Mutations of barley ß-amylase that improve substrate -binding affinity and thermostability
Ma, YF and Evans, E and Logue, SJ and Langridge, P, Mutations of barley s-amylase that improve substrate -binding affinity and thermostability, Molecular Genetics and Genomics: An International Journal, 266, (3) pp. 345-352. ISSN 1617-4615 (2001) [Refereed Article]
Three allelic forms of barley Î²-amylase (Sd1, Sd2H and Sd2L) exhibit different thermostability and kinetic properties. These differences critically influence the malting quality of barley varieties. To understand the molecular basis for the different properties of these three allelic forms, Sd1 and Sd2L Î²-amylase cDNAs were cloned, and the effects of the amino acid substitutions between them were evaluated by site-directed mutagenesis. The results showed that an R115C mutation is responsible for the difference in kinetic properties. This substitution resulted in an additional hydrogen bond which may create a more favourable environment for substrate-binding. The different thermostabilities of the Î²-amylase forms are due to two amino acid substitutions (V233A and L347S), which increased the enzyme's thermostability index T 50 by 1.9Â°C and 2.1Â°C, respectively. The increased thermostability associated with these two mutations may be due to relief of steric strain and the interaction of the protein surface with solvent water. Although both V233A and L347S mutations increased thermostability, they affected the thermostability in different ways. The replacement of L347 by serine seems to increase the thermostability by slowing thermal unfolding of the protein during heating, while the replacement of V233 by alanine appears to cause an acceleration of the refolding after heating. Because the different Î²-amylase properties determined by the three mutations (R115C, V233A and L347S) are associated with malting quality of barley variety, a mutant with high thermostability and substrate-binding affinity was generated by combining the three preferred amino acid residues C115, A233 and S347 together. A possible approach to producing barley varieties with better malting quality by genetic engineering is discussed.