Biologia

, Volume 66, Issue 1, pp 27–32 | Cite as

Biochemical characterization of a glucoamylase from Saccharomycopsis fibuligera R64

  • Dessy Natalia
  • Keni Vidilaseris
  • Pasjan Satrimafitrah
  • Wangsa T. Ismaya
  • Purkan
  • Hjalmar Permentier
  • Guntur Fibriansah
  • Fernita Puspasari
  • Zeily Nurachman
  • Bauke W. Dijkstra
  • Soetijoso Soemitro
Section Cellular and Molecular Biology
  • 60 Downloads

Abstract

Glucoamylase from the yeast Saccharomycopsis fibuligera R64 (GLL1) has successfully been purified and characterized. The molecular mass of the enzyme was 56,583 Da as determined by mass spectrometry. The purified enzyme demonstrated optimum activity in the pH range of 5.6–6.4 and at 50°C. The activity of the enzyme was inhibited by acarbose with the IC50 value of 5 μM. GLL1 shares high amino acid sequence identity with GLU1 and GLA1, which are Saccharomycopsis fibuligera glucoamylases from the strains HUT7212 and KZ, respectively. The properties of GLL1, however, resemble that of GLU1. The elucidation of the primary structure of GLL1 contributes to the explanation of this finding.

Key words

glucoamylase Saccharomycopsis fibuligera R64 variation between strains thermostable raw starch binding 

Abbreviations

Aspni

Aspergillus niger

GLA1

glucoamylase from Saccharomycopsis fibuligera KZ

GLL1

glucoamylase from Saccharomycopsis fibuligera R64

GLM1

glucoamylase from Saccharomycopsis fibuligera IFO0111

GLU1

glucoamylase from Saccharomycopsis fibuligera HUT7212

MES

2-(N-morpholino)ethanesulfonic acid

MMT

L-malic-acid-MES-Tris-HCl

Sacfi

Saccharomycopsis fibuligera

SDS-PAGE

sodium dodecyl-sulphate polyacrylamide gel electrophoresis

TLC

thin-layer chromatography

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Bradford M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.CrossRefPubMedGoogle Scholar
  2. Chi Z.M., Chi Z., Liu G.L., Wang F., Ju L. & Zhang T. 2009. Saccharomycopsis fibuligera and its applications in biotechnology. Biotechnol. Adv. 27: 423–431.CrossRefPubMedGoogle Scholar
  3. DeLano W. L. 2008. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC., Palo Alto, CA, USA.Google Scholar
  4. Emsley P. & Cowtan K. 2004. Coot: model-building tools for molecular graphics. Acta Crystallogr. D60: 2126–2132.Google Scholar
  5. Futatsugi M., Ogawa T. & Fukuda H. 1993. Purification and properties of 2 forms of glucoamylase from Saccharomycopsis fibuligera. J. Ferment. Bioeng. 76: 521–523.CrossRefGoogle Scholar
  6. Fuwa H. 1954. A new method for microdetermination of amylase activity by the use of amylose as the substrate. J. Biochem. 41: 583–603.Google Scholar
  7. Gasperik J. & Hostinova E. 1993. Glucoamylases encoded by variant Saccharomycopsis fibuligera genes: structure and properties. Curr. Microbiol. 27: 11–14.CrossRefGoogle Scholar
  8. Gasperik J., Hostinova E. & Sevcik J. 2005. Acarbose binding at the surface of Saccharomycopsis fibuligera glucoamylase suggests the presence of a raw starch-binding site. Biologia 60(Suppl. 16): 167–170.Google Scholar
  9. Gonzalez C.F., Farina J.I. & de Figueroa L.I.C. 2008. Optimized amylolytic enzymes production in Saccharomycopsis fibuligera DSM-70554: an approach to efficient cassava starch utilization. Enzyme Microb. Tech. 42: 272–277.CrossRefGoogle Scholar
  10. Hasan K., Ismaya W.T., Kardi I., Andiyana Y., Kusumawidjaya S., Ishmayana S., Subroto T. & Soemitro S. 2008. Proteolysis of α-amylase from Saccharomycopsis fibuligera: characterization of digestion products. Biologia 63: 1044–1050.CrossRefGoogle Scholar
  11. Hostinova E. 2002. Amylolytic enzymes produced by the yeast Saccharomycopsis fibuligera. Biologia 57(Suppl. 11): 247–251.Google Scholar
  12. Hostinova E., Balanova J. & Gasperik J. 1991. The nucleotidesequence of the glucoamylase gene GLA1 from Saccharomycopsis fibuligera KZ. FEMS Microbiol. Lett. 83: 103–108.CrossRefGoogle Scholar
  13. Hostinova E. & Gasperik J. 2010. Yeast glucoamylases: molecular-genetic and structural characterization. Biologia 65: 559–568.CrossRefGoogle Scholar
  14. Hostinova E., Solovicova A., Dvorsky R. & Gasperik J. 2003. Molecular cloning and 3D structure prediction of the first raw-starch-degrading glucoamylase without a separate starchbinding domain. Arch. Biochem. Biophys. 411: 189–195.CrossRefPubMedGoogle Scholar
  15. Itoh T., Ohtsuki I., Yamashita I. & Fukui S. 1987. Nucleotide sequence of the glucoamylase gene GLU1 in the yeast Saccharomycopsis fibuligera. J. Bacteriol. 169: 4171–4176.PubMedGoogle Scholar
  16. Norouzian D., Akbarzadeh A., Scharer J.M. & Young M.M. 2006. Fungal glucoamylases. Biotechnol. Adv. 24: 80–85.CrossRefPubMedGoogle Scholar
  17. Pandey A. 1995. Glucoamylase research: an overview. Starch 47: 439–445.CrossRefGoogle Scholar
  18. Sauer J., Sigurskjold B.W., Christensen U., Frandsen T.P., Mirgorodskaya E., Harrison M., Roepstorff P. & Svensson B. 2000. Glucoamylase: structure/function relationships, and protein engineering. Biochim. Biophys. Acta 1543: 275–293.PubMedGoogle Scholar
  19. Sevcik J., Hostinova E., Solovicova A., Gasperik J., Dauter Z. & Wilson K. S. 2006. Structure of the complex of a yeast glucoamylase with acarbose reveals the presence of a raw starch binding site on the catalytic domain. FEBS J. 273: 2161–2171.CrossRefPubMedGoogle Scholar
  20. Solovicova A., Christensen T., Hostinova E., Gasperik J., Sevcik J. & Svensson B. 1999. Structure-function relationships in glucoamylases encoded by variant Saccharomycopsis fibuligera genes. Eur. J. Biochem. 264: 756–764.CrossRefPubMedGoogle Scholar
  21. Truscheit E., Frommer W., Junge B., Müller L., Schmidt D.D. & Wingender, W. 1981. Chemistry and biochemistry of microbial α-glucosidase inhibitors. Angew. Chem. Int. Ed. 20:744–761.CrossRefGoogle Scholar
  22. Xiao Z., Storms R. & Tsang A. 2006. A quantitative starch-iodine method for measuring α-amylase and glucoamylase activities. Anal. Biochem. 351: 146–148.CrossRefPubMedGoogle Scholar
  23. Yamashita I., Itoh T. & Fukui S. 1985. Cloning and expression of the Saccharomycopsis fibuligera glucoamylase gene in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 23: 130–133.Google Scholar

Copyright information

© © Versita Warsaw and Springer-Verlag Wien 2011

Authors and Affiliations

  • Dessy Natalia
    • 1
  • Keni Vidilaseris
    • 1
  • Pasjan Satrimafitrah
    • 1
  • Wangsa T. Ismaya
    • 2
    • 3
  • Purkan
    • 1
  • Hjalmar Permentier
    • 5
  • Guntur Fibriansah
    • 2
  • Fernita Puspasari
    • 1
  • Zeily Nurachman
    • 1
  • Bauke W. Dijkstra
    • 2
  • Soetijoso Soemitro
    • 3
  1. 1.Division of Biochemistry, Faculty of Mathematics and Natural SciencesBandung Institute of TechnologyBandungIndonesia
  2. 2.Protein X-ray Crystallography, Laboratory of BiochemistryUniversity of GroningenGroningenThe Netherlands
  3. 3.Laboratory of Biochemistry, Department of ChemistryPadjajaran UniversityBandungIndonesia
  4. 4.Department of Biochemistry and Cell Biology, Veterinary MedicineUniversity of UtrechtUtrechtThe Netherlands
  5. 5.Mass Spectrometry Core FacilityUniversity of GroningenGroningenThe Netherlands

Personalised recommendations