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.