Characterization of a novel recombinant D-mannose isomerase from Bifidobacterium bifidum and its catalytic mechanism.

Due to the increasing demand for health-conscious and environmentally friendly products, D-mannose has gained significant attention as a natural, low-calorie sweetener. The use of D-mannose isomerases (D-MIases) for D-mannose production has emerged as a prominent area of research, offering superior advantages compared with conventional methods such as plant extraction and chemical synthesis. In this study, a gene encoding D-MIase was cloned from Bifidobacterium and expressed in E. coli BL21 (DE3). The heterologously expressed enzyme, Bifi-mannose, formed a trimer with a molecular weight of 146.3 kDa and a melting temperature (T m) of 63.39 ± 1.3 °C. Bifi-mannose exhibited optimal catalytic activity at pH 7.5 and 55 °C, and retained more than 80% of its activity after a 3-hour incubation at 55 °C, demonstrating excellent thermal stability. The K m , V max , and k cat / K m values of Bifi-mannose for D-fructose isomerization were determined as 538.7 ± 62.5 mM, 11.7 ± 0.9 μmol·mg 1·s 1, and 1.02 ± 0.3 mM 1·s 1, respectively. Notably, under optimized conditions, catalytic yields of 29.4, 87.1, and 148.5 mg·mL 1 were achieved when using 100, 300, and 500 mg·mL 1 of D-fructose as substrates, resulting in a high conversion rate (29%). Furthermore, kinetic parameters and molecular docking studies revealed that His387 residue primarily participates in the opening of the pyranose ring, while His253 acts as a basic catalyst in the isomerization process. [Display omitted] • A novel D-mannose isomerase from Bifidobacterium was biochemically characterized. • Bifi-MIase had a homo-trimeric structure and was quite stable at 55 °C. • A high conversion rate (29%) was achieved using Bifi-MIase to produce d-mannose. • Molecular simulation depicted the presumed catalysis mechanism of Bifi-MIase. [ABSTRACT FROM AUTHOR]