Applied Microbiology and Biotechnology

, Volume 100, Issue 7, pp 3125–3135 | Cite as

Molecular characterization and heterologous expression of a Xanthophyllomyces dendrorhous α-glucosidase with potential for prebiotics production

  • Patricia Gutiérrez-Alonso
  • María Gimeno-Pérez
  • Mercedes Ramírez-Escudero
  • Francisco J. Plou
  • Julia Sanz-Aparicio
  • María Fernández-LobatoEmail author
Biotechnologically relevant enzymes and proteins


Basidiomycetous yeast Xanthophyllomyces dendrorhous expresses an α-glucosidase with strong transglycosylation activity producing prebiotic sugars such as panose and an unusual tetrasaccharides mixture including α–(1–6) bonds as major products, which makes it of biotechnological interest. Initial analysis pointed to a homodimeric protein of 60 kDa subunit as responsible for this activity. In this study, the gene Xd-AlphaGlu was characterized. The 4131-bp-long gene is interrupted by 13 short introns and encodes a protein of 990 amino acids (Xd-AlphaGlu). The N-terminal sequence of the previously detected 60 kDa protein resides in this larger protein at residues 583–602. Functionality of the gene was proved in Saccharomyces cerevisiae, which produced a protein of about 130 kDa containing Xd-AlphaGlu sequences. All properties of the heterologously expressed protein, including thermal and pH profiles, activity on different substrates, and ability to produce prebiotic sugars were similar to that of the α-glucosidase produced in X. dendrorhous. No activity was detected in S. cerevisiae containing exclusively the 1256-bp from gene Xd-AlphaGlu that would encode synthesis of the 60 kDa protein previously detected. Data were compatible with an active monomeric α-glucosidase of 990 amino acids and an inactive hydrolysis product of 60 kDa. Protein Xd-AlphaGlu contained most of the elements characteristic of α-glucosidases included in the glycoside hydrolases family GH31 and its structural model based on the homologous human maltase-glucoamylase was obtained. Remarkably, the Xd-AlphaGlu C-terminal domain presents an unusually long 115-residue insertion that could be involved in this enzyme’s activity against long-size substrates such as maltoheptaose and soluble starch.


Xanthophyllomyces dendrorhous Alpha-glucosidase GH31 family Maltooligosaccharides Panose 



Projects BIO2013-48779-C4-1/-3/-4 from the Spanish Ministry of Economy and Competitiveness supported this research. We thank Fundación Ramón Areces for the institutional grant to the Centro de Biología Molecular Severo Ochoa. M.G.P. was supported by a Spanish FPU fellowship from the Ministry of Economy and Competitiveness.

Compliance with ethical standards


This study was funded by the Spanish Ministry of Economy and Competitiveness (BIO2013-48779-C4-1/-3/-4).

Conflict of interests

The authors declare that they have no competing interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2015_7171_MOESM1_ESM.pdf (340 kb)
Additional file 1 Table S1. Characteristics of the Xd-AlphaGlu introns. Figure S1. Positioning of peptide masses generated by the MALDI-TOF analysis of the protein Xd-AlphaGlu expressed in X. dendrorhous (a) and S. cerevisiae (b). The peptide m/z is indicated. (PDF 339 kb)


  1. Arnold K, Bordoli L, Koop J, Schwede T (2006) The Swiss-model workspace: a web-based environment for protein structure homology modeling. Bioinformatics 22:195–201. doi: 10.1093/bioinformatics/bti770 CrossRefPubMedGoogle Scholar
  2. Baeza M, Alcaíno J, Barahona S, Sepulveda D, Cifuentes V (2015) Codon usage and codon context bias in Xanthophyllomyces dendrorhous. BMC Genomics 16:293. doi: 10.1186/s12864-015-1493-5 CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bang ML, Villadsen I, Sandal T (1999) Cloning and characterization of an endo-β-1,3(4)glucanase and an aspartic protease from Phaffia rhodozyma CBS 6938. Appl Microbiol Biotechnol 51:215–222. doi: 10.1007/s002530051384 CrossRefPubMedGoogle Scholar
  4. Bon E, Casaregola S, Blandin G, Llorente B, Neuveglise C, Münsterkotter M, Güldener U, Mewes HW, Van Helden J, Dujon B, Gaillardin C (2003) Molecular evolution of eukaryotic genomes: hemiascomycetous yeast spliceosomal introns. Nucleic Acids Res 31:1121–1135. doi: 10.1093/nar/gkg213 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Burke D, Dawson D, Stearns T (2000) Methods in yeast genetics: A Cold Spring Harbour Laboratory course manual. Cold Spring Harbor Laboratory Press, Plainview, N.YGoogle Scholar
  6. Cartharius K, Frech K, Grote K, Klocke B, Haltmeier M, Klingenhoff A, Frisch M, Bayerlein M, Werner T (2005) MatInspector and beyond: promoter analysis based on transcription factor binding sites. Bioinformatics 21:2933–42. doi: 10.1093/bioinformatics/bti473 CrossRefPubMedGoogle Scholar
  7. Chiba S (1997) Molecular mechanism in α-glucosidase and glucoamylase. Biosci Biotechnol Biochem 61:1233-1239. 10.1271/bbb.61.1233
  8. Fernández-Arrojo L, Marín D, Gómez de Segura A, Linde D, Alcalde M, Gutiérrez-Alonso P, Ghazi I, Plou FJ, Fernández-Lobato M, Ballesteros A (2007) Transformation of maltose into prebiotic isomaltooligosaccharides by a novel α-glucosidase from Xantophyllomyces dendrorhous. Process Biochem 42:1530–1536. doi: 10.1016/j.procbio.2007.08.007 CrossRefGoogle Scholar
  9. Goffin D, Delzenne N, Blecker C, Hanon E, Deroanne C, Paquot M (2011) Will isomalto-oligosaccharides, a well-established functional food in Asia, break through the European and American market? The status of knowledge on these prebiotics. Crit Rev Food Sci and Nutr 51:394–409. doi: 10.1080/10408391003628955 CrossRefGoogle Scholar
  10. Henrissat B, Davies G (1997) Structural and sequence-based classification of glycoside hydrolases. Curr Opin Struct Biol 7:637-644. Google Scholar
  11. Hermans MM, Kroos MA, van Beeumen J, Oostra BA, Reuser AJ (1991) Human lysosomal α-glucosidase. Characterization of the catalytic site. J Biol Chem 266:13507–13512PubMedGoogle Scholar
  12. Janecek S, Svensson B, MacGregor EA (2014) α-Amylase: an enzyme specificity found in various families of glycoside hydrolases. Cell Mol Life Sci 71:1149–1170. doi: 10.1007/s00018-013-1388-z CrossRefPubMedGoogle Scholar
  13. Kato N, Suyama S, Shirokane M, Kato M, Kobayashi T, Tsukagoshi N (2002) Novel α-glucosidase from Aspergillus nidulans with strong transglycosylation activity. Appl Environ Microbiol 68:1250–1256. doi: 10.1128/AEM.68.3.1250-1256.2002 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Kofod LV, Kauppinen S, Christgau S, Andersen LN, Heldt-Hansen HP, Dörreich K, Dalbøge H (1994) Cloning and characterization of two structurally and functionally divergent rhamnogalacturonases from Aspergillus aculeatus. J Biol Chem 269:29182–29189PubMedGoogle Scholar
  15. Kupfer DM, Drabenstot SD, Buchanan KL, Lai H, Zhu H, Dyer DW, Roe BA, Murphy JW (2004) Introns and splicing elements of five diverse fungi. Eukaryot Cell 3:1088-1100. doi: 10.1128/EC.3.5.1088-1100
  16. Linde D, Macias I, Fernández-Arrojo L, Plou FJ, Jiménez A, Fernández-Lobato M (2009) Molecular and biochemical characterization of a β-fructofuranosidase from Xanthophyllomyces dendrorhous. Appl Environ Microbiol 75:1065–1073. doi: 10.1128/AEM.02061-08 CrossRefPubMedPubMedCentralGoogle Scholar
  17. Loto I, Gutiérrez MS, Barahona S, Sepúlveda D, Martínez-Moya P, Baeza M, Cifuentes V, Alcaíno J (2012) Enhancement of carotenoid production by disrupting the C22-sterol desaturase gene (CYP61) in Xanthophyllomyces dendrorhous. BMC Microbiol 12:235. doi: 10.1186/1471-2180-12-2351471-2180-12-235 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Lovering AL, Lee SS, Kim YW, Withers SG, Strynadka NC (2005) Mechanistic and structural analysis of a family 31 α-glycosidase and its glycosyl-enzyme intermediate. J Biol Chem 280:2105-2115. doi: 10.1074/jbc.M410468200
  19. Marín D, Linde D, Fernández Lobato M (2006) Purification and biochemical characterization of an α-glucosidase from Xanthophyllomyces dendrorhous. Yeast 23:117–125. doi: 10.1002/yea.1345S0378-1119(11)00511-7 CrossRefPubMedGoogle Scholar
  20. Melo EB, Gomes AS, Carvalho I (2006) α- and β-Glucosidase inhibitors: chemical structure and biological activity. Tetrahedron 62:10277–10302. doi: 10.1016/j.tet.2006.08.055 CrossRefGoogle Scholar
  21. Mohan S, Eskandari R, Pinto BM (2014) Naturally occurring sulfonium-ion glucosidase inhibitors and their derivatives: a promising class of potential antidiabetic agents. Acc Chem Res 47:211–225. doi: 10.1021/ar400132g CrossRefPubMedGoogle Scholar
  22. Pal K, Kumar S, Sharma S, Garg SK, Alam MS, Xu HE, Agrawal P, Swaminathan K (2010) Crystal structure of full-length Mycobacterium tuberculosis H37Rv glycogen branching enzyme: insights of N-terminal β-sandwich in substrate specificity and enzymatic activity. J Biol Chem 285:20897–20903. doi: 10.1074/jbc.M110.121707M110.121707 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Pan YC, Lee WC (2005) Production of high-purity isomalto-oligosaccharides syrup by the enzymatic conversion of transglucosidase and fermentation of yeast cells. Biotechnol Bioeng 89:797–804. doi: 10.1002/bit.20402 CrossRefPubMedGoogle Scholar
  24. Raben N, Plotz P, Byrne BJ (2002) Acid α-glucosidase deficiency (glycogenosis type II, Pompe disease). Curr Mol Med 2:145–166. doi: 10.2174/1566524024605789#sthash.KQI1znTD.dpuf CrossRefPubMedGoogle Scholar
  25. Rehm J, Willmitzer L, Heyer AG (1998) Production of 1-kestose in transgenic yeast expressing a fructosyltransferase from Aspergillus foetidus. J Bacteriol 180:1305–1310PubMedPubMedCentralGoogle Scholar
  26. Ren LM, Qin XH, Cao XF, Wang LL, Bai F, Bai G, Shen Y (2011) Structural insight into substrate specificity of human intestinal maltase-glucoamylase. Protein Cell 2:827–836. doi: 10.1007/s13238-011-1105-3 CrossRefPubMedGoogle Scholar
  27. Rosenberg AH, Goldman E, Dunn JJ, Studier FW, Zubay G (1993) Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system. J Bacteriol 175:716–722PubMedPubMedCentralGoogle Scholar
  28. Sharma R, Gassel S, Steiger S, Xia X, Bauer R, Sandmann G, Thines M (2015) The genome of the basal agaricomycete Xanthophyllomyces dendrorhous provides insights into the organziation of its acetyl-CoA derived pathways and the evolution of Agaricomycotina. BMC Genomics 16:233. doi: 10.1186/s12864-015-1380-0 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Shen X, Saburi W, Gai ZQ, Komoda K, Yu J, Ojima-Kato T, Kido Y, Matsui H, Mori H, Yao M (2014) Crystallization and preliminary x-ray crystallographic analysis of α-glucosidase HaG from Halomonas sp. strain H11. Acta Crystallogr F Struct Biol Commun 70:464–466. doi: 10.1107/S2053230X14001940S2053230X14001940 CrossRefPubMedGoogle Scholar
  30. Shirai T, Hung VS, Morinaka K, Kobayashi T, Ito S (2008) Crystal structure of GH13 α-glucosidase GSJ from one of the deepest sea bacteria. Proteins 73:126–133. doi: 10.1002/prot.22044 CrossRefPubMedGoogle Scholar
  31. Sim L, Quezada-Calvillo R, Sterchi EE, Nichols BL, Rose DR (2008) Human intestinal maltase–glucoamylase: crystal structure of the N-terminal catalytic subunit and basis of inhibition and substrate specificity. J Mol Biol 375:782–792. doi: 10.1016/j.jmb.2007.10.069 CrossRefPubMedGoogle Scholar
  32. Tagami T, Yamashita K, Okuyama M, Mori H, Yao M, Kimura A (2013) Molecular basis for the recognition of long-chain substrates by plant α-glucosidases. J Biol Chem 288:19296–19303. doi: 10.1074/jbc.M113.465211jbc.M113.465211 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Tagami T, Yamashita K, Okuyama M, Mori H, Yao M, Kimura A (2015) Structural advantage of sugar beet α-glucosidase to stabilize the Michaelis complex with long-chain substrate. J Biol Chem 290:1796–1803. doi: 10.1074/jbc.M114.606939M114.606939 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Wang YH, Jiang Y, Duan ZY, Shao WL, Li HZ (2009) Expression and characterization of an α-glucosidase from Thermoanaerobacter ethanolicus JW200 with potential for industrial application. Biologia 64:1053–1057. doi: 10.2478/s11756-009-0197-1 Google Scholar
  35. Wu KY, Huang SH, Ding S, Zhang YK, Chen GG, Liang ZQ (2010) Expression, purification and characterization of recombinant α-glucosidase in Pichia pastoris. Folia Microbiol 66:582–587. doi: 10.1007/s12223-010-0093-7 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Patricia Gutiérrez-Alonso
    • 1
  • María Gimeno-Pérez
    • 1
  • Mercedes Ramírez-Escudero
    • 2
  • Francisco J. Plou
    • 3
  • Julia Sanz-Aparicio
    • 2
  • María Fernández-Lobato
    • 1
    Email author
  1. 1.Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular (CSIC-UAM)Nicolás Cabrera 1. Universidad Autónoma MadridMadridSpain
  2. 2.Departamento de Cristalografía y Biología EstructuralInstituto de Química-Física Rocasolano, CSICMadridSpain
  3. 3.Instituto de Catálisis y Petroleoquímica, CSICMadridSpain

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