Introduction of a synthetic Thermococcus-derived α-amlyase gene into barley genome for increased enzyme thermostability in grains

Publication date: Available online 15 August 2017
Source:Electronic Journal of Biotechnology
Author(s): Daniel Mihálik, Marcela Gubišová, Ján Kraic, Martina Hudcovicová, Michaela Havrlentová, Jana Moravčíková, Miroslav Glasa, Ildikó Matušíková
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 ~50kDa 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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