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Updated: Jul 20, 2026

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
Published on: April 1, 2016
Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the
Lili Kandra1, Maher Abou Hachem, Gyöngyi Gyémánt
1Department of Biochemistry, Faculty of Sciences, University of Debrecen, Debrecen H-4010, Hungary.
Abstract:
Subsite affinity maps of long substrate binding clefts in barley alpha-amylases, obtained using a series of maltooligosaccharides of degree of polymerization of 3-12, revealed unfavorable binding energies at the internal subsites -3 and -5 and at subsites -8 and +3/+4 defining these subsites as binding barriers. Barley alpha-amylase 1 mutants Y105A and T212Y at subsite -6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (-2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile, converting barriers to binding areas. These findings highlight the dynamic binding energy distribution and the versatility of long maltooligosaccharide derivatives in mapping extended binding clefts in alpha-amylases.
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