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

Protein Expression and Purification
Fina Amreta LaksmiMatsujiro Ishibashi

Abstract

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 p...Continue Reading

References

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Oct 5, 2018·Biochimica Et Biophysica Acta. Proteins and Proteomics·Fina Amreta LaksmiMatsujiro Ishibashi

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Citations

Feb 9, 2021·Biotechnology and Applied Biochemistry·Isabela Victorino da Silva AmattoHamilton Cabral

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