Generation and analyses of the transgenic potatoes expressing heterologous thermostable β-amylase

beta-Amylase hydrolyzes the alpha-1,4-glycosidic linkages of starch resulting in the release of maltose. This reaction is of industrial importance for maltose production and for the preparation process of fermented foods and alcoholic beverages. A demand for an acceleration of the rate of enzymatic cleavage of the starch macro-molecule is a prerequisite for large-scale and highly efficient production. Increasing the temperature up to the optimum of approximately 60 degrees C can significantly speed up the reaction. However, at higher temperatures, the effect on protein denaturation becomes dominant, and the conversion rate decreases. The primary objective of this study was to generate transgenic plants of the Kennebec potato variety for production of thermostable beta-amylase using Agrobacterium-mediated transformation. Four chimeric genes encoding the beta-amylase with or without signal peptide sequences for targeting expression in cytoplasm, amyloplasts, or vacuoles were constructed and driven by high tuber expression promoter from Sucrose synthetase gene Sus4. Forty-two transgenic lines were selected for this study. Transgenic lines with various beta-amylase constructs were verified for the existence and expression of the transgenes by PCR approaches. The expression level of the introduced beta-amylase protein was estimated by immunoblot analyses using polyclonal antibodies. Recombinant beta-amylase was successfully expressed in Escherichia coli B21 (DE3), and temperature ranges of these inducible recombinant proteins were found to be between 40 and 90 degrees C. This enzymatic complex produced in the in vitro cultured microtubers and field-grown tubers from transgenic potatoes were proved to be stable and active at 60 degrees C. The relative activities of beta-amylase in tubers of field-grown potatoes were compared, and the maximum increase was found with transgenic line #6A of the pSUS4-AMY construct which has an 11 fold greater increase than the untransformed Kennebec. Variations of the chemical compositions were found in the selected transgenic lines. Results of this study suggest the feasibility of utilizing thermostable beta d-amylase in transgenic potatoes for the starch-processing industries. (C) 2008 Elsevier Ireland Ltd. All rights reserved.