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Cloning and expression of a gene for an alpha-glucosidase from Saccharomycopsis fibuligera homologous to family GH31 of yeast glucoamylases

Abstract

Cloning of cDNA encoding an α-glucosidase from the dimorphous yeast Saccharomycopsis fibuligera and characterization of the gene product were performed. The cDNA of the putative α-glucosidase gene consists of 2,886 bp, which includes an open reading frame encoding a 19 amino acid signal peptide at the N-terminal end and a 944 amino acid mature protein with a predicted molecular mass of 105.4 kDa and pI value of 4.52. The deduced amino acid sequence shows a high degree of identity (70%) with two yeast glucoamylases, namely, the extracellular glucoamylase Gam from Schwanniomyces occidentalis and the cell surface glucoamylase Gca from Candida albicans. The recombinant product, synthesized in Saccharomyces cerevisiae, is localized on the cell surface and hydrolyses maltooligosaccharides exclusively without the ability to digest soluble starch, which is consistent with the specificity characteristic of α-glucosidase, EC. 3.2.1.20.

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References

  1. Beggs JD (1978) Transformation of yeast by a replicating hybrid plasmid. Nature (Lond) 275:104–109

  2. Chiba S (1988) Handbook of amylases and related enzymes. Pergamon, Oxford, pp 104–116

  3. Chiba S (1997) Molecular mechanism in α-glucosidase and glucoamylase. Biosci Biotechnol Biochem 61:1233–1239

  4. DeMot R, Andries K, Verachtert H (1984) Comparative study of starch degradation and amylase production by ascomycetous yeast species. Syst Appl Microbiol 5:106–118

  5. Dohmen RJ, Strasser AWM, Dahlems UM, Hollenberg CP (1990) Cloning of the Schwanniomyces occidentalis glucoamylase gene (GAM1) and its expression in S. cerevisiae. Gene 95:111–121

  6. Eksteen JM, Van Rensburg P, Coredoero Otero RR, Pretorius IS (2003) Starch fermentation by recombinant Saccharomyces cerevisiae strains expressing α-amylase and glucoamylase genes from Lipomyces kononenkoae and Saccharomycopsis fibuligera. Biotechnol Bioeng 84:639–646

  7. Frandsen TP, Svensson B (1998) Plant α-glucosidases of the glycoside hydrolase family 31. Molecular properties, substrate specificity, reaction mechanism, and comparison with family members of different origin. Plant Mol Biol 37:1–13

  8. Frandsen TP, Palcic MM, Svensson B (2002) Substrate recognition by three family 13 yeast α-glucosidases. Eur J Biochem 269:728–734

  9. Gašperík J, Hostinová E (1990) α-glucosidase accompanying extracellular amylolytic enzymes of Saccharomycopsis fibuligera. Biologia (Bratisl) 45:1013–1019

  10. Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarity. Biochem J 280:309–316

  11. Hostinová E, Balanová J, Zelinka J (1990) The molecular cloning and expression of the Saccharomycopsis fibuligera glucoamylase and α-amylase genes in Saccharomyces cerevisiae. Biologia (Bratisl) 45:301–306

  12. Hostinová E, Balanová J, Gašperík J (1991) The nucleotide sequence of the glucoamylase gene GLA1 from Saccharomycopsis fibuligera KZ. FEMS Microbiol Lett 83:103–108

  13. Hostinová E, Solovicová A, Dvorský R, Gašperík J (2003) Molecular cloning and 3D structure prediction of the first raw-starch-degrading glucoamylase without a separate starch-binding domain. Arch Biochem Biophys 411:189–195

  14. Hulseweh B, Dahlems UM, Dohmen J, Strasser WM, Hollenberg CP (1997) Characterization of the active site of Schwanniomyces occidentalis glucoamylase by in vitro mutagenesis. Eur J Biochem 244:128–133

  15. Itoh T, Ohtsuki I, Yamashita I, Fukui S (1987a) Nucleotide sequence of the glucoamylase gene GLU 1 in the yeast Saccharomycopsis fibuligera. J Bacteriol 169:4171–4176

  16. Itoh T, Yamashita I, Fukui S (1987b) Nucleotide sequence of the α-amylase gene (ALP1) in the yeast Saccharomycopsis fibuligera. FEBS Lett 219:339–342

  17. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond) 227:680–685

  18. Machida M, Ohtsuki I, Fukui S, Yamashita I (1988) Nucleotide sequence of Saccharomycopsis fibuligera genes for extracellular beta-glucosidase as expressed in Saccharomyces cerevisiae. Appl Environ Microbiol 54:3147–3155

  19. Okuyama M, Okuno A, Shimizu N, Mori H, Kimura A, Chiba S (2001) Carboxyl group of residue Asp 647 as possible proton donor in catalytic reaction of α-glucosidase from Schizosaccharomyces pombe. Eur J Biochem 268:2270–2280

  20. Reiser V, Gašperík J (1995) Purification and characterization of the cell-wall-associated and extracellular α-glucosidases from Saccharomycopsis fibuligera. Biochem J 308:753–760

  21. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor

  22. Sauer J, Sigurskjold BW, Christensen U, Frandsen TP, Mirgorodskaya E, Harrison M, Roepstorff P, Svensson B (2000) Glucoamylase: structure/function relationships, and protein engineering. Biochim Biophys Acta 1548:275–293

  23. Sturtevant J, Dixon F, Wadsworth E, Latge JP, Zhao XJ, Calderone R (1999) Identification and cloning of GCA1, a gene that encodes a cell surface glucoamylase from Candida albicans. Med Mycol 37:357–366

  24. Teague WM, Brumm PJ (1992) Commercial enzymes for starch hydrolysis products. In: Schenck FW, Hebeda RE (eds) Starch hydrolysis products: worldwide technology, production, and application. VCH, New York, pp 45–77

  25. Ueda S, Saha BC (1983) Behaviour of Endomycopsis fibuligera glucoamylase towards raw starch. Enzyme Microb Technol 5:196–198

  26. Vernet T, Dignard D, Thomas DY (1987) A family of yeast expression vectors containing the phage f1 intergenic region. Gene 52:225–233

  27. Yamashita I, Itoh T, Fukui S (1985a) Cloning and expression of the Saccharomycopsis fibuligera α-amylase gene in Saccharomyces cerevisiae. Agric Biol Chem 49:3089–3091

  28. Yamashita I, Itoh T, Fukui S (1985b) Cloning and expression of the Saccharomycopsis fibuligera glucoamylase gene in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 23:130–133

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Acknowledgements

This work was supported by grants 2/1092/21 and 1/010/03 from the Slovak Grant Agency VEGA.

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Correspondence to Eva Hostinová.

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Hostinová, E., Solovicová, A. & Gašperík, J. Cloning and expression of a gene for an alpha-glucosidase from Saccharomycopsis fibuligera homologous to family GH31 of yeast glucoamylases. Appl Microbiol Biotechnol 69, 51–56 (2005). https://doi.org/10.1007/s00253-005-1971-9

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Keywords

  • Glycoside Hydrolase Family
  • Amylolytic Enzyme
  • Isomaltose
  • Mitotic Stability
  • Ascomycetous Yeast Species