A new process for (6S)-tetrahydrofolate production from dihydrofolate was designed that used dihydrofolate reductase and an NADPH regeneration system. Glucose dehydrogenase from Gluconobacter scleroides KY3613 was used for recycling of the cofactor. The reaction mixture contained 200mM dihydrofolate, 220mM glucose, 2mM NADP, 14.4U/ml dihydrofolate reductase, and 14.4U/ml Glucose dehydrogenase, and the reaction was complete after incubation at pH 8.0, and 40°C for 2.5hr. With (6S)-tetrahydrofolate as the starting material, l-leucovorin was synthesized via a methenyl derivative. The purity of the l-leucovorin was 100%, and its diastereomeric purity was > 99.5% d.e. as the (6S)-form.

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