Characterization of a Novel Mannose Isomerase from Stenotrophomonas rhizophila and Identification of Its Possible Catalytic Residues.

D-Mannose has great value in the treatment of chronic diseases. D-Mannose isomerase can catalyze the bioconversion of D-fructose to D-mannose. Therefore, a novel D-mannose isomerase gene (Strh-MIase) from Stenotrophomonas rhizophila strain IS26 was expressed, purified, and characterized for the industrial production of D-mannose. The specific activities of the Strh-MIase for D-mannose and D-fructose were 437.5 ± 0.8 U/mg and 267.2 ± 0.7 U/mg. Its optimal temperature and pH were 50 °C and 7.0. The enzymatic bioconversion produced 25 g/L D-mannose from concentration D-fructose (100 g/L) in 6 h by recombinant Strh-MIase, resulting in a final yield of 25%. Sodium phosphate inhibition has little influence on D-mannose production when a high concentration of D-fructose is used as substrate. We demonstrate that the metal ions improve the efficiency of D-mannose isomerase because of the enhancement of its thermostability. Moreover, the possible catalytic residues of Strh-MIase were identified by site-directed mutagenesis.

Molecular Characterization of a Mesophilic Cellobiose 2-Epimerase That Maintains a High Catalytic Efficiency at Low Temperatures.

Cellobiose 2-epimerase (CE) can catalyze bioconversion of lactose to its prebiotic derivative epilactose. The catalytic property of a novel CE from Treponema brennaborense (Trbr-CE) was investigated. Trbr-CE showed the highest catalytic efficiency of epimerization toward lactose among all of the previously reported CEs. This enzyme's specific activity could reach as high as 208.5 ± 5.3 U/mg at its optimum temperature, which is 45 °C. More importantly, this enzyme demonstrated a considerably high activity at low temperatures, suggesting Trbr-CE as a promising enzyme for industrial low-temperature production of epilactose. This structurally flexible enzyme exhibited a comparatively high binding affinity toward substrates, which was confirmed by both experimental verification and computational analysis. Molecular dynamics (MD) simulations and binding free energy calculations were applied to provide insights into molecular recognition upon temperature changes. Compared with thermophilic CEs, Trbr-CE presents a more negative enthalpy change and a higher entropy change when the temperature drops.