, Volume 54, Issue 11–12, pp 717–723 | Cite as

Use of immobilised glucoamylase G2 for separation of enantiomers

  • A. Karlsson
  • I. Arfwidsson
  • Z. Husovic
  • B. Svensson
Originals Column Liquid Chromatography


The aim of this study was to compare the isolated glucoamylase G2 domain as chiral selector with previously presented data obtained using immobilised native glucoamylase as chiral stationary phase. The glucoamylase G2 was isolated from a commercial preparation ofAspergillus niger glucoamylase and immobilised onto silica particles. Enantioselectivity was tested for several amino alcohols including the β-receptor blocking agents metoprolol and alprenolol. Separation factors greater than 2 were observed. Mobile phase pH was varied to optimise the enantioselective recognition and several different uncharged additives were included to examine their effect on retention. For some of the tested solutes increased retention times were observed when increasing the content of uncharged modifier. Increased mobile phase concentration of several of the tested organic modifiers resulted in increased separation factors. The effect of column temperature was studied. Retention and enantioselectivity increased at higher temperatures. Addition to the mobile phase of acarbose, an inhibitor that binds to the catalytic site of G2 with picomolar affinity, resulted in total loss of enantioselectivity. Comparison of the present results with those obtained previously with the glucoamylase G1 form also containing a starch binding domain, shows that the catalytic domain is essential for chiral recognition of amino alcohols.

Key Words

Column liquid chromatography Column temperature Enantiomer separation Glucoamylase G2 chiral stationary phase Amino alcohols 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. [1]
    Lanchote, V.L.; Bonato, P.S.; Cerqueira, P.M.; Pereira, V.A.; Cesarino E.J.J. Chromatogr. B 2000,738, 27.Google Scholar
  2. [2]
    Krstulovic, A.M. (editor). Chiral separation by HPLC: Applications to pharmaceutical compounds. Ellis Horwood Ltd, John Wiley & Sons,1989.Google Scholar
  3. [3]
    Karlsson, A.; Karlsson, O.J. Chromatogr. A 2001,905, 329.CrossRefGoogle Scholar
  4. [4]
    Berthod, A.; Yu. T.; Kullman, JP; Armstrong, DW.; Gasparrini, F.; D'Acquarica, I.; Misiti, D.; Carotti, A.J. Chromatogr. A 2000,897, 113.CrossRefGoogle Scholar
  5. [5]
    Tang, LY; Xiang, D; Blackwell, JAEnantiomer 2000,5, 345.Google Scholar
  6. [6]
    Vaccher, C.; Wallez, V.; Carato, P.; Vaccher, MP; Bonte, J.P.Chromatographia 2000,52, 513.Google Scholar
  7. [7]
    Karlsson, C.; Wikstrom, H.; Armstrong, DW; Owens, PKJ. Chromatogr. A 2000,897, 349.CrossRefGoogle Scholar
  8. [8]
    Kim, K.H.; Shin, S.D.; Lee, J.H.; Lee, S.C.; Kang, J.S.; Mar, W.; Hong, S.P.; Kim, H.J.Arch. Pharm. Res. 2000,23, 230.Google Scholar
  9. [9]
    Bertucci, C.; Andrisano, V.; Gotti, R.; Cavrini, V.Chromatographia 2000,52, 319.CrossRefGoogle Scholar
  10. [10]
    Karlsson, A.; Aspegren, A.J. Chromatogr. A 2000,866, 15.CrossRefGoogle Scholar
  11. [11]
    Wainer, I.W.; Jadaud, P.; Schombaum, G.R.; Kadodkar, S.V.; Henry, M.P.Chromatographia 1988,25, 903.CrossRefGoogle Scholar
  12. [12]
    Götmar, G.; Fornstedt, T.; Andersson, M.; Guiochon, G.J. Chromatogr. A 2001,905, 3.CrossRefGoogle Scholar
  13. [13]
    Haginaka, J.; Matsunaga, H.; Kakehi, K.J. Chromatogr. B 2000,745, 149.Google Scholar
  14. [14]
    Nyström, A.; Strandberg, A.; Aspegren, A.; Behr, S.; Karlsson, A.Chromatographia 1999,50, 209.CrossRefGoogle Scholar
  15. [15]
    Ceccato, A.; Hubert, P.; Crommen, J.J. Chromatogr. A 1997,760, 193.CrossRefGoogle Scholar
  16. [16]
    Konishi, T.; Nishikawa, H.; Kitamura, S.; Tatsumi, K.J. Chromatogr. B 1998,709, 105.Google Scholar
  17. [17]
    Gottfries, J.; Johansson, P.; Karlsson, A.J. Chromatogr. A 1997,763, 115.CrossRefGoogle Scholar
  18. [18]
    Strandberg, A.; Nystrom, A.; Behr, S.; Karlsson, A.Chromatographia 1999,50, 215.CrossRefGoogle Scholar
  19. [19]
    Svensson, B.; Pedersen, T.G.; Svendsen, I.; Sakai, T.; Ottesen, M.Carlsberg Res. Commun. 1982,47, 55.Google Scholar
  20. [20]
    Stoffer, B.; Frandsen, T.P.; Busk, P.K.; Schneider, P.; Svendsen, I.; Svensson B.Biochem. J. 1993,292, 197.Google Scholar
  21. [21]
    Jackson, E.L. in Adams, R.; Bachmann, W.E.; Fieser, L.F.; Johnson, J.R.; Snyder, H.R. Eds.Organic reactions 1944,2, Chapter 8.Google Scholar
  22. [22]
    Fitos, I.; Visy, J.; Simonyi, M.; Hermansson, J.J. Chromatogr. A 1995,709, 265.CrossRefGoogle Scholar
  23. [23]
    Karlsson, A.; Nyström, A.Chromatographia 2001,53, 135.CrossRefGoogle Scholar
  24. [24]
    Jönsson, S.; Schön, A.; Isaksson, R.; Pettersson, C.; Pettersson, G.Chirality 1992,4, 505.CrossRefGoogle Scholar
  25. [25]
    Svensson, B.; Sierks, M.R.Carbohydr. Res. 1992,228, 28.Google Scholar
  26. [26]
    Sigurskjold, B.W.; Berland, C.R.; Svensson, B.Biochemistry 1994,33, 10191.CrossRefGoogle Scholar
  27. [27]
    Clarke, A.J.; Svensson, B.Carlsberg Res. Commun. 1984,49, 559.CrossRefGoogle Scholar
  28. [28]
    Aleshin, A.E.; Stoffer, B.; Firsov, L.M.; Svensson, B.; Honzatko, R.B.Biochemistry 1996,35, 8319.CrossRefGoogle Scholar

Copyright information

© Friedr. Vieweg & Sohn Verlagsgesellschaft mbH 2001

Authors and Affiliations

  • A. Karlsson
    • 1
  • I. Arfwidsson
    • 1
  • Z. Husovic
    • 1
  • B. Svensson
    • 2
  1. 1.Analytical R&DAstraZeneca R&D MölndalMölndalSweden
  2. 2.Carlsberg LaboratoryDepartment of ChemistryCopenhagenDenmark

Personalised recommendations