Plant Molecular Biology

, Volume 38, Issue 3, pp 379–391

Expression of enzymatically active, recombinant barley α-glucosidase in yeast and immunological detection of α-glucosidase from seed tissue

  • Brian K. Tibbot
  • Cynthia A. Henson
  • Ronald W. Skadsen
Article

DOI: 10.1023/A:1006006203372

Cite this article as:
Tibbot, B.K., Henson, C.A. & Skadsen, R.W. Plant Mol Biol (1998) 38: 379. doi:10.1023/A:1006006203372

Abstract

An α-glucosidase cDNA clone derived from barley aleurone tissue was expressed in Pichia pastoris and Escherichia coli. The gene was fused with the N-terminal region of the Saccharomyces cerevisiae α-factor secretory peptide and placed under control of the Pichia AOX1 promoter in the vector pPIC9. Enzymatically active, recombinant α-glucosidase was synthesized and secreted from the yeast upon induction with methanol. The enzyme hydrolyzed maltose > trehalose > nigerose > isomaltose. Maltase activity occurred over the pH range 3.5–6.3 with an optimum at pH 4.3, classifying the enzyme as an acid α-glucosidase. The enzyme had a Km of 1.88 mM and Vmax of 0.054 µmol/min on maltose. The recombinant α-glucosidase expressed in E. coli was used to generate polyclonal antibodies. The antibodies detected 101 and 95 kDa forms of barley α-glucosidase early in seed germination. Their levels declined sharply later in germination, as an 81 kDa α-glucosidase became prominent. Synthesis of these proteins also occurred in isolated aleurones after treatment with gibberellin, and this was accompanied by a 14-fold increase in α-glucosidase enzyme activity.

Abbreviations: AGL, barley seed α-glucosidase; rAGL, recombinant barley seed α-glucosidase; BMGY, buffered glycerol-complex medium; BMMY, buffered methanol-complex medium; GA, gibberellic acid; UTR, untranslated region.

germination heterologous expression maltase Pichia recombinant protein starch hydrolysis 

Copyright information

© Kluwer Academic Publishers 1998

Authors and Affiliations

  • Brian K. Tibbot
    • 1
  • Cynthia A. Henson
    • 1
  • Ronald W. Skadsen
    • 2
  1. 1.Department of AgronomyUniversity of WisconsinMadisonUSA
  2. 2.Cereal Crops Research Unit, USDA, Agricultural Research ServiceMadisonUSA

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