Development of yeast strains for the efficient utilisation of starch: evaluation of constructs that express alpha-amylase and glucoamylase separately or as bifunctional fusion proteins

Applied Microbiology and Biotechnology
Lídia Maria Pepe de MoraesS G Oliver

Abstract

Eight constructions involving the Bacillus subtilis alpha-amylase gene (amyE), a mouse pancreatic alpha-amylase cDNA (AMY2) and an Aspergillus awamori glucoamylase cDNA (glaA) were prepared: three fusion genes, involving one alpha-amylase and the glucoamylase, two double-cassette plasmids (expressing one or other alpha-amylase and the glucoamylase) and three single-cassette plasmids, expressing the individual coding sequences. Following transformation of each plasmid into Saccharomyces cerevisiae, a plate test revealed that the largest starch hydrolysis halo was produced by the strain bearing the B. subtilis alpha-amylase/glucoamylase fusion (BsAAase/GAase), and the smallest halo by the one expressing the mouse pancreatic alpha-amylase/glucoamylase fusion (MAAase/GAase). When assayed for enzymatic activity in liquid medium, the strains bearing the fusion and the double-cassette plasmids involving B. subtilis alpha-amylase and the glucoamylase exhibited both enzymic activities. Moreover, the BsAAase/GAase hybrid was able to adsorb and digest raw starch. The MAAse/GAase fusion protein was found to exhibit only alpha-amylase activity. Finally, the capacity to grow on soluble and corn starch was tested in liquid medium for the stra...Continue Reading

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