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.
Plant Molecular Biology – Springer Journals
Published: Oct 6, 2004
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