:189–195CrossRefGoogle Scholar
  21. 21.
    Iefuji H, Chino M, Kato M, Iimura Y (1996) Biochem J 318:989–996Google Scholar
  22. 22.
    Itoh T, Ohtsuki I, Yamashita I, Fukui S (1987) J Bacteriol 169:4171–4176Google Scholar
  23. 23.
    Itoh T, Yamashita I, Fukui S (1987) FEBS Lett 219:339–342CrossRefGoogle Scholar
  24. 24.
    Janecek S (1994) Eur J Biochem 224:519–524CrossRefGoogle Scholar
  25. 25.
    Janecek S (1997) Progr Biophys Mol Biol 67:67–97CrossRefGoogle Scholar
  26. 26.
    Janecek S (2002) Biologia 57(Suppl. 11):29–41Google Scholar
  27. 27.
    Janecek S, Leveque E, Belarbi A, Haye B (1999) J Mol Evol 48:421–426CrossRefGoogle Scholar
  28. 28.
    Janecek S, Sevcik J (1999) FEBS Lett 456:119–125CrossRefGoogle Scholar
  29. 29.
    Kato S, Shimizu-Ibuka A, Mura K, Takeuchi A, Tokue C, Arai S (2007) Biosci Biotechnol Biochem 71:3007–3013CrossRefGoogle Scholar
  30. 30.
    Kelley LA, Sternberg MJE (2009) Nat Protoc 4:363–371CrossRefGoogle Scholar
  31. 31.
    Kelly RM, Dijkhuizen L, Leemhuis H (2009) J Biotechnol 140:184–193CrossRefGoogle Scholar
  32. 32.
    MacGregor EA, Janecek S, Svensson B (2001) Biochim Biophys Acta 1546:1–20Google Scholar
  33. 33.
    Machovic M, Janecek S (2006) Cell Mol Life Sci 63:2710–2724CrossRefGoogle Scholar
  34. 34.
    Matsubara T, Ammar YB, Anindyawati T, Yamamoto S, Ito K, Iizuka M, Minamiura N (2004) J Biochem Mol Biol 37:422–428Google Scholar
  35. 35.
    Matsui I, Matsui E, Ishikawa K, Miyairi S, Honda K (1990) Agric Biol Chem 54:2009–2015Google Scholar
  36. 36.
    Matsui I, Yoneda S, Ishikawa K, Miyairi S, Fukui S, Umeyama H, Honda K (1994) Biochemistry 18:451–458CrossRefGoogle Scholar
  37. 37.
    Matsuura Y, Kusunoki M, Harada W, Kakudo M (1984) J Biochem 95:697–770Google Scholar
  38. 38.
    McCann AK, Barnett JA (1986) Yeast 2:109–115CrossRefGoogle Scholar
  39. 39.
    Nielsen MM, Bozonet S, Seo ES, Motyan JA, Andersen JM, Dilokpimol A, Abou Hachem M, Gyemant G, Naested H, Kandra L, Sigurskjold BW, Svensson B (2009) Biochemistry 18:7686–7697CrossRefGoogle Scholar
  40. 40.
    Page RD (1996) Comput Appl Biosci 12:357–358Google Scholar
  41. 41.
    Pandey A, Nigam P, Soccol CR, Soccol VT, Singh D, Mohan R (2000) Biotechnol Appl Biochem 31:135–152CrossRefGoogle Scholar
  42. 42.
    Ragunath C, Manuel SGA, Kasinathan C, Ramasubbu N (2008) Biologia 63:1028–1034CrossRefGoogle Scholar
  43. 43.
    Ragunath C, Manuel SG, Venkataraman V, Sait HB, Kasinathan C, Ramasubbu N (2008) J Mol Biol 384:1232–1248CrossRefGoogle Scholar
  44. 44.
    Ramachandran N, Pretorius IS, Cordero Otero RR (2005) Biologia 60(Suppl. 16):103–110Google Scholar
  45. 45.
    Robert X, Haser R, Gottschalk TE, Ratajczak F, Driguez H, Svensson B, Aghajari N (2003) Structure 11:973–984CrossRefGoogle Scholar
  46. 46.
    Rodriguez-Sanoja R, Oviedo N, Sanches S (2005) Curr Opin Microbiol 8:260–267CrossRefGoogle Scholar
  47. 47.
    Saitou N, Nei M (1987) Mol Biol Evol 4:406–425Google Scholar
  48. 48.
    Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  49. 49.
    Shatsky M, Nussinov R, Wolfson HJ (2004) Proteins 56:143–156CrossRefGoogle Scholar
  50. 50.
    Somogyi M (1952) J Biol Chem 195:19–23Google Scholar
  51. 51.
    Southall SM, Simpson PJ, Gilbert HJ, Williamson G, Williamson MP (1999) FEBS Lett 447:58–60CrossRefGoogle Scholar
  52. 52.
    Stam MR, Danchin EG, Rancurel C, Coutinho PM, Henrissat B (2006) Protein Eng Des Sel 19:555–562CrossRefGoogle Scholar
  53. 53.
    Steyn AJC, Marmur J, Pretorius IS (1995) Gene 166:65–71CrossRefGoogle Scholar
  54. 54.
    Strikovsky A, Hradil J, Wulff G (2003) React Funct Polymers 54:49–61CrossRefGoogle Scholar
  55. 55.
    Sun H, Zhao P, Ge X, Xia Y, Hao Z, Liu J, Peng M (2010) Appl Biochem Biotechnol 160:988–1003CrossRefGoogle Scholar
  56. 56.
    Ševčík J, Hostinová E, Solovicová A, Gašperík J, Dauter Z, Wilson KS (2006) FEBS J 273:2161–2172CrossRefGoogle Scholar
  57. 57.
    Thompson JD, Higgins D, Gibson TJ (1994) Nucleic Acids Res 22:4673–4680CrossRefGoogle Scholar
  58. 58.
    Tibbot BK, Wong DWS, Robertson GH (2002) Biologia 57(Suppl. 11):229–238Google Scholar
  59. 59.
    Tranier S, Deville K, Robert X, Bozonnet S, Haser R, Svensson B, Aghajari N (2005) Biologia (Suppl. 16):37–46Google Scholar
  60. 60.
    van der Kaaij RM, Janecek S, van der Maarel MJ, Dijkhuizen L (2007) Microbiology 153:4003–4015CrossRefGoogle Scholar
  61. 61.
    van der Maarel M, van der Veen B, Uitdehaag J, Leemhuis H, Dijkhuizen L (2002) J Biotechnol 94:137–155CrossRefGoogle Scholar
  62. 62.
    Vernet T, Dignard D, Thomas DY (1987) Gene 52:225–233CrossRefGoogle Scholar
  63. 63.
    Vujicic-Zagar A, Dijkstra BW (2006) Acta Crystallogr Sect F Struct Biol Cryst Commun 62:716–721CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Eva Hostinová
    • 1
    Email author
  • Štefan Janeček
    • 1
  • Juraj Gašperík
    • 1
  1. 1.Institute of Molecular BiologySlovak Academy of SciencesBratislavaSlovakia

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