Development of a recombinant d-mannose isomerase and its characterizations for d-mannose synthesis

Abstract

d-Mannose isomerase (MIase) catalyzes the conversion of d-fructose to d-mannose. In this study, the MIase encoding gene (yihS) from Escherichia coli BL21 contains an ORF of 1242bp, was cloned and expressed in Bacillus subtilis WB800. This heterologous expression resulted in a hexamer with a molecular weight of 274.5kDa and Tm of 61.4°C. Efficient MIase secretory expression by the robust recombinant B. subtilis was achieved with activity of 51.2U/ml (d-mannose forming). Its optimal temperature and pH were 45.0°C and 7.0, respectively. Using d-fructose as the substrate, Km, kcat and catalytic efficiency value of kinetic reaction were 203.7±6.7mM, 27.7±0.7s(-1) and 136.0±2.9M(-1)s(-1), respectively. The production of d-mannose reached about 150g/l with approximately 25% turnover yield under the optimum conditions. These results demonstrated that B. subtilis was a promising candidate of MIase expression system for d-mannose production.

Keywords: Biotransformation; Gene expression; d-mannose isomerase.