Expression and characterization of l-arabinose isomerase from Geobacillus stearothermophilus for improved activity...

A low-calorie sugar-substituting sweetener, d-tagatose, can be produced by l-arabinose isomerase (l-AI) from the substrate d-galactose. However, this process suffers from a Maillard reaction when performed at alkaline pH and high temperature. For industrial applications, therefore, a reaction under slightly acidic conditions is desirable to minimize the Maillard reaction. Previously, we obtained a mutant of l-AI, H18T, from Geobacillus stearothermophilus with greater substrate specificity. Although H18T possessed excellent thermostability, its activity under acidic conditions was not optimal. Here, we successfully obtained a potential variant of the H18T protein, H18T-Y234C, which achieved improved activity at pH 6.0, based on random mutagenesis using error-prone PCR around the binding pocket area of H18T. This double H18T-Y234C mutant possessed 1.8-fold and 3-fold higher activity at pH 6.0 than the parent H18T and the wild type, thereby broadening the optimal pH range to 6.0-8.0. Mutation from Tyr to Cys at residue 234 had little effect on the secondary structure of L-AI. Furthermore, the formation of disulfide bonds was not detected. Thus, the improvement of activity at pH 6.0 is probably caused by the change in the binding pocket area involving residue 234. This study offers insight into the importance of residue 234 in improving the activity under acidic conditions.