Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-01-19
2001-04-24
Crouch, Deborah (Department: 1632)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S105000, C435S148000, C435S041000, C536S001110, C536S124000, C536S127000
Reexamination Certificate
active
06221634
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to novel microorganisms having an ability to produce xylitol or D-xylulose, and a method for producing xylitol or D-xylulose by using a microorganism having an ability to produce xylitol or D-xylulose. D-Xylulose is useful as a material for the production of xylitol, and xylitol is useful as a sweetener in the field of food industry and the like.
2. Description of the Related Art
The demand of xylitol which is a naturally occurring sugar alcohol is expected to increase in future. Xylitol is a promising low-calorie sweetener because it has lower calories and exhibits comparable sweetness compared with sucrose. In addition, because of its anti-dental caries property, it is utilized as a dental caries preventive sweetener. Furthermore, because xylitol does not elevate glucose level in blood, it is utilized for fluid therapy in the treatment of diabetes. For these reasons, it is expected that the demand of xylitol will increase in future.
The current industrial production of xylitol mainly relies on hydrogenation of D-xylose as disclosed in U.S. Pat. No. 4,008,825. D-Xylose used as a raw material is obtained by hydrolysis of plant materials such as trees, straws, corn cobs, oat hulls and other xylan-rich materials.
However, such D-xylose produced by hydrolysis of plant materials suffers a drawback that it is rather expensive, and it is arisen from high production cost. For example, the low yield of the hydrolysis treatment of plant materials leads to low purity of the produced D-xylose. Therefore, the acid used for the hydrolysis and the dyes must be removed by ion exchange treatment after the hydrolysis treatment, and the resulting D-xylose must be further crystallized to remove other hemicellulosic saccharides. In order to obtain D-xylose suitable for foodstuffs, further purification would be required. Such ion exchange treatment and crystallization treatment invite the increase of production cost.
Therefore, several methods for producing xylitol have been developed, which utilize readily available raw materials and generate little waste. For example, there have been developed methods for producing xylitol utilizing other pentitols as a starting material. One of such readily available pentitols is D-arabitol, and D-arabitol can be produced by using yeast (
Can. J. Microbiol
., 31, 1985, 467-471
; J. Gen. Microbiol
., 139, 1993, 1047-54). As a method for producing xylitol by utilizing D-arabitol as a raw material, there can be mentioned the method reported in
Applied Microbiology
., 18, 1969, 1031-1035, which comprises producing D-arabitol from glucose by fermentation using
Debaryomyces hansenii
ATCC20121, then converting the D-arabitol into D-xylulose using
Acetobacter suboxydans
, and converting D-xylulose into xylitol by the action of
Candida guilliermondii
var. soya.
EP 403 392A and EP421 882A disclose methods comprising producing D-arabitol by fermentation using an osmosis-resistant yeast, then converting D-arabitol into D-xylulose using a bacterium belonging to the genus Acetobacter, the genus Gluconobacter, or the genus Klebsiella, forming a mixture of xylose and D-xylulose from the D-xylulose by the action of glucose (xylose) isomerase, and converting the obtained mixture of xylose and D-xylulose into xylitol by hydrogenation. There is also disclosed the production of xylitol comprising preliminarily concentrating xylose in the mixture of xylose and D-xylulose and converting the xylose into xylitol by hydrogenation.
However, those methods for the production of xylitol mentioned above utilize D-arabitol produced by fermentation as a starting material, and convert it by multiple process steps. Therefore, the processes are complicated, and less satisfactory ones in view of process economy compared with the methods based on extraction.
Accordingly, there has been desired a microorganism which has an ability to produce xylitol or D-xylulose through a single step by fermentation starting from glucose as used in the production of other saccharides a nd sugar alcohols. However, such a bacterium having an ability to produce xylitol or D-xylulose has not been reported so far.
On the other hand, breeding of xylitol fermenting bacteria has been attempted by using gene manipulation techniques. International Publication WO94/10325 discloses production of xylitol from glucose by fermentation by using a recombinant microorganism obtained by introducing an arabitol dehydrogenase gene derived from a bacterium belonging to the genus Klebsiella and a xylitol dehydrogenase gene derived from the genus Pichia into an arabitol fermenting microorganism (yeast belonging to the genus Candida, the gunus Torulopsis, or the genus Zygosaccharomyces). However, while production of 15 g/L of xylitol from 400 g/L of glucose has been reported for the aforementioned recombinant microorganism, it does not reach a practically useful accumulation level. Moreover, the aforementioned recombinant microorganism is introduced with a gene derived from a different species, and ther efore information about its safety cannot be considered sufficient.
SUMMARY OF THE INVENTION
The present invention has been accomplished in view of the aforementioned state of the art, and an object of the present invention is to provide a method for producing xylitol or D-xylulose by utilizing a microorganism having an ability to produce xylitol or D-xylulose from glucose by fermentation.
In order to achieve the aforementioned object, the inventors of the present invention searched for a microorganism having an ability to produce xylitol or D-xylulose from glucose by fermentation. As for the direct production of sugar alcohols by fermentation of microorganisms such as yeasts, there have also been reported production of glycerol by using
Zygosaccharomyces acidifaciens
(Arch. Biochem., 7, 257-271 (1945)), production of erythritol by using a yeast belonging to the genus Trychosporonoides (Trychosporonoides sp., Biotechnology Letters, 15, 240-246 (1964)) and so on, in addition to the aforementioned arabitol fermentation. All of these yeasts having an ability to produce sugar alcohol show osmophilicity, i.e., good growth in a culture medium of high osmotic pressure. Therefore, while any microorganisms having an ability to produce xylitol had not been found among the osmophilic yeasts, the inventors of the present invention considered that a novel microorganism having an ability to produce xylitol should exist among osmophilic microorganisms, and extensively screened osmophilic microorganisms. As a result, the inventors already found microorganisms having an ability to produce xylitol and D-xylulose from glucose among osmophilic microorganisms, and those microorganisms are analogous to known acetic acid bacteria (Japanese Patent Application No. 10-193472).
Based on the above findings, the inventors of the present invention further searched for a microorganism having an ability to produce xylitol or D-xylulose among acetic acid bacteria and analogous bacteria. As a result, they found microorganisms belonging to the genus Gluconobacter, Acetobacter, or Frateuria, and having an ability to produce xylitol or D-xylulose from glucose. Thus, they accomplished the present invention.
That is, the present invention provides a method for producing xylitol or D-xylulose, which comprises culturing a microorganism belonging to the genus Gluconobacter, Acetobacter or Frateuria and having an ability to produce xylitol or D-xylulose from glucose in a suitable medium to accumulate xylitol or D-xylulose in the medium, and collecting xylitol or D-xylulose from the medium.
In a preferred embodiment of the aforementioned method for producing xylitol or D-xylulose according to the present invention, the microorganism belonging to the genus Gluconobacter is selected from
Gluconobacter cerinus
or
Gluconobacter oxydans
, the microorganism belonging to the genus Acetobacter is selected from
Acetobacter aceti, Acetobacter liquefaciens
or
Acetobacter pasteurianus
, and the mic
Takeuchi Sonoko
Tonouchi Naoto
Yokozeki Kenzo
Ajinomoto Co. Inc.
Crouch Deborah
Nguyen Quang
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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