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

Area of Science:

• Biochemistry
• Molecular Biology
• Plant Science

Background:

• Barley beta-amylase exists in three allelic forms (Sd1, Sd2H, Sd2L) with varying thermostability and kinetic properties.
• These enzymatic differences significantly affect the malting quality of barley varieties.

Purpose of the Study:

• To elucidate the molecular basis for the differing properties of barley beta-amylase allelic forms.
• To identify specific amino acid substitutions responsible for variations in enzyme kinetics and thermostability.

Main Methods:

• Cloning of barley beta-amylase (Sd1 and Sd2L) cDNAs.
• Site-directed mutagenesis to evaluate the effects of amino acid substitutions.
• Analysis of enzyme kinetics and thermostability (T50 index).

Main Results:

• The R115C mutation is responsible for altered kinetic properties, potentially enhancing substrate binding via an additional hydrogen bond.
• V233A and L347S mutations increase thermostability by 1.9°C and 2.1°C, respectively, likely through relief of steric strain and improved solvent interaction.
• These mutations affect thermostability differently: L347S slows unfolding, while V233A accelerates refolding.

Conclusions:

• Specific amino acid substitutions (R115C, V233A, L347S) dictate the distinct kinetic and thermostability profiles of barley beta-amylase allelic forms.
• A combined mutant (C115, A233, S347) exhibits enhanced thermostability and substrate-binding affinity.
• Genetic engineering strategies targeting these mutations offer a pathway to improve barley malting quality.