Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2000-04-11
2002-01-22
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S170000, C435S190000, C435S822000, C435S823000
Reexamination Certificate
active
06340582
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method of producing xylitol. Xylitol is useful in the field of food, medicines, and the like.
BACKGROUND ART
The demand for xylitol, which is a sugar alcohol existing in nature, is expected to increase from now on. Xylitol has a lower caloric value than that of sucrose but is sweet as comparable to sucrose. Thus, it is promising as a low caloric sweetener. Furthermore, xylitol is anticariogenic and can be a dental caries-preventing sweetener. Since xylitol does not raise the blood glucose level, it has been used for infusion liquids for treating diabetes.
At present, xylitol is mainly produced in an industrial scale by hydrogenation of D-xylose as described in U.S. Pat. No. 4,008,825. The raw material, D-xylose, can be obtained by hydrolyzing a starting material such as hardwoods, straws, ear stems of corns, crusts of oats, or the other plant-derived materials rich in xylan.
However, D-xylose that is obtained by hydrolyzing the plant materials is disadvantageously expensive because of the high production cost. For example, the yield of the plant material-hydrolyzed product is low, which makes purity of produced D-xylitol low. After the hydrolysis, it is thus necessary to remove the acid used in the hydrolysis and the pigment by the ion exchange treatment. Furthermore, D-xylitol is crystallized to remove other hemicelluloses. Further purification is required to obtain D-xylose that can be used for food. The ion exchange treatment and crystallization results in an increase of the production cost.
In order to solve the above problems, a method of producing xylitol that uses a readily available starting material and that produces a reduced amount of waste matters has been desired. For example, a method of producing xylitol using pentitol as a starting material has been developed. One of the readily available pentitols is D-arabitol that can be produced using yeast (Can. J.
Microbiol
. 31, 1985, 467-471, J.
Gen. Microbiol
. 139, 1993, 1047-1054).
Several methods have been developed for producing xylitol using D-arabitol as a starting material.
Applied Microbiology
, 18, 1969, 1031-1035 reported a method that comprises producing D-arabitol from glucose by fermentation using
Debaryomyces hansenii
ATCC20121, converting D-arabitol thus obtained to D-xylulose using
Acetobacter suboxydans
, and converting D-xylulose to xylitol using
Candida guilliermondii
var. Soya.
EP-A-403392 (applicant: Roquette Freres) and EP-A-421882(applicant: Roquette Freres) each discloses a method which comprises producing D-arabitol by fermentation using an osmotic pressure-resistant yeast, converting D-arabitol thus produced to D-xylulose using a microorganism belonging to the genus Acetobacter, Gluconobacter, or Klebsiella, reacting xylulose thus obtained with glucose (xylose) isomerase to produce a mixture of xylose and xylulose, and converting the thus-formed xylose/xylulose to xylitol by hydrogenation. These publications also disclose a method of preliminarily concentrating xylose in the xylose/xylulose mixture and converting concentrated xylose to xylitol by hydrogenation.
The above-described method of producing xylitol using the D-arabitol above as a starting material enables a high yield production of xylitol. However, it is disadvantageous in requiring plural reaction steps, which makes the process complicated. Thus, the method is not economically satisfactory.
To solve these problems, the present inventors have discovered microorganisms having an ability to converting D-arabitol to xylitol directly and developed a method for producing xylitol characterized by allowing the microorganism to act on D-arabitol to produce xylitol and collecting it (Japanese Patent Application Nos. 9-285455 and 10-9598). This method is excellent in that it can efficiently convert D-arabitol to xylitol in a simple one-step process by a fermentation method. However, it calls for further improvements in the stability of reaction and yield of xylitol.
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide a method of producing xylitol using D-arabitol as a starting material and the method achieved by a simple process.
The present inventors have analyzed microorganisms having an ability to directly converting D-arabitol to xylitol to elucidate that the conversion reaction involves D-arabitol dehydrogenase activity and D-xylulose reductase (xylitol dehydrogenase) activity and found that addition of a carbon source or NADH (a reduced type nicotinamide adenine dinucleotide) in a reaction of converting D-arabitol to xylitol permits xylitol production with high stability and high yield, thus completing the present invention.
That is, the present invention relates to a method for producing xylitol comprising the step of reacting a microorganism having an ability to convert D-arabitol to xylitol with D-arabitol to produce xylitol, wherein a carbon source or NADH to is added to a reaction system.
The above-described method is more specifically a method in which when the microorganism is a microbial cell, the carbon source is added to the reaction system and when the microorganism is a preparation product of cells, the NADH is added to the reaction system. The microorganism includes those which have an D-arabitol dehydrogenase activity and D-xylulose reductase (xylitol dehydrogenase) activity. Specifically, the microorganism includes those which have an ability of metabolizing a carbon source to produce NADH and more specifically those which belong to the genera Gluconobacter or Acetobacter, and in particular
Gluconobacter oxydans
and
Acetobacter xylinum.
The carbon source includes sugars, sugar derivatives, alcohols, aldehydes, organic acids and mixtures thereof. More specifically, the carbon source includes glucose, fructose, sucrose, lactose, sorbitol, glycerol, gluconic acid, methanol, ethanol, propanol, isopropyl alcohol, 1,4-butanediol, 2,3-butanediol, formaldehyde, acetaldehyde, propionaldehyde, isobutyraldehyde, glyceraldehyde, formic acid, acetic acid, citric acid, fumaric acid, malic acid, and mixtures thereof.
According to the present invention, xylitol can be produced from D-arabitol as a raw material in high yields by a simple process.
Hereinafter, the present invention will be described in detail.
<1> Microorganisms Having an Ability to Convert D-arabitol to Xylitol
The microorganisms which can be used in the present invention are microorganisms which have an ability to convert D-arabitol to xylitol. Hereafter, those microorganisms which have such an ability are sometimes referred to as “the microorganisms of the present invention.” As the microorganisms of the present invention, there are cited those microorganisms which have both a D-arabitol dehydrogenase activity which oxidizes D-arabitol to D-xylitol and a D-xylulose reductase (xylitol dehydrogenase) activity which deoxidizes D-xylulose to xylitol. Further, it is preferred that the microorganisms of the present invention have an ability to metabolize a suitable carbon source to produce NADH.
Specific examples of the microorganisms which have both a D-arabitol dehydrogenase activity and a D-xylulose reductase activity as well as an ability to metabolize a suitable carbon source to produce NADH include those bacteria belonging to the genera Gluconobacter or Acetobacter. The Gluconobacter bacteria include
Gluconobacter oxydans
, and the Acetobacter bacteria include
Acetobacter xylinum
. More specifically, there are cited the following strains:
Gluconobacter oxydans
ATCC621
Gluconobacter oxydans
IAM 1839
Gluconobacter oxydans
IAM 1842
Acetobacter xylinum
ATCC14851
Note that the microorganisms which can be used in the present invention may be any one that has the both enzyme activities described above but not limited to the above-described microorganisms.
IAM 1839 and IAM 1942 are available to any person from Institute of Molecular and Cellular Bioscience (formerly, Institute of Applied Microbiology), located at The university of Tokyo, Yayoi 1-chome, Bunkyo-ku, Tokyo, Japan.
T
Mihara Yasuhiro
Mori Maiko
Sugiyama Masakazu
Suzuki Shunichi
Yokozeki Kenzo
Ajinomoto Co. Inc.
Lilling Herbert J.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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