Biological process for L-fructose synthesis

#Present dietetic needs, predilections, and perceptions have led to the increased use of artificial sweeteners as a replacement for the "natural" sugars, including sucrose and fructose. Such artificial sweeteners are highly imperfect, including being under continual review for their long term physiological effects, yet their demand has grown unabated. Accompanying their growth as a commercial area with substantial economic impact has been a renewed emphasis on discovering and supplying new artificial sweeteners. Recently the class of L-sugars has been actively explored as a source of new sweeteners, with particular emphasis on L-fructose.

#Exploitation of the favorable properties of L-sugars is hindered by their relative unavailability. L-fructose, for example, is not found to any significant extent in nature. This unavailability has spurred recent efforts in developing commercially feasible methods for preparing L-sugars in amounts necessary for their use as a staple of commerce. U.S. Pat. Nos. 4,371,616 and 4,421,568 describe a method of producing L-sugars, including L-idose and L-glucose, from the readily available D-glucose. Although the preparation of a number of L-sugars is described in U.S. Pat. No. 4,262,032 the focus seems to be on typical laboratory methods wholly unsuited for economical industrial production, in contrast to the process herein. U.S. Pat. No. 4,440,855 uses glucose oxidase to convert L-glucose to L-glucosone. To the extent that there are suitable procedures adaptable to the large scale production of L-glucose as well as the conversion of L-glucosone to L-fructose, the teachings of the patentee relate to the preparation of L-fructose. The subject matter of U.S. Pat. No. 4,207,413 is L-sucrose, the enantiomer of ordinary table sugar, which can be hydrolyzed to afford L-fructose. More recently U.S. Pat. No. 4,467,033 described the oxidation of L-sorbitol to L-fructose using D-iditol dehydrogenase from certain mutants of the genus Pseudomonas.

#The oxidation of D-mannitol to D-fructose using a polyoldehydrogenase is a known process. However, the analogous conversion of L-mannitol to L-fructose has not been described, no doubt because bacterial enzyme systems are constructed to operate largely, if not exclusively, on the naturally occurring D-sugars. It is unclear from the prior art whether a suitable polyoldehydrogenase for L-mannitol oxidation exists, and even if it does the art gives no hint where to find such an enzyme. We have now discovered that species of the genera Gluconobacter and Acetobacter produce a polyoldehydrogenase which readily converts L-mannitol to L-fructose, a discovery leading to our invention which is a method of making L-fructose by oxidizing L-mannitol with said enzyme.

#The example given below is merely illustrative of this invention and is not intended to limit it thereby.

#An aqueous solution (132 g) containing 5.8 wt. % L-glucose and 10.9% L-mannose was hydrogenated in a sealed rotating autoclave at 2000 psig hydrogen at 100.degree. C. for 4 hours using as a catalyst 3.1 g of 1% ruthenium dispersed on carbon. After the reduced mixture was cooled, catalyst was removed from the product by filtration. The filtrate was concentrated by evaporation under vacuum at 50.degree. C. to approximately onehalf of its original volume, at which point a precipitate formed. This precipitate was washed with methanol and was recrystallized twice more from water to yield 2.2 g of material composed of 89.9% L-mannitol, 7.8% L-sorbitol and 2.3% unknown.

#The conversion of L-mannitol to L-fructose by fermentation can be accomplished using such microorganisms as Gluconobacter oxydans ssp. suboxydans, ATCC 23777, and Acetobacter pasteurianus, ATCC 23767. The procedure to produce L-fructose by these bacteria consists of two steps, the preparation of cells followed by fermentation.