Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Reexamination Certificate
2001-08-23
2004-08-17
Lilling, Dr. Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
C435S187000, C435S252100
Reexamination Certificate
active
06777212
ABSTRACT:
FIELD OF INVENTION
The present invention relates to a novel microbial process for producing L-ascorbic acid and D-erythorbic acid and salts thereof. More specifically, the present invention relates to a process for producing L-ascorbic acid or D-erythorbic acid from 2-keto-L-gulonic acid or 2-keto-D-gluconic acid, respectively, using a thermoacidophilic microorganism. The present invention also relates to a process for producing salts of L-ascorbic acid or D-erythorbic acid from salts of L-gulonic acid or 2-keto-D-gluconic acid, respectively, using a thermoacidophilic microorganism.
BACKGROUND OF THE INVENTION
L-Ascorbic acid (vitamin C) is widely used in health care as well as in preparing food and animal feed, such as, for example, fish feed, and in cosmetics. D-Erythorbic acid is mainly used as an antioxidant for food additives.
L-Ascorbic acid has been produced from D-glucose by the well-known Reichstein method (Helv. Chim. Acta 17, 311-328, 1934). In this multi-step method, L-ascorbic acid is produced chemically from the intermediate 2-keto-L-gulonic acid. The method has been used commercially for more than 60 years, during which time many chemical and technical modifications have been made to improve the efficiency of the steps that produce the intermediates D-sorbitol, L-sorbose, diacetone-L-sorbose, diacetone-2-keto-L-gulonic acid, 2-keto-L-gulonic acid, and methyl 2-keto-L-gulonate, as well as improving the efficiency of the final product, L-ascorbic acid. The conversion of D-sorbitol to L-sorbose is the sole microbial step, the others being chemical steps. The conversion of diacetone-2-keto-L-gulonic acid into L-ascorbic acid has been performed by two different procedures: 1) deprotection to give 2-keto-L-gulonic acid, followed by esterification with methanol and base-catalyzed cyclization; and 2) acid-catalyzed cyclization to L-ascorbic acid directly from the protected or deprotected 2-keto-L-gulonic acid. These conversion processes must be performed in non-aqueous or low-aqueous reaction media. Environmentally and economically, carrying out the reaction in the absence of organic solvents is preferred.
D-erythorbic acid has been produced from D-glucose via 2-keto-D-gluconic acid. 2-keto-D-gluconic acid can be produced by fermentation using a strain belonging to the genus Pseudomonas, and via methyl 2-keto-D-gluconate.
Much time and effort has been devoted to finding other methods of producing L-ascorbic acid using microorganisms. Most studies on the microbial production of L-ascorbic acid have focused on the production of the intermediate 2-keto-L-gulonic acid, particularly from L-sorbose (G. Z. Yin et al., Sheng Wu Hsueh Pao. 20, 246-251, 1980; A. Fujiwara et al., European Patent Publication No.213 591; T. Hoshino et al., U.S. Pat. No. 4,960,695; and I. Nogami et al., European Patent Publication No. 221 707), from D-sorbitol (A. Fujiwara et al., European Patent Publication No. 213 591; T. Hoshino et al., U.S. Pat. No. 5,312,741; M. Niwa et al., W.I.P.O. Publication No. 95/23220; and S. F. Stoddard et al., WO 98/17819), or from D-glucose via 2,5-diketogluconic acid, with a single, mixed, or recombinant culture (T. Sonoyama et al., Appl. Environ. Microbiol. 43, 1064-1069, 1982; and S. Anderson et al., Science 230, 144-149, 1985). The 2-keto-L-gulonic acid can then be converted into L-ascorbic acid by chemical means, as described above.
The use of a biological process for the conversion of 2-keto-L-gulonic acid ester into L-ascorbic acid has recently been reported in Hubbs, U.S. Pat. No. 6,022,719 (“'719 patent”). This patent discloses a process for producing L-ascorbic acid by contacting 2-keto-L-gulonic acid, or an ester thereof, with a hydrolase enzyme catalyst, such as, for example, a protease, an esterase, a lipase, or an amidase. The '719 patent discloses the formation of L-ascorbic acid from an ester of 2-keto-L-gulonic acid, such as, for example, butyl 2-keto-L-gulonate, but not the formation of L-ascorbic acid from 2-keto-L-gulonic acid itself. For example, it discloses that a
Candida antartica
B lipase catalyzed reaction formed 413 to 530 mg/l of methyl 2-keto-L-gulonate, but no L-ascorbic acid, from 1% (w/v) 2-keto-L-gulonic acid in the presence of 8.6% methanol, at a pH of from 3.1 to 3.2, at 38° C. The ester synthetic activity of
Candida antartica
B lipase on 2-keto-L-gulonic acid, an &agr;-keto-carboxylic acid, at an acidic pH, was apparently positive. However, intramolecular ester formation by this lipase was negligible.
In addition to the hydrolase reaction, ester bond synthesis reactions, such as those used for the formation of proteins (amino-esters), fatty acid esters (carboxyl-esters), and nucleotide chains (phospho-esters), are all highly functional in cells. Even in the aqueous phase, these ester bond synthesis reactions proceed unidirectionally, and are seldom inhibited by the product, particularly when compared with the reverse reaction of a hydrolase. These reaction systems require a supply of activated esters, such as, for example, activated transfer ribonucleic acid (tRNA), adenosine triphosphate (ATP), acyl coenzyme A (acyl-CoA), and the like, which are generated by energy-converting metabolism in cells. The “in vitro” reconstitution of these reactions requires a stoichiometric supply, or a regeneration system, of energy donors (e.g., ATP). Such energy donors are expensive to use in the commercial production of vitamins, as well as other chemicals, such as L-ascorbic acid and D-erythorbic acid. Thus, the utilization of intact cells is one of the more preferred commercial methods.
The chemical conversion of 2-keto-L-gulonic acid to L-ascorbic acid via 2-keto-L-gulonic acid &ggr;-lactone is an acid-catalyzed reaction accompanied by the elimination of a water molecule. The principle step in the reaction is a carboxyl ester bond formation to form a &ggr;-lactone ring in a 2-keto-L-gulonic acid molecule. Therefore, especially in the aqueous phase, the final state of the equilibrium reaction is determined by physico-chemical conditions. The productivity of L-ascorbic acid from 2-keto-L-gulonic acid by chemical conversion is considerable, even in the aqueous phase, but it is not sufficient for commercial application. However, performing the process in the aqueous phase, or in an aqueous phase with a low content of an organic solvent, is highly desirable due to its cost effectiveness, and for complying with environmental demands. Accordingly, the biological enhancement of the chemical conversion would be desirable for production in the aqueous phase.
Both high temperature and acidic (i.e., low) pH are desirable reaction parameters for improving the efficiency of the chemical reaction. However, in general, such physico-chemical conditions are known to be biologically incompatible with the cell survival and/or cellular activity of most microorganisms viable under mesophilic conditions. The utilization of thermophilic or acidophilic microorganisms is well known. However, there have been few examples using thermoacidophilic microorganisms which have tolerance to both heat and acid.
SUMMARY OF THE INVENTION
It has now been found that the conversion of 2-keto-L-gulonic acid, as the free acid or as its sodium potassium or calcium salt, to L-ascorbic acid, or the respective salt, in the aqueous phase, can be directly and favorably performed by thermoacidophilic microorganisms under biologically extreme conditions, such as, for example, at high temperature and low (i.e., acidic) pH. It has further been found that the conversion of 2-keto-D-gluconic acid, as the free acid or as its sodium, potassium or calcium salt, to D-erythorbic acid, or the respective salt, in the aqueous phase, can also be directly and favorably performed by thermoacidophilic microorganisms under biologically extreme conditions.
One embodiment of the present invention is a process for producing L-ascorbic acid, or a sodium, potassium or calcium salt thereof from 2-keto-L-gulonic acid, or a sodium, potassium or calcium salt of 2-keto-L-gulonic acid involving: incubating in
Asakura Akira
Hoshino Tatsuo
Shinjoh Masako
Bryan Cave LLP
Lilling Dr. Herbert J.
Roche Vitamins Inc.
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