WO2005003356A1 - Endo-$g(b)-(1?6)-galactanase - Google Patents

Endo-$g(b)-(1?6)-galactanase Download PDF

Info

Publication number
WO2005003356A1
WO2005003356A1 PCT/JP2004/000334 JP2004000334W WO2005003356A1 WO 2005003356 A1 WO2005003356 A1 WO 2005003356A1 JP 2004000334 W JP2004000334 W JP 2004000334W WO 2005003356 A1 WO2005003356 A1 WO 2005003356A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
enzyme
galactanase
endo
dna
Prior art date
Application number
PCT/JP2004/000334
Other languages
English (en)
Japanese (ja)
Inventor
Takahisa Kotake
Yoichi Tsumuraya
Original Assignee
Japan Science And Technology Agency
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Japan Science And Technology Agency filed Critical Japan Science And Technology Agency
Publication of WO2005003356A1 publication Critical patent/WO2005003356A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01164—Galactan endo-1,6-beta-galactosidase (3.2.1.164)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a novel end- ⁇ - (1 ⁇ 6) -galactanase ⁇ the endo-1) 3 -— (1 ⁇ 6) -galactanase encoding a novel endogen derived from Trichoderma viride, a kind of soil fungus. And a recombinant vector or transformed plant containing the gene.
  • Plant cell membranes and cell walls are composed of glycoproteins with arabinogalactan structure.
  • This arapinogalactan protein (AGP) is a type of high M r proteodalican, usually composed of less than 10% protein and more than 90% carbohydrates. They are known as extracellular matrix and water-soluble components of the cell wall (Phytochemistry 18, 521-540, 1979, Annu. Rev. Plat Physiol. 34, 47-70, 1983, Int. Rev. Cytol. 174, 195- 291, 1997), or immobilized on the plasma membrane via daricosylphosphatidylinositol (GPI) anchor (Trends Plant Sci. 3, 426-431, 1998, J. Biol. Chem. 274, 14724-).
  • GPI daricosylphosphatidylinositol
  • the protein component of AGP is generally H? It is rich in 1 "0, 8 1 & and 36.
  • the carbohydrate component is mainly composed of L-arabinose (A ra) and galactose (G al).
  • ⁇ 6) jS— (1 ⁇ 3) has a highly branched structure consisting of a single galactan skeleton in which the bond is replaced by a galactosyl side chain. Side chain It is added to O-3 of some galactosyl residues.
  • the alteration serves to label cell identity and signal neighboring cells; cell proliferation, cell hypertrophy, somatic embryogenesis, pollen tube growth, cell death, plant growth, stress response, It fulfills a variety of physiological functions, including reproductive control, and has shown a role for AGPs in some aspects of growth and development (Int. Rev. Cytol. 174, 195-291, 1997).
  • exo-iS— (1 ⁇ 3) galactanase (without EC number) from Irpex lacteus was replaced by radish AGP / 3 / 3— (3 ⁇ 6) —j3 in galactan core. It has been reported that it specifically cleaves one (1 ⁇ 3) one galactoside bond (J. Biol. Chem. 265, 7207-7215, 1990). After the exo action of this enzyme, which can avoid branch points, the side chains can be released as a variety of oligomers with G a1 at the reducing end, generated from the linear backbone of AGP. .
  • enzymes that decompose plant polysaccharides are known to be cellulase, xylase, zincase, mannanase, galaxase, etc. It is used as an enzymatic enzyme.
  • An object of the present invention is to elucidate the functions of AGP, which has various physiological functions such as plant growth, stress response, and reproductive control such as pollen tube growth control Yes, it can be applied to transformed plants such as male sterility, which can be an effective means for breeding of self-fertile plants by heterosis, as well as food processing, pharmaceuticals, and enzymes for producing protoplasts. (1 ⁇ 6) — to provide galactanase and its genes.
  • the present inventors are intensively studying the function elucidation of AGP, but in the course of research aimed at modifying the function of the sugar chain portion of AGP by an external gene, the ⁇ -1 (1 ⁇ 6) 1 galactanase gene Has been found that it can be introduced as an active enzyme gene into plants, and can be used to improve the characteristics of agricultural and horticultural plants. Therefore, ⁇ - (1 ⁇ 6) -galactanase activity was investigated for many commercially available enzymes, and ⁇ - (1 ⁇ 6)-was added to Onozuka R—10, a commercial preparation of cellulase derived from Trichoderma viride.
  • the present invention relates to (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, or (b) one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2
  • a DNA consisting of an amino acid sequence and encoding a protein having endo- 1-(1-6) -galactanase activity (Claim 1), or the base sequence shown in SEQ ID NO: 1 or its complement I8 — (1 ⁇ 6) consisting of a DNA comprising a partial sequence or a sequence comprising part or all of the sequence of the base sequence shown in SEQ ID NO: 1 or its complementary sequence.
  • the present invention relates to a protein (claim 5) comprising the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2 wherein one or several amino acids are deleted, substituted, or added.
  • a protein comprising the amino acid sequence described above and having endo-1— (1 ⁇ 6) -galactanase activity; the protein according to claim 5 or 6; the protein according to claim 5 or 6; Alternatively, the present invention relates to a fusion protein or a fusion peptide (Claim 7) bound to a peptide tag.
  • the present invention provides a recombinant vector containing the DNA of any one of claims 1 to 4 (claim 8) and a transformed plant containing the DNA of any one of claims 1 to 4 (claim 9).
  • Fig. 1 shows the column chromatograph of endo-jS_ (1 ⁇ 6) -galactanase. It is a figure showing the result of the first toography. In the figure, A and CM cellulose; B and Sephadex G-100 are shown, and the column was calibrated using blue dextran (V.) and G1c (Vi). ⁇ Ichio, enzyme activity;, absorbance at 280 nm; ⁇ , concentration of KC 1. Par indicates pooled fractions.
  • FIG. 2 is a photograph showing the results of native PAGE (A) and SDS-PAGE (B) of purified endo] 3_ (1 ⁇ 6) -galactose.
  • Figure Lane (1) evening protein staining; (2) a carbohydrate staining; (3) M r Ma one force one protein; (4) an enzyme.
  • FIG. 3 is a graph showing the time-dependent change of methyl / 3-daricoside in hydrolysis of endo-1 / 3_ (1 ⁇ 6) -galactanase] 3— (1 ⁇ 6) -galactohexaose.
  • the released reducing sugars were derivatized with ABE E and analyzed by HPLC.
  • the arrows in the figure indicate the elution positions of the standard ABEE-derivatized G a1 and DP 2 to 4] 3 — (1 ⁇ 6) galactoligomers.
  • the peak around 23 min is presumed to be ABE derivatized j3 — (1 ⁇ 6) — galactopene. Peaks without diagonal lines around 2-4 minutes are impurities remaining after derivatization.
  • the intensity of UV absorbance at the reaction time of 30 minutes and 120 minutes was reduced to 1Z4 and 1-5, respectively, as compared with the cases of 0 minutes and 3 minutes.
  • Fig. 4 is a photograph showing the results of TLC analysis of the hydrolysis product generated by the action of i3— (1 ⁇ 6) —galacto-oligoside. It is. See the description in Table 4 for reaction conditions.
  • lanes (1) Standard G a1 and iS — (1 ⁇ 6) —galactobiose, ⁇ — (1 ⁇ 6) —galacto triose, and ⁇ — (1 ⁇ 6) —galactotetraose (top) (2) ⁇ - (1 ⁇ 6) galactobiose; (3) ⁇ — (1 ⁇ 6) galactobiose + enzyme; (4) and (5) ⁇ - (1 ⁇ 6) -galactotriose and its hydrolyzate; (6) and (7) / 3- (1 ⁇ 6) 1-galactotetraose and its hydrolyzate.
  • Fig. 5 shows the results of ⁇ -L-arabinofuranosidase-treated AG ⁇ (A), endo- ⁇ - (1 ⁇ 6) -degradation product by galactanase ( ⁇ ), and exo- ⁇ - ( 1 ⁇ 3)
  • This figure shows the results of chromatography of Sephadex G-100 on the degradation product (C) of one galactanase.
  • ⁇ total sugar
  • ⁇ — ⁇ peronic acid
  • ... protein.
  • FIG. 6 shows the full-length end / 3 / 3- (1 ⁇ 6) -galactanase cDNA obtained by 3 ′ and 5 ′ RAC E—PCR, and the full-length end 1 / 3— (1 ⁇ 6)
  • This is a figure showing the amino acid sequence of -galactanase.
  • the underlined region in the figure corresponds to the N-terminal amino acid sequence of end 1 (1 ⁇ 6) -galactanase purified from a commercially available enzyme (Trichoderma viride cellulase Onozuka R-10, Yakult) are doing. Double lines indicate possible sites of glycosylation (N-glycosylation).
  • This gene is very weakly homologous to the family 5 glycolytic enzymes, and the catalytic residues (two dalamic acids) of the common enzyme are boxed.
  • FIG. 7 is a photograph showing the result of electrophoresis of purified recombinant endo-J3- (1 ⁇ 6) -galactanase.
  • Lanes 1 and 5 in the figure are molecular weight markers
  • 2 is a crude extract of recombinant E. coli
  • 3 is a single purification on a chelate column.
  • 4 indicates the recombinant endo-i3_ (1 ⁇ 6) -galactanase also purified twice. As shown in Lane 4, the recombinant enzyme was completely purified.
  • FIG. 8 is a photograph showing the effect of recombinant endo-3 / 3 (1 ⁇ 6) -galactanase on galactooligosaccharides in Examples of the present invention.
  • lane 1 is for galactose and polymerization degree 2 and 3] 3_1,3-galacto-oligosaccharide marker 1
  • lane 2 is for galactose and polymerization degree 2, 3 and 4.
  • 3-1 4-galacto-oligosaccharide marker
  • lane 3 is galactose and the degree of polymerization 2, 3, and 4) 3- (1 ⁇ 6) -galacto-oligosaccharide marker.
  • the recombinant enzyme was used for the polymerization of ⁇ 1,3-galacto-oligosaccharide with a degree of polymerization of 2, 3 / 1,3-galacto-oligosaccharide with a degree of polymerization of 3, and 3-1, 4-galacto-oligosaccharide, degree of polymerization 3/3 / 1,4-galacto-oligo, degree of polymerization 4 3-1,4-galacto-oligosaccharide, degree of polymerization 2 iS— (1 ⁇ 6) -galacto-oligosaccharide, polymerization Degree 3] — (1 ⁇ 6) -galacto-oligo, polymerization degree 4 ⁇ — (1 ⁇ 6) -galacto-oligosaccharide.
  • DNA of the present invention (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or (b) one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2 are deleted, substituted or added.
  • Amino acid sequence, and has endo- / 3- (1 ⁇ 6) -galactanase activity DNA encoding the protein, the DNA consisting of the base sequence shown in SEQ ID NO: 1 or its complementary sequence, or the base sequence shown in SEQ ID NO: 1 or a part or all of the sequence of its complementary sequence DNA that encodes a protein that has a sequence of 1/3 and (1 ⁇ 6) -galactanase activity, or DNA that has the base sequence shown in SEQ ID NO: 1 or its complementary sequence.
  • the DNA is not particularly limited as long as it is a DNA that hybridizes under stringent conditions and encodes a protein having endo- / 3- (1 ⁇ 6) monogalactanase activity. Can be prepared, for example, by the method described in Example 2 described later.
  • a protein that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 or a sequence complementary thereto and has end iS— (1 ⁇ 6) —galactanase activity is coded.
  • a DNA encoding a target protein that is as effective as endo-j3- (1 ⁇ 6) -galactanase can also be obtained.
  • the conditions of the hybridization for obtaining the DNA of the present invention include, for example, hybridization at 42 ° C, and 42 ° C with a buffer containing 1 XSSC and 0.1% SDS.
  • the washing treatment at 65 ° C is more preferable, and the washing treatment at 65 ° C with a buffer containing 0.1 XSSC and 0.1% SDS is more preferable.
  • the protein of the present invention comprises an endo-3- (1 ⁇ 6) single galactanase ⁇ consisting of the amino acid sequence shown in SEQ ID NO: 2, and the amino acid sequence shown in SEQ ID NO: 2 lacking one or several amino acids.
  • the protein is not particularly limited as long as it is composed of a lost, substituted or added amino acid sequence and has an endo-1 / 3- (1 ⁇ 6) monogalactanase activity.
  • the degree of “deletion or addition” and their positions are determined by the fact that the modified protein has the same structure as the endo-] 3 -— (1 ⁇ 6) -galactanase having the amino acid sequence represented by SEQ ID NO: 2.
  • the alteration (mutation) of the amino acid sequence may be caused by, for example, mutation or post-translational modification, but may also be artificially altered.
  • the present invention encompasses all modified proteins having the above-mentioned properties, irrespective of the cause and means of such modification and mutation.
  • the fusion protein of the present invention may be any protein as long as the protein of the present invention is linked to the marker—protein and Z or peptide tag.
  • the marker is not particularly limited as long as it is a marker protein, and examples thereof include alkaline phosphatase, the Fc region of an antibody, HRP, GFP, and the like.
  • Specific examples of peptide tags include peptide tags such as HA, FLAG and Myc, and affinity tags such as GST, maltose binding protein, biotinylated peptide, and oligohistidine.
  • Illustrative such c- fusion proteins can be made by conventional methods, and N i —NT Endo —— (1 ⁇ 6) —Galactanase purification and Affinity of A-His tag. )-It is also useful for quantification of antibodies to galactanase and other research reagents in this field.
  • the recombinant vector of the present invention is not particularly limited, as long as it is a vector containing the DNA of the present invention.
  • Ti recombinant plasmid (Tumor inducing plasmid), pSPORTl, pT7Blue-T vector Specific examples include vectors in which the gene DNA of the present invention is incorporated into a vector such as a plasmid such as tobacco mosaic virus, cauliflower mosaic virus, or a plant virus such as geminivirus.
  • a method for producing the recombinant vector of the present invention a well-known method in the field of genetic engineering can be applied.
  • a base at a predetermined position of the vector whose structure has already been analyzed The recombinant vector of the present invention can be obtained by cutting the sequence with a restriction enzyme and then enzymatically introducing the gene of the present invention into the end of the cut vector.
  • an appropriate control sequence such as a promoter and a terminator may be linked to the DNA of the present invention in advance.
  • a promoter may include, for example, the force reflower mosaic virus 35S promoter
  • a terminator may include, for example, one of the nopaline synthase gene terminators.
  • the recombinant vector of the present invention can be introduced easily and with high probability into a plant cell (receptor cell) to be transformed, and by incorporating the gene DNA of the present invention into the genomic DNA of the plant cell.
  • the transformation can be effected so that the plant cells produce end ⁇ - (1 ⁇ 6) -galactanase. That is, by introducing the recombinant vector of the present invention into cultured cells of various plants, it is possible to obtain cultured plant cells having the ability to produce endo-] 3- (1 ⁇ 6) -galactanase.
  • the present invention By transforming various crops and horticultural plants using the recombinant vector, AGPs with various physiological functions such as plant growth, stress response, and reproductive control, such as the control of pollen tube growth in the original plant, By changing the structure, it is possible to obtain transformed plants such as male sterile plants.
  • the transformed plant of the present invention is not particularly limited as long as it contains the gene DNA of the present invention.
  • the function of controlling the growth of pollen tubes in the original plant is improved.
  • Transgenic plants such as male sterile, in which the structure of AGP, which has various physiological functions such as plant growth, stress response, and reproductive control, have changed, can be obtained.
  • the type of plant used as the transformed plant of the present invention is not particularly limited. Plants such as flowers, fruit plants, vegetables, root vegetables, cereals, houseplants, trees including fruit trees, and cultured cells of these plants Can be selected as appropriate.
  • the recombinant vector of the present invention containing the gene DNA of the present invention is used.
  • the recombinant vector is introduced into a plant cell, and the genomic DNA in the plant cell is transformed into the genomic DNA of the plant cell.
  • the method of introducing the gene DNA can be adopted. Transformation of a plant can be appropriately performed by a known method such as a leaf disk co-culturing method, an electroporation method, an agrobacterium method, and a particle gun method, depending on the type of the plant.
  • a method of physically or chemically increasing the permeability of a plant cell to directly incorporate the gene DNA of the present invention into a receptor cell to prepare a transformed plant may be employed.
  • the present invention will be described more specifically with reference to examples, but the technical scope of the present invention is not limited to these examples.
  • Onozuka R-10 a commercially available cellulase preparation from Trichoderma viride (lot No. 201570), was purchased from Yakult Pharmaceutical Ind. Co., Ltd. Exo—] 3_ (1 ⁇ 3) -galactanase was manufactured by Driselase Power; If (J. Biol. Chem. 265, 7207-7215, 1990). i3-galactosidase (grade III, derived from E. coli) and endo-glycosidase H were obtained from Sigma-Aldrich Japan.
  • Radish AGP (referred to in the literature as AGP-IV), and enzymatically modified products of such radish AGP obtained by degradation with L-arabinofuranosidase from Rhodotorula flava was prepared as previously reported (Plant Physiol. 86, 155-160, 1988).
  • ⁇ - (1 ⁇ 3) -galactan and / 3 -— (1 ⁇ 4) -galactanan were prepared by Smith decomposition of Acacia gam and partial acid hydrolysis of soybean arabinogalactan (Plant Physiol. 90, 567). -574, 1989).
  • the sugar beet Arabinan was prepared by the method of Tagawa and Kaji (Carbohydr. Res. 11, 293-301, 1969).
  • 3-Gal— (1 ⁇ 6) -Gal were prepared from gum acacia.
  • One Gal— (1 ⁇ 3) —Gal was prepared from the sap of a lacquer tree, Rhus vernicifera (Ursi) (Carbohydr. Res.
  • Prototheca zopfii ATCC 1653 3 3 a unicellular organism belonging to Chlorophyceae, was cultured in 3% (w / v) Sabouraud dextrose culture (p. Manners et al. (Carbohydr. Res. 29, 63-77) on agar (2%, wZv) plates containing H6.8; Difco Laboratories) for 5 days under fluorescent light at 25 ° C. , 1973). The cells (15 g by dry weight) were crushed and broken with force-porundum. According to the method of Roy et al. (Mol. Immunol.
  • the polysaccharide fraction was extracted from the mixture with boiling water, precipitated with Et ⁇ H, and 1,1,2- After deproteination by shaking with 1,2,2-trifluoretane, Triclo-mouth, dialyzed against water and lyophilized (yield 2.9 g).
  • 100 mL of 20 mM phosphate buffer (pH 6.8) containing 6 mM NaCl, alpha-amylase (1.3 mg; type I-A, derived from pig kidney) Sigma-Aldrich) was used to decompose the crude polysaccharide at 37 ° C for 1.5 hours (Mol. Immunol. 18, 79-84, 1981).
  • the mixture was heat inactivated, dialyzed against water, and lyophilized (1 (1 ⁇ 6) g).
  • D EAE cellulose - For such polysaccharides, D EAE cellulose -;
  • the chromatography was carried out one (HC 0 3 Serva Feinbiochemica GmbH & Co. , Inc.) column (3. 6 X 2 8 cm) , total sugars (see below) and 2 The absorbance at 80 nm was monitored.
  • the column was washed with water, N a HC 0 3 linear gradient (0 ⁇ 0. 5 M, 2. 2 L) during the period, as a broad peak in the range of 5 0 to 2 5 0 mM, galactosyltransferase Tan Fractions were eluted.
  • the galactan fraction (700 mg) was loaded on a column of Sephadex G-50 (2.6 x 120 cm) and eluted with 1% (v / V) HOAc. The fraction that appeared near the void volume of the column was collected (590 mg) and used for enzyme analysis.
  • a TSQ 70 triple quadrupole mass spectrometer (Finnigan) equipped with a Varian 4300 gas chromatograph was used.
  • XI 50 mm was used to analyze AB EE-derivatized monosaccharides by reversed-phase high-performance liquid chromatography (HPLC).
  • HPLC reversed-phase high-performance liquid chromatography
  • the column was eluted at 45 ° C using 0.2 M potassium borate buffer (pH 9.0) containing 6% MeCN at a flow rate of 1. OmLZ.
  • the fluorescence in the eluate was monitored at 350 nm (excitation) and 360 nm (emission) (Biosci. Biotech. Biochem. 61, 1944-1946, 1997).
  • Endo-3- (1 ⁇ 6) activate galactanase by the enzyme, 0.5% (wZv) of algal galactan, and 0.01% (w / v) of serum albumin (BSA) , And 50 mM acetate buffer (pH 4.3) (0.1 mL). After incubating at 30 ° C for 5 to 20 minutes, the released sugars was determined by reduction analysis. One unit of enzymatic activity liberates 1 ⁇ mo 1 of reducing sugars (as G a 1 equivalent) per minute. Other activities against various polysaccharides were similarly tested in 50 mM acetate buffer (pH 4.6) at 30 ° C. for 4 hours.
  • This enzyme analysis was performed at least twice and the average was recorded.
  • the enzyme was eluted with a linear KC1 gradient (0 to 0.4 M) in the buffer (total volume: 1.4 L, 10 mL / fraction). .
  • the pooled fractions were concentrated to 8 mL using an Amicon ultrafiltration device equipped with a YM-3 membrane. This enzyme solution was equilibrated and eluted with 50 M acetate buffer (PH 5.0) (3 mLZ fraction).
  • PH 5.0 acetate buffer
  • the active fractions were combined, concentrated, and purified using an LKB 8101 column (11 OmL) and a carrier ampholyte (Ampholine; manufactured by LKB Japan) having a pH range of 5 to 8 at a voltage of 600 V.
  • the cells were subjected to isoelectric focusing for 48 hours at a constant voltage. One mL fractions were collected. The active fraction that appeared in the pH range of 5.2 to 5.8 was pooled, concentrated, and equilibrated and eluted with 20 M acetate buffer (pH 5.0). Desalting was carried out by passing through an X 5 lcm Bio-Gel G-10 force ram. The purified active fractions containing endo-1- (1 ⁇ 6) -galactanase were pooled, concentrated and stored at 120 ° C.
  • the Bakerbond QUAT column 1.5 x 21 cm
  • the CBX column (1 5 X 1 lcm)
  • purification of the enzyme is facilitated because the higher binding capacity of these resins to the enzyme protein allows the use of larger amounts of OnozukaR-10 (30 g).
  • the working time for chromatographic analysis can be reduced.
  • Enzyme proteins were measured by the method of Bradford (Anal. Biochem. 72, 248-254, 1976). Protein in radish AGP and algal galactan was measured by the method of McGrath (Anal. Biochem. 49, 95-102, 1972). BSA was used as a standard in both procedures.
  • Enzyme (7 ⁇ g) in 5 O mM citrate-phosphate buffer (PH 5.2, 60 L) containing endo-glycosidase (0.1 milliunit) for 24 hours at 37 ° C And analyzed by SDS-PAGE.
  • Amino acid analysis was performed using the Jasco 880 HPLC system used for dansylated amino acid derivatives.
  • the sample was hydrolyzed with 6 M HC1 for 24 hours at 110 ° C in a vacuum-sealed test tube, and the hydrolyzate was lysed with Dansilui-Dango (Amino-chrome; Ciba Corning Diagnostics Ltd. ). After peroxyformate oxidation, cysteine analysis was performed (Biochem. J. 57, 33-37, 1954). Amino terminal sequence analysis of this enzyme was performed using Shimadzu PSQ-2 Protein Sequencer.
  • the mode of action of the purified galactanase was converted to ⁇ - (1 ⁇ 6) -galactat with 50 mM acetate buffer (pH 4.3, 50 L) containing this enzyme (0.1 fg).
  • Xaose methyl i3-glycoside 250 ig was analyzed by incubating at 30 ° C. for 0-120 minutes. At appropriate intervals, a portion was removed and heat inactivated. Liberated reduction
  • Exo—j3— (1 ⁇ 3) basically the same procedure used for the product obtained by digestion with galactanase alone (J. Biol. Chem. 265, 7207-7215, 1990) The mobilities in single-particle chromatography using A and B, sugar composition, Escherichia coli) The sensitivity to 3-galactosidase and the form of glycosidic bonds were measured and compared with a standard substance. The isolated oligosaccharides were identified. (Result)
  • the galactan produced by Rzopfii contains a small amount ( ⁇ 10%) consisting of a (1 ⁇ 6) -linked i3-GalP residue as the main chain and a single non-reducing terminal Gal al residue. It has a branched structure with side chains (Carbohdr. Res. 29, 63-77, 1973, Mol. Immunol. 18, 79-84, 1981). Therefore, this galactan could be used to study (1 ⁇ 6) binding / 3—G a1p It has been thought that this would be an excellent substrate for measuring the enzymatic activity of hydrolyzing. However, it may be necessary to know more detailed properties of galactan and its degradation products in order to achieve its purpose.
  • the peronic acid content of this fraction was as high as 9% (w / w).
  • the ratio of 0_ 6 binding G a 1 p residue is reduced, the non-reducing end, branching, and O-3 ratio of G a 1 p residues of binding is correspondingly (Table 1).
  • galactan is hardly hydrolyzed by exo_] 3- (1 ⁇ 3) -galactanase (J. Biol. Chem. 265, 7207-7215, 1990), and galactan is transformed into Escherichia coli jS-galactan. It was resistant to tosidases. These results support the idea that galactan is a suitable substrate for the enzyme assay of endo-3_ (1 ⁇ 6) -galactinase.
  • the purified enzyme also did not hydrolyze the following polysaccharides: dextran, arabinan, starch, laminarin, ⁇ - (1 ⁇ 4) galactan and / 3- (1-3) galactan, karamazarappinogalactan Acid-insoluble polygalactic acid (Biosci. Biotech. Biochem. 65, 1519-1527, 2001). (Table 2)
  • Kiyaribure child tio Baiogeru emission was performed P - was measured 1 apparent M r values at 0 0, M r values of the native enzyme is rather low, it was 1 7, 0 0 0.
  • Canon even size exclusion HPLC under denaturing conditions using Asahipak column was calibration, similar value (M r value 1 7, 5 0 0) was obtained.
  • the M r values of the enzyme do not match, the interaction with glycosylated characteristics ⁇ Pi Z or gel matrix of the enzyme It seems to be due to. Similar discrepancies have been observed in endo- (1 ⁇ 6) galactanase isolated from A. niger.
  • the M r values in that case were 60,000 (SDS-PAGE) and 29,000 (gel filtration).
  • the isoelectric point (pi) of this enzyme is pH 5.4 and contains a high proportion of Ser, A1a, Glx, Gly, Thr, and Leu (Table 3).
  • (a) indicates that it was determined as cysteinic acid, and (b) indicates that it was not measured.
  • the amino terminal sequence (11 residues) of this enzyme was DTTL SIDPTSN—.
  • Table 4 summarizes the activities of the galacto-oligosaccharides galactanases and their acidic derivatives with different glycosidic bond types and chain lengths.
  • A In Table 4, hydrolysis was performed by mixing a mixture containing this enzyme (28 ng), various substrates (5 mgZmL each), and acetate buffer (PH4.3) for 10 hours at 30 ° C. After incubation, the mixture was heated in a boiling water path for 3 minutes to terminate the reaction, and analyzed by TLC or paper chromatography. Unreacted and degraded oligomers are indicated by 1 and 10, respectively.
  • the relative rate (%) of the hydrolysis (b) in Table 4 is based on the standard analysis conditions including the enzyme (5 w 120 ng) and the degraded substrate (galactan, 5 mgZmL; each oligomer, 5 mL). Incubated below. At appropriate time intervals, the initial hydrolysis rate was determined reductively. Values were corrected for each time zero blank. The relative velocities are calculated with the algal galactan at 100%. As shown in Table 4, the enzyme hydrolyzes ⁇ _ (1 ⁇ 6) -linked galactoligomers longer than DP3, while ⁇ - (1 ⁇ 3) -linked oligomers and i3- (1 ⁇ 4) The bound oligomer did not act as a substrate.
  • ⁇ — (1 ⁇ 6) The relative rate of hydrolysis of galactotetraose is faster than that of) 6_ (1 ⁇ 6) -galactotriose. With the increase in However, reducing the reducing end group of ⁇ - (1 ⁇ 6) -galactoligomers with sodium borohydride significantly reduced the rate of hydrolysis. (Table 4)
  • galactopene aose indicates the cleavage of the fifth i6— (1 ⁇ 6) —galactosidic bond from the non-reducing end of the substrate molecule, resulting in galactopeneose. Leads to release of ose and methyl / 3-galactoside.
  • Ga1 and galactobiose accumulated as the only hydrolysates. This indicates that this enzyme is end-acting galactanase.
  • G a 1 It also appears to cleave at the linkage of non-reducing and / or reducing terminal galactosyl residues that produce lactobiose. This pattern of action is not very inconsistent with the patterns of some other end-type daricanases.
  • Cryptococcus endo-1 / 3— (1 ⁇ 4) -xylanase is known to cleave its smallest substrate, xylotriose, into xylobiose and xylose.
  • the decomposition of xylotetraose produces not only xylobiose but also xylose. The reason for this is that some xylotrateoses first become xylotriose and xylose by cleavage (Methods Enzymol— (1 ⁇ 6) 0, 638-648, 1988).
  • AGP The relative rate of hydrolysis of native AGP and radish AGP treated with galactanases is based on the relative rate of hydrolysis of algal galactan (100%) as a single body. As 41% and 101%, respectively. The limits of hydrolysis of these two substrates were 8% and 26%, respectively (as G a 1 equivalent to total sugars).
  • ⁇ — 3, 6 A large amount of ⁇ attached to the side chain of galactan skeleton; — L — A raf residue (represents 19% of total sugars in native AGP (Plant Physiol. 86, 155-160, 1988) After removal of)), the sensitivity of native AGP to enzymatic hydrolysis seems to have become even stronger. This behavior is similar to the behavior of A. niger-derived endo-1
  • (b) is the value obtained after reduction of calpoxyl
  • (c) is the value obtained without carboxyl reduction
  • the inside of kazuko is the content of peronic acid determined colorimetrically.
  • d) is less than 1%, and (e) indicates that it could not be detected.
  • (a) shows the percentage of natural AGF based on the sugar content (total 76%)
  • (b) shows the DP number of each
  • (c) shows that the number was not determined. ing. o O
  • Uronic acid content determined colorimetrically in d Less than 1%.
  • the ratio of non-reducing terminal residues, end one jS - (1 ⁇ 6) one junk for high M r fractions obtained after proteinase, methylation data for local Pokishiru reduced samples from unmodified radish AGP It is higher than the ratio of branched Gal residues in Table 5), and the degree of difference tends to increase with the stage of enzymatic degradation.
  • This may reflect the microstructure, chain length, and number of glycan chains along a single polypeptide backbone of AGP.
  • NicotianaA GP there are 9 candidate chains (Carbohydr. Res. 277, 67-85, 1995) with about 95 sugar residues. Has proposed a large number of chains with 30-150 residues (Plant Physiol.
  • Exo-i3— (1 ⁇ 3) The morphology of daricoside bonds in the high-M r fraction recovered after degradation by galactanase was compared with ⁇ -L-arabinosidase and exo_) 3— (1 ⁇ 3) one galactosyltransferase Yunaichize inventors 'previous results for the high M r fractions obtained after continuously decompose radish AGP' at two enzymes that (J. Biol. Chem. 265, 7207-7215, 1990). Previous results showed that such a fraction was recovered in a yield of 7.3% (based on native AGP), with a high proportion of O-6-linked Gal residues (in this study).
  • the enzyme purified from Onozuka R-1 10 is considered to be classified as an end-type galactanase specific to the continuous ⁇ 1 (1 ⁇ 6) -linked G a 1 residue longer than DP3, and the non-reducing end
  • i3— (1 ⁇ 6) -galactoligosaccharides with or without Me—G1cA group Con AGP can release its side chains.
  • E a high M r products key saw ⁇ - (1 ⁇ 3) - Garakutana by applying an Ze, end one ⁇ - (1 ⁇ 6) This effect Pas evening one galactanase was firm for Ichin Evidence was obtained.
  • N-terminal amino acid sequence of Endo ⁇ -(1 ⁇ 6) 1-galactanase purified by the method described in Example 1 was determined from a commercially available enzyme preparation (Trichoderma viride-derived cellulase Onozuka R-10, Yakult). Obtained sequence, N—DTTLTIDPTS NWG TWE GWG VSL AWWAK AFGNRDDL-C (SEQ ID NO: 4), two primers, F—1 (5 ′ one AAYTGGGG NA CNTGGGARGG—3 ′: SEQ ID NO: 5, SEQ ID NO: No.
  • R-2 (5'-GAGT C TGGC CATTG ACTGC-3 ': SEQ ID NO: 7) and R-3 (5,1-GGC CAGAT CA TCTC GGTT G-3': SEQ ID NO: 8)
  • a primer was prepared, and the upstream region was amplified by PCR using a commercially available 5'-RACE kit (Clontech). The PCR reaction was performed under the same conditions as above. As a result, a full-length endo-i3- (1 ⁇ 6) -galactanase cDNA containing 5'-side and 3'-side untranslated regions was obtained.
  • Tv6 GAL cDNA
  • Asp21-Gln439 corresponding to the mature protein (region excluding the signal peptide) was converted to EX- F—2 primer (5′—GGATC CATG GACAC CAC GCTTAC CAT C-3 ′: SEQ ID NO: 9, GG AT CC is restriction enzyme BamHI site) and EX—R—1 primer (5′—GAGCTC AT TGCAACACAACGC— 3 :: SEQ ID NO: 10, GAGCT C is amplified by the restriction enzyme Sac I site and inserted into the pET32a vector (Novagen), a fusion protein expression vector, by the restriction enzyme site introduced by the primers. did.
  • the resulting recombinant protein expression vector was introduced into Escherichia coli BL21 strain, cultured at 10 ° C for 1 week, and then treated with 0.5 mM IPTG for 24 hours to induce the recombinant protein.
  • the recombinant protein was purified by Chelating Sepharose FF column (Amersham Biosciences). Extraction of the recombinant protein and purification by column were performed according to the manufacturer's manual. The purity of the purified recombinant protein was confirmed by SDS-PAGE (SDS _ polyacrylamide gel electrophoresis) (Fig. 7). [Method for evaluating recombinant protein]
  • AGPs arabinogalactan protein
  • the use of i-ze is useful for elucidating the functions of AGPs that have various physiological functions such as plant growth control, stress response, and reproductive control, such as pollen tube growth control. It can be expected to be applied to transformed plants such as male sterility, which can be an effective means in this process, and to food processing, pharmaceuticals, and enzymes for producing protoplasts.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne une nouvelle endo-β-(1?6)-galactanase, un gène codant pour celle-ci, etc. qui sont susceptibles d'être utiles pour clarifier les fonctions d'AGP ayant différentes fonctions physiologiques telles que la régulation de la croissance du tube pollinique et la régulation de la reproduction des végétaux, et de ce fait, susceptibles de s'appliquer à la production de végétaux modifiés (stérilité mâle, etc.), au traitement alimentaire, agents pharmaceutiques et enzymes pour la production de protoplastes. La β-(1?6)-galactanase est purifiée par repli 57 à partir de Onozuka R-10 qui est une préparation de cellulase marquée, et analysée par utilisation de galactane provenant de Prototheca zopfii. En conséquence, elle a une activité maximale à pH 4,3 et hydrolyse spécifiquement des β-(1?6)-galactooligosaccharides avec un degré de polarisation d'au moins 3 et des dérivés présentant un groupe 4-O-méthyl-glucosylurone ou glucosylurone à leur extrémité non réductrice. Ensuite, l'ADNc pleine longueur de cette enzyme est préparée par 3'- ou 5'-RACE-PCR avec utilisation d'ARN provenant des spores de T. viride en tant que matrice.
PCT/JP2004/000334 2003-06-30 2004-01-16 Endo-$g(b)-(1?6)-galactanase WO2005003356A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2003188293A JP2005021046A (ja) 2003-06-30 2003-06-30 エンド−β−（１→６）−ガラクタナーゼ
JP2003-188293 2003-06-30

Publications (1)

Publication Number Publication Date
WO2005003356A1 true WO2005003356A1 (fr) 2005-01-13

Family

ID=33562271

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2004/000334 WO2005003356A1 (fr) 2003-06-30 2004-01-16 Endo-$g(b)-(1?6)-galactanase

Country Status (2)

Country Link
JP (1) JP2005021046A (fr)
WO (1) WO2005003356A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015185689A1 (fr) * 2014-06-04 2015-12-10 Novozymes A/S Composition détergente

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4997487B2 (ja) * 2005-08-03 2012-08-08 独立行政法人農業・食品産業技術総合研究機構 液−液抽出あるいは固−液抽出のための装置およびそれを用いた抽出方法
CN110184379B (zh) * 2019-06-06 2022-12-27 武汉市农业科学院 一种中型无绿藻分子生物学鉴定方法及应用

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
BRILLOUET, J.M. ET AL.: "Purification and Some Properties of a Novel Endo-beta-(1-->6)-D-galactanase from Aspergillus niger", AGRIC. BIOL. CHEM., vol. 55, no. §, 1991, pages 1565 - 1571, XP002984937 *
DATABASE GENBANK [online] 5 September 2003 (2003-09-05), KOTAKE, T. ET AL.: "Trichoderma viride 6GAL mRNA for endo-beta-1,6-galactanase, complete cds", XP002984940, Database accession no. 104898 *
DATABASE GENBANK [online] 5 September 2003 (2003-09-05), KOTAKE, T. ET AL: "endo-beta-1,6-galactanase [Trichoderma viride]", XP002984939, Database accession no. 84995 *
KOTAKE, T. ET AL.: "AGP Tosa Bunkai Kassei o motsu endo-beta-1, 6-galactanase no cloning", DAI 24 KAI THE JAPANESE SOCIETY OF CARBOHYDRATE RESEARCH NENKAI YOSHISHU, 3 July 2003 (2003-07-03), pages 80 - P1-16, XP002984938 *
KOTAKE, T. ET AL.: "Molecular cloning and expression in Escherichia coli of a Trichoderma viride endo-beta-(1-->6)-galactanase gene", BIOCHEM. J., vol. 377, 1 February 2004 (2004-02-01), pages 749 - 755, XP002984941 *
LUONTERI, E. ET AL.: "Purification and characterization of Aspergillus beta-D-galactanases acting on beta-1,4-and beta-1, 3/6-linked arabinogalactans", CARBOHYDRATE POLYMERS, vol. 53, no. 2, 1 August 2003 (2003-08-01), pages 155 - 168, XP004421212 *
OKEMOTO, K. ET AL.: "Purification and characterization of an endo-beta-(1-->6)-galactanase from Trichoderma viride", CARBOHYD. RES., vol. 338, no. 3, 31 January 2003 (2003-01-31), pages 219 - 230, XP004402782 *
SAKURAI, M.H. ET AL.: "Characterization of antigenic epitopes in anti-ulcer pectic polysaccharides from Bupleurum falcatum L. using several carbohydrases", CARBOHYDR. RES., vol. 311, 1998, pages 219 - 229, XP004141933 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015185689A1 (fr) * 2014-06-04 2015-12-10 Novozymes A/S Composition détergente
CN106414698A (zh) * 2014-06-04 2017-02-15 诺维信公司 洗涤剂组合物
EP3152290A1 (fr) * 2014-06-04 2017-04-12 Novozymes A/S Composition détergente
US10647947B2 (en) 2014-06-04 2020-05-12 Novozymes A/S Detergent composition

Also Published As

Publication number Publication date
JP2005021046A (ja) 2005-01-27

Similar Documents

Publication Publication Date Title
Voiniciuc et al. MUCILAGE-RELATED10 produces galactoglucomannan that maintains pectin and cellulose architecture in Arabidopsis seed mucilage
Xu et al. Functional genomic analysis of Arabidopsis thaliana glycoside hydrolase family 1
McNeil et al. Structure and function of the primary cell walls of plants
Tan et al. An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein
Schröder et al. Biochemical and molecular characterisation of xyloglucan endotransglycosylase from ripe kiwifruit
Kotake et al. Molecular cloning of a β-galactosidase from radish that specifically hydrolyzes β-(1→ 3)-and β-(1→ 6)-galactosyl residues of arabinogalactan protein
Voiniciuc et al. Identification of key enzymes for pectin synthesis in seed mucilage
Ogawa‐Ohnishi et al. Identification of three potent hydroxyproline O‐galactosyltransferases in Arabidopsis
Van Damme et al. The NeuAc (α‐2, 6)‐Gal/GalNAc‐Binding Lectin from Elderberry (Sambucus Nigra) Bark, a type‐2 Ribosome‐Inactivating Protein with an Unusual Specificity and Structure
Catalá et al. Characterization of a tomato xyloglucan endotransglycosylase gene that is down-regulated by auxin in etiolated hypocotyls
Sonnewald et al. Targeting and glycosylation of patatin the major potato tuber protein in leaves of transgenic tobacco
Marx et al. Hydrolysis of fructan in grasses: a β-(2-6)-linkage specific fructan-β-fructosidase from stubble of Lolium perenne
Okemoto et al. Purification and characterization of an endo-β-(1→ 6)-galactanase from Trichoderma viride
Doong et al. Cell-free synthesis of pectin (Identification and partial characterization of polygalacturonate 4-[alpha]-galacturonosyltransferase and its products from membrane preparations of tobacco cell-suspension cultures)
Escribano et al. Isolation and cytotoxic properties of a novel glycoconjugate from corms of saffron plant (Crocus sativus L.)
WO2007084711A2 (fr) Utilisation de protéines gr2 pour modifier des matériaux cellulosiques et pour améliorer la modification enzymatique et chimique de cellulose
Hrmova et al. Hydrolysis of (1, 4)-β-D-mannans in barley (Hordeum vulgare L.) is mediated by the concerted action of (1, 4)-β-D-mannan endohydrolase and β-D-mannosidase
JP2898499B2 (ja) エンド型キシログルカン転移酵素遺伝子
Pfeifer et al. The cell walls of different Chara species are characterized by branched galactans rich in 3‐O‐methylgalactose and absence of AGPs
Peyer et al. Purification and characterization of a β-xylosidase from potatoes (Solanum tuberosum)
Zhu et al. Characterization of cell wall polysaccharides from the medicinal plant Panax notoginseng
WO2005003356A1 (fr) Endo-$g(b)-(1?6)-galactanase
Gleeson et al. Alteration in the composition of β-lectins caused by chemical and enzymic attack
Coleman et al. Characterization of a fungal glycoprotein that elicits a defence response in French bean
JP3914348B2 (ja) 植物の細胞壁成分を改変する方法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DPEN Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed from 20040101)
122 Ep: pct application non-entry in european phase