Introduction of a synthetic Thermococcus-derived α-amlyase gene into barley genome for increased enzyme thermostability in grains |
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| Institution: | 1. Department of Biotechnology, Faculty of Natural Sciences, University of Ss., Cyril and Methodius in Trnava, Nám. J. Herdu 2, SK-917 01 Trnava, Slovakia;2. Research Institute of Plant Production, National Agricultural and Food Centre, SK-921 68 Pie??any, Slovakia;3. Institute of Plant Genetics and Biotechnology, Plant Science and Biodiversity Center SAS, Akademická 2, P.O. Box 39A, 950 07 Nitra, Slovakia;4. Institute of Virology, Biomedical Research Centre, Slovak Academy of Sciences, Dúbravská cesta 9, 84505 Bratislava, Slovakia;5. Department of Ecochemistry and Radioecology, Faculty of Natural Science, University of Ss. Cyril and Methodius, SK-917 01, Trnava, Slovakia |
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| Abstract: | BackgroundThe enzymes utilized in the process of beer production are generally sensitive to higher temperatures. About 60% of them are deactivated in drying the malt that limits the utilization of starting material in the fermentation process. Gene transfer from thermophilic bacteria is a promising tool for producing barley grains harboring thermotolerant enzymes.ResultsGene for α-amylase from hydrothermal Thermococcus, optimally active at 75–85°C and pH between 5.0 and 5.5, was adapted in silico to barley codon usage. The corresponding sequence was put under control of the endosperm-specific promoter 1Dx5 and after synthesis and cloning transferred into barley by biolistics. In addition to model cultivar Golden Promise we transformed three Slovak barley cultivars Pribina, Levan and Nitran, and transgenic plants were obtained. Expression of the ~ 50 kDa active recombinant enzyme in grains of cvs. Pribina and Nitran resulted in retaining up to 9.39% of enzyme activity upon heating to 75°C, which is more than 4 times higher compared to non-transgenic controls. In the model cv. Golden Promise the grain α-amylase activity upon heating was above 9% either, however, the effects of the introduced enzyme were less pronounced (only 1.22 fold difference compared with non-transgenic barley).ConclusionsExpression of the synthetic gene in barley enhanced the residual α-amylase activity in grains at high temperatures. |
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