Efficient conversion of ᴅ-fructose to ᴅ-mannose enabled by coupled reactions of mannose isomerase and sugar kinase with co-factor regeneration

Abstract

ᴅ-Mannose is a functional monosaccharide that is difficult to prepare on a large scale via chemical or enzymatic synthetic routes due to low conversion rates and complex downstream isolation. In this study, a two-step enzymatic route was established to achieve highly efficient conversion of ᴅ-fructose to ᴅ-mannose without the need for a tedious isomer separation process. Firstly, a mannose isomerase from Xanthomonas phaseoli was coupled with a sugar 1-kinase from Bifidobacterium infantis and a polyphosphate kinase to synthesize mannose 1-phosphate (Man-1-P) from ᴅ-fructose and polyphosphate (PolyP), using adenosine 5′-triphosphate or adenosine 5′-monophosphate (AMP) as the regenerating co-factor. The reaction was optimized to achieve 99 % conversion of ᴅ-fructose at substrate concentrations of 300 mmol/L for ᴅ-fructose, with only 0.5 mmol/L AMP (1/600 equiv.) as the co-factor. The solution from the first-step reaction, containing Man-1-P, was simply clarified by centrifugation and then precipitated with calcium salt. Subsequently, Man-1-P was hydrolyzed by a phosphatase to yield ᴅ-mannose, which was purified by desalting with activated carbon adsorption. As a demonstration of this strategy, 11.1 g of ᴅ-mannose was produced with an overall yield of 85.6 %. This synthesis strategy is expected to be applicable for large-scale preparation.