Mutations of barley beta-amylase that improve substrate-binding affinity and thermostability

Three allelic forms of barley beta -amylase (Sd1, Sd2H and Sd2L) exhibit di fferent thermostability and kinetic properties. These differences criticall y influence the malting quality of barley varieties. To understand the mole cular basis for the different properties of these three allelic forms, Sd1 and Sd2L beta -amylase cDNAs were cloned, and the effects of the amino acid substitutions between them were evaluated by site-directed mutagenesis. Th e results showed that an R115C mutation is responsible for the difference i n 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 beta -amylase forms are due to two a mino acid substitutions (V233A and L347S), which increased the enzyme's the rmostability index T-50 by 1.9 degreesC and 2.1 degreesC, respectively. The increased thermostability associated with these two mutations may be due t o relief of steric strain and the interaction of the protein surface with s olvent water. Although both V233A and L347S mutations increased thermostabi lity, 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 a lanine appears to cause an acceleration of the refolding after heating. Bec ause the different beta -amylase properties determined by the three mutatio ns (R115C, V233A and L347S) are associated with malting quality of barley v ariety, a mutant with high thermostability and substrate-binding affinity w as generated by combining the three preferred amino acid residues C115, A23 3 and S347 together. A possible approach to producing barley varieties with better malting quality by genetic engineering is discussed.