Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-04-08
2001-11-13
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Vector, per se
C435S183000, C435S207000, C435S224000, C435S320100, C435S209000, C536S023100, C536S023200, C536S023740
Reexamination Certificate
active
06316251
ABSTRACT:
TECHNICAL FIELD
This invention relates to a gene encoding cellulose synthase complex originating in
Acetobacter xylinum
subsp.
sucrofermentans
, a gene encoding cellulase, a gene encoding &bgr;-glucosidase (G3ase), and gene group comprising these genes, as well as a novel &bgr;-glucosidase (G3ase) itself.
BACKGROUND ART
It is well known that UDP-glucose is a direct substrate in cellulose biosynthesis of Acetobacter, which is linked together by a membrane protein complex called “cellulose synthase”, and released out of cells. This complex has been reported to consist of four proteins encoded by an operon of cellulose synthase gene, being named bcs A, B, C and D, respectively (H. C. Wong, et al., P.N.A.S., Vol.87, pgs.8130-8134 (1990)). Among these genes, bcsA, B and C genes are known to be essential for cellulose synthesis since their destruction would lose cellulose-producing capacity. It has been reported that bcsd gene also plays an important role since its destruction would cause a significant change of the structure of cellulose (I. M. Saxena, et al., J.Bacteriol., Vol.176, pgs.5735-5752 (1994)). Recently, it has been reported that the second cellulose synthase gene operon was obtained (I. M. Saxena, et al., J.Bacteriol., Vol.177, pgs.5276-5283 (1995)).
The cellulose synthase complex needs di-GMP as a cofactor. di-GMP is synthesized by cyclase and a gene encoding this enzyme has also been reported (R. Tal and D. H. Gelfand, PCT WO93/11244 (1994)).
It has been reported that upstream of the cellulose synthase gene operon, there are a cellulase gene(CMCase) and another gene (R. Standal, et al., J. Bacteriol., Vol.176, pgs.665-672 (1994)).
The present inventors have studied the cellulose synthase complex gene operon of Actobacter, and now succeeded in determinating the base sequences of a series of genes comprising a novel cellulose synthase complex gene operon and cellulase gene, which originate in
Acetobacter xylinum
subsp.
sucrofermentans
. According to our examination of the base sequence of a novel gene downstream of the novel cellulose synthase complex gene operon, we have also found that the nobel gene conserves well the sequence/region that are maintained in &bgr;-glucosidase of various organisms (Y. Kashiwagi, et al., J.Ferment.Bioeng., Vol.78, pgs.394-398 (1994)) and therefore confirmed that this gene is &bgr;-glucosidase.
Further, we have actually purified a protein encoded by the above &bgr;-glucosidase gene, and examined its various properties.
DISCLOSURE OF INVENTION
The present invention relates to a gene encoding a protein constituting a cellulose synthase complex originating in
Acetobacter xylinum
subsp.
sucrofermentans
, particularly to a gene encoding a protein having an amino acid sequence represented by one of SEQ ID NO:2~SEQ ID NO:5.
This invention also relates to a gene encoding a variant protein having cellulose synthase activity and having an amino acid sequence that has been partially changed from that represented by one of SEQ ID NO:2~SEQ ID NO:5 by deletion, replacement or addition of one or a few amino acids. The genes of the present invention are therefore not limited to those originating in
Acetobacter xylinum
subsp.
sucrofermentans.
There may be exemplified as the base sequences of said genes DNA those shown as bcsA, bcsB, bcsC, and bcsD in SEQ ID NO:1. Furthermore, the present invention includes any base sequence or any part thereof prepared by a chemical synthesis and genetic engineering method by taking degeneracy of a genetic codon into consideration, which base sequence encodes the same amino acid sequence.
Also the present invention includes a gene comprising DNA that may hybridize with the above base sequences under stringent conditions, and encode the protein having cellulose synthase activity.
Further, the present invention relates to to a gene encoding cellulose originating in
Acetobacter xylinum
subsp.
sucrofermentans
, particularly to a gene encoding a protein having an amino acid sequence represented by SEQ ID NO:6.
This invention also relates to a gene encoding a variant protein having cellulase activity and having an amino acid sequence that has been partially changed from that represented by SEQ ID NO:6 by deletion, replacement or addition of one or a few amino acids. The gene of the present invention is not limited to that originating in
Acetobacter xylinum
subsp.
sucrofermentans.
There may be exemplified as the base sequence of said gene's DNA that shown as CMCase in SEQ ID NO:1. Furthermore, the present invention includes any base sequence or any part thereof prepared by a chemical synthesis and genetic engineering method by taking degeneracy of a genetic codon into consideration, which base sequence encodes the same amino acid sequence.
Also the present invention includes a gene comprising DNA that may hybridize with the above base sequences under stringent conditions, and encode the protein having cellulase activity.
Further, the present invention relates to &bgr;-glucosidase (G3ase) originating in Acetobacter microorganisms such as
Acetobacter xylinum
subsp.
sucrofermentans
, particularly to a protein having an amino acid sequence represented by SEQ ID NO:7.
The amino acid sequence of this protein is not limited to that of SEQ ID NO:7, but may include that of a variant protein having &bgr;-glucosidase activity and having an amino acid sequence that has been partially changed from that represented by SEQ ID NO:7 by deletion, replacement or addition of one or a few amino acids.
This invention also relates to a gene encoding &bgr;-glucosidase. One of its example is the base sequence shown as &bgr;-glucosidase in SEQ ID NO:1. Furthermore, the present invention includes any base sequence or any part thereof prepared by a chemical synthesis and genetic engineering method by taking degeneracy of a genetic codon into consideration, which encodes the same amino acid sequence.
Also the present invention includes a gene comprising DNA that may hybridize with the above base sequence under stringent conditions, and encode the protein having &bgr;-glucosidase activity.
One of the representatives of
Acetobacter xylinum
subsp.
sucrofermentans
of the present invention is BPR 2001, which has been deposited at the National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305 Japan) on Feb. 24, 1993 under the accession number FERM P-13466, and then transferred on Feb. 7, 1994 to the deposit under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and Regulation under the accession number FERM BP-4545.
Other examples of the microorganisms belonging to Acetobacter may be
Acetobacter xylinum
ATCC23768,
Acetobacter xylinum
ATCC23769,
Acetobacter pasteurianus
ATCC10245,
Acetobacter xylinum
ATCC14851,
Acetobacter xylinum
ATCC11142,
Acetobacter xylinum
ATCC10821; and the like.
The present invention further relates to a gene group comprising the gene (operon) encoding the cellulose synthase complex of the present invention, and the gene encoding &bgr;-glucosidase originating in Acetobacter microorganisms downstream (on 3′-terminal side) of the cellulose synthase gene.
The present gene group may include the cellulase gene of the present invention and/or glucanase gene upstream of the cellulose synthase complex gene (operon). The present gene group may include further various structural genes and regulating genes such as a promoter and operator. Each of these genes is separated by an appropriate number of bases apart from the other genes. For example, &bgr;-glucosidase gene of BPR 2001 strain is located 214-bp downstream of the gene encoding bcsD of the cellulose synthase complex. One embodiment of the base sequence of the present gene group is shown as SEQ ID NO:1. The genes comprised in said gene group, their locations in the base sequence, and intervals therebetween are shown in FIG.
1
.
There exists an open reading frame (ORF2)
Hayashi Takahisa
Tahara Naoki
Tonouchi Naoto
Tsuchida Takayasu
Yano Hisato
Bio-Polymer Research Co., Ltd.
Hutson Richard
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Prouty Rebecca E.
LandOfFree
Gene, group of genes, and novel &bgr;-gluclosidase does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Gene, group of genes, and novel &bgr;-gluclosidase, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Gene, group of genes, and novel &bgr;-gluclosidase will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2572121