Mutations of barley β-amylase that imporve substrate-binding affinity and thermostability

Abstract

Abstract Three allelic forms of barley fJ-amylase (Sd I, 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, Sd I and Sd2L f3-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 R 115C 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 f3-amylase forms are due to two amino acid substitutions (V233A and L347S), which increased the enzyme's thermostability index T50 by 1.9°C and 2.1°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 ofV233 by alanine appears to cause an acceleration ofthe refolding after heating. Because the different fJ-amylase properties determined by the three mutations (RI15C, 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 C 115, A233 and S347 together. A possible approach to producing barley varieties with better malting quality by genetic engineering is discussed.