Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1999-07-29
2001-06-05
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S155000
Reexamination Certificate
active
06242228
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a novel xylitol dehydrogenase of acetic acid bacteria, gene coding for the same, method for producing xylitol dehydrogenase, and method for producing xylitol. Xylitol is useful in the fields of food industry, drug industry and the like.
BACKGROUND ART
Xylitol, which is a naturally occurring sugar alcohol, is a promising low-calorie sweetener because it has lower calories but exhibits comparable sweetness compared with sucrose. In addition, because of its anti-dental caries property, it can be a dental caries preventive sweetener. Furthermore, because xylitol does not elevate glucose level, it has been utilized for fluid therapy in the treatment of diabetes mellitus. 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. 4,008,285. 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 from hydrolysis of plant materials suffers from 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-xylitol. 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 hemicellulose 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; and
J. Gen. Microbiol.,
139, 1993, 1047-54).
Thus, several methods for producing xylitol that utilize D-arabitol as a raw material have been developed. One method has been reported in
Applied Microbiology,
18, 1969, 1031-1035, wherein D-arabitol is produced from glucose by fermentation using
Debaryomyces hansenli
ATCC20121, then converted into D-xylulose using
Acetobacter suboxydans
, and the D-xylulose is converted into xylitol by the action of
Candida guilliermondii
var. soya.
EP 403 392A and EP421 882A disclose methods which comprise 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, Gluconobacter, or Klebsiella, forming a mixture of xylose and D-xylulose from the D-xylulose by the action of glucose (xylose) isomerase, and converting the produced 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 concentrated xylose into xylitol by hydrogenation.
While those methods for the production of xylitol utilizing D-arabitol as a starting material mentioned above can produce xylitol with a relatively high yield, however, they suffer from a drawback that they requires multiple reaction steps, and hence the processes should become complicated. Therefore, they have not been economically acceptable.
On the other hand, breeding of xylitol fermenting microorganisms has been attempted by using genetic manipulation techniques. International Publication WO94/10325 discloses production of xylitol from glucose through 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 a bacterium belonging to the genus Pichia into an arabitol fermenting microorganism (yeast belonging to the genus Candida, Torulopsis, or Zygosaccharomyces).
However, such breeding of xylitol fermenting microorganisms by using genetic manipulation techniques as mentioned above is not considered to be completed as a practical means.
By the way, xylitol dehydrogenase is an enzyme that catalyzes the reaction producing xylitol from xylulose, and its presence has been known in various organisms. For example, there has been known the presence of xylitol dehydrogenase in yeast species such as
Pichia stipitis
(
J. Ferment. Bioeng.,
67, 25 (1989)),
Pachysolen tannophilus
(
J. Ferment. Technol.,
64, 219 (1986)),
Candida shehatae
(
Appl. Biochem. Biotech.,
26, 197 (1990)),
Candida parapsilosis
(
Biotechnol. Bioeng.,
58, 440 (1998)),
Debaryomyces hansenli
(
Appl. Biochem. Biotech.,
56, 79 (1996)), and
Pullularia pullulans
(
An. Acad. Brasil. Cienc.,
53, 183 (1981)), filamentous bacteria such as
Aspergillus niger
(
Microbiology,
140, 1679 (1994)) and
Neurospora crassa
(
FEMS Microbiol. Lett.,
146, 79 (1997)), algae such as
Galdieria sulphuraria
(
Planta,
202, 487 (1997)), bacteria such as
Morgannela morganil
(
J. Bacteriol.,
162, 845 (1985)), and the like.
As for the xylitol dehydrogenase gene, there have been reported nucleotide sequences of the gene derived from
Pichia stipitis
(
FEBS Lett.,
324, 9 (1993)) and
Morgannela morganii
(DDBJ/GenBank/EMBL accession No. L34345).
However, xylitol dehydrogenase derived from acetic acid bacteria and its gene have not been known so far even for their presence itself.
SUMMARY OF THE INVENTION
The object of the present invention is to provide an enzyme involved in the xylitol biosynthesis of microorganisms excellent in xylitol production ability, genes thereof, and use thereof in order to establish a technique for efficiently producing xylitol or breeding of xylitol fermenting bacteria.
To achieve the aforementioned object, the present inventors searched microorganisms having ability to directly convert D-arabitol to xylitol. As a result, they found that certain bacteria belonging to the genus Gluconobacter or Acetobacter have such ability. Further, they succeeded in purifying two kinds of xylitol dehydrogenase from one of such bacteria,
Gluconobacter oxydans
, and also succeeded in isolating genes coding for these enzymes and determining their structures. Thus, the present invention has been accomplished.
That is, the present invention provides:
(1) a protein defined in the following (A) or (B):
(A) a protein which has the amino acid sequence of SEQ ID NO: 4 in Sequence Listing;
(B) a protein which has the amino acid sequence of SEQ ID NO: 4 in Sequence Listing including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and has xylitol dehydrogenase activity; and
(2) a protein defined in the following (C) or (D):
(C) a protein which has the amino acid sequence of SEQ ID NO: 6 in Sequence Listing;
(D) a protein which has the amino acid sequence of SEQ ID NO: 6 in Sequence Listing including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and has xylitol dehydrogenase activity.
The present invention also provides:
(3) a DNA which codes for a protein defined in the following (A) or (B):
(A) a protein which has the amino acid sequence of SEQ ID NO: 4 in Sequence Listing;
(B) a protein which has the amino acid sequence of SEQ ID NO: 4 in Sequence Listing including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and has xylitol dehydrogenase activity;
(4) a DNA which codes for a protein defined in the following (C) or (D):
(C) a protein which has the amino acid sequence of SEQ ID NO: 6 in Sequence Listing;
(D) a protein which has the amino acid sequence of SEQ ID NO: 6 in Sequence Listing including substitution, deletion, insertion, addition, or inver
Sugiyama Masakazu
Suzuki Shunichi
Tonouchi Naoto
Yokozeki Kenzo
Ajinomoto Co. Inc.
Monshipouri Maryam
Oblon, Spivak, McClelland, Maier & Neustadt, P. C.
Prouty Rebecca E.
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