|Tibbot, Brian - UNIVERSITY OF WISCONSIN|
Submitted to: Plant Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 19, 1998
Publication Date: N/A
Interpretive Summary: Germinating seeds initially require nutrients stored in the starchy part of the seed, the starchy endosperm. The growing root and shoot require small sugars, which are supplied by the process of starch digestion. The large starch molecules are first cleaved into two-sugar units by the enzyme alpha-amylase. These are then cleaved into single sugar units by the enzyme alpha-glucosidase (AGL). The combination of these two enzymes working together also has the potential to greatly increase the rate of starch digestion in industrial applications, such as maltodextrin sweetener production. To develop this process, the gene for AGL must first be cloned and then used to produce the authentic enzyme by expressing it in large quantities in a heterologous system, such as yeast cells. We have previously produced the first plant clone (barley) for the AGL gene. Because this gene is similar to a wide variety of other genes which encode sugar-modifying enzymes, it was necessary to put the gene into yeast cells and coax the cells to produce the AGL enzyme. The identity of the enzyme would then be judged by its ability to perform the same reactions which the native barley seed enzyme performs. In this research, we have accomplished these goals and produced AGL enzyme activity in yeast cells. We also produced the protein in E. coli cells and used this protein to produce AGL antibodies. These were used to react with the seed enzyme during germination. This revealed that the enzyme is produced 3 days after seed imbibition and appears first as a large precursor, which is then processed to a smaller sized enzyme. This research will impact the sweetner, syrup and brewing industries by providing AGL enzyme in plentiful quantities.
Technical Abstract: The carbohydrase enzyme alpha-glucosidase (AGL) aids the process of starch hydrolysis by hydrolyzing maltose, a reaction product of alpha-amylase, to its component glucose subunits. This provides nutrients to the developing seedling during germination. An AGL cDNA clone was produced from barley aleurone messenger RNA. The gene was fused with the N-terminal region of the Saccharomyces cerevisiae alpha-factor secretory peptide gene and placed under control of the Pichia pastoris AOX1 promoter in the expression vector pPIC9. Enzymatically active, recombinant AGL was synthesized and secreted from the yeast upon induction with methanol. The enzyme preferentially hydrolyzed maltose and (in order of preference) trehalose, nigerose and isomaltose. Maltase activity occurred over the pH range 3.5 to 6.3 with an optimum of 4.3. This classifies the enzyme as an acid AGL. The enzyme had a Km of 1.88 mM and Vmax of 0.054 umol/min on maltose. Recombinant AGL was also expressed in E. coli and used to generate polyclonal antibodies. The antibodies detected 101 and 95 kDa forms of AGL early in seed germination. Their levels declined sharply later in germination, as an 81 kDa AGL became prominant. Synthesis of these proteins also occurred in isolated aleurones after treatment with gibberellin, and this was accompanied by a 14-fold increase in AGL enzyme activity.