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Introduction of a synthetic Thermococcus -derived α-amlyase gene into barley genome for increased enzyme thermostability in grains
Mihálik, Daniel; Gubišová, Marcela; Kraic, Ján; Hudcovicová, Martina; Havrlentová, Michaela; Moravčíková, Jana; Glasa, Miroslav & Matušíková, Ildikó
Abstract
Background: The 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.
Results: Gene 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).
Conclusions: Expression of the synthetic gene in barley enhanced the residual α-amylase activity in grains at
high temperatures.
Keywords
Amylopectin; Amylose; Beer; Fermentation; Gene transfer; Glutenin; Hordeum; Promoter; Seed-directed expression; Synthetic gene; Thermotolerant bacteria
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