Efficient biosynthesis of d-allose from d-psicose by cross-linked recombinant l-rhamnose isomerase: Separation of product by ethanol crystallization

Mass production of a rare aldohexose d-allose from d-psicose was achieved in a batch reaction by crude recombinant l-rhamnose isomerase (l-RhI) cross-linked with glutaraldehyde. The d-psicose substrate was, in turn, mass produced from a naturally abundant ketohexose d-fructose by immobilized recombinant d-tagatose 3-epimerase (d-TE). At an equilibrium state, 25% of d-psicose was isomerized to d-allose, that is, 25 g of d-allose was obtained from 100 g of d-psicose. The d-allose product was easily separated and crystallized from the reaction mixture that contains 25% d-allose, 8% d-altrose and 67% d-psicose using ethanol. Empirically, approximately 338 g, that is, 90% of a theoretical overall yield for the purification of pure d-allose crystals was produced from 1.5 kg of d-psicose within 30 d using a constructed bioreactor. The cross-linked enzyme had an operative half-life of two months after repeated usages.