Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/60—Sugars; Derivatives thereof
  • A61K8/606—Nucleosides; Nucleotides; Nucleic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
  • A61K8/645—Proteins of vegetable origin; Derivatives or degradation products thereof
• • 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
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
  • C12N15/8246—Non-starch polysaccharides, e.g. cellulose, fructans, levans
• • 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)
  • C12N9/2477—Hemicellulases not provided in a preceding group
  • C12N9/2488—Mannanases
  • C12N9/2494—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
• • 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)
  • C12Y302/01078—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to the use of DNA fragments of coffee coding for at least one enzyme involved in the hydrolysis of polysaccharides consisting of at least simple or branched mannan molecules and linked together by a ⁇ bond (l- > 4).
• polysaccharides which contain mannose are frequently present in the cell walls of higher plants in particular in legumes and are considered as a reserve of carbohydrates in seeds.
• galactomannans In the coffee bean, there are notably galactomannans. The latter represent around 24% of the dry weight of the grain (Bradbury and Halliday, J Agric Food Chem 3_8_, 389-392, 1990). These polysaccharides are formed of a linear chain of mannosyl residues which are linked to each other by ⁇ -l-> 4 type bonds and to which monomers of ⁇ -galactosyl residues are attached.
• endo- ⁇ -mannanase (EC 3.2.1.78) is a hydrolase which degrades the polymers of (l-> 4) - ⁇ -mannanes, thus facilitating the exit of the rootlet during germination and releasing small oligosaccharides which are then used as an energy source for the growth of the young plant.
• the invention is therefore intended to provide new means for controlling, modifying and / or restoring the hydrolysis of coffee polysaccharides consisting of at least simple or branched mannan molecules and linked together by a ⁇ bond (l-> 4) .
• the present invention relates to any DNA fragment originating from coffee coding for at least one enzyme involved in the hydrolysis of such polysaccharides.
• the present invention also relates to the use of all or part of such DNA fragments as a primer for carrying out a PCR or as a probe for detecting in vitro or modifying in vivo at least one coffee gene coding for at least one endo- ⁇ - mannanase.
• the present invention also relates to any protein originating from the coffee seed coded by a coffee gene and involved in the hydrolysis of polysaccharides consisting of at least simple or branched mannan molecules and linked together by a ⁇ bond (l-> 4 ) and having the amino acid sequence SEQ ID NO: 2 or any amino acid sequence homologous to the latter.
• Another subject of the invention relates to any microorganism and any plant cell comprising, integrated into its genome or by means of a replicable plasmid, a DNA fragment according to the present invention.
• the invention relates to a food, cosmetic or pharmaceutical composition comprising a DNA fragment or a protein according to the invention.
• FIG. 1 represents the plasmid pMAN1 of 3.58 kb.
• FIG. 2 represents the plasmid pMAN2 of 2.98 kb.
• FIG. 3 represents the plasmid pMAN3 of 3.78 kb.
• FIG. 4 represents the plasmid pMAN4 of 4.56 kb.
• homologous sequence means any nucleic acid or amino acid sequence having an identical function, differing from the sequences according to the invention only by substitution, deletion or addition of a small number of nucleic bases or amino acids, for example
• homologous sequence that which has more than 70% homology with the sequences according to the invention, in particular more than 80% or 90%.
• the homology is determined by the ratio between the number of bases or amino acids of a homologous sequence which are identical to those of a sequence according to the invention, and the total number of bases or d amino acids of said sequence according to the invention.
• fragment which hybridizes means any fragment capable of hybridizing to the fragments according to the invention by the Southern-Blot method (Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, USA, 1989, chapters 9.31 to 9.58).
• the hybridization is carried out under stringent conditions so as to avoid non-specific or unstable hybridizations.
• fragment or "DNA fragment” must be understood as a double stranded DNA of chromosomal origin, which can be synthesized, reproduced in-vitro for example by the known method called "Polymerase Chain
• sequences SEQ ID NO: refer to the sequences presented in the list of sequences below.
• the synthetic oligonucleotides SEQ ID NO: 3 to SEQ ID NO: 7, and SEQ ID NO: 11 to SEQ ID NO: 12 mentioned in the description and presented in the list of sequences below, are provided by Eurogentec (Science Park of Sait Tilman-4102 Seraing-Belgium).
• the present invention relates to any DNA fragment from coffee encoding at least one enzyme involved in the hydrolysis of polysaccharides consisting of at least pure or branched mannan molecules linked together by a ⁇ bond (l-> 4).
• the DNA fragment from the coffee according to the invention codes for at least one endo- ⁇ -mannanase.
• the invention relates to any DNA fragment having the nucleic sequence SEQ ID NO: 1 or any DNA fragment homologous or hybridizing to this nucleic sequence.
• the invention relates to the DNA fragment delimited by nucleotides lia 1294 of the nucleic sequence SEQ ID NO: 1.
• the invention also relates to the new enzymes coded by the genes of the sequence SEQ ID NO: 1, in particular the sequences which are homologous to them. It is thus possible to envisage using them to modify or degrade in vitro such polysaccharides, for example. For this, it is preferable to purify at least one of these enzymes, by conventionally overexpressing their gene in a bacterium and by isolating them conventionally, by precipitation and / or chromatography from the culture medium, for example.
• the invention also relates to the use of all or part of DNA fragments.
• the subject of the present invention is a protein derived from the coffee seed coded by a coffee gene and involved in the hydrolysis of polysaccharides consisting of at least pure or branched mannan molecules linked together by a ⁇ bond (l -> 4) and having the amino acid sequence SEQ ID NO: 2 or any amino acid sequence homologous to the latter.
• the endo- ⁇ -mannanase can contain at least one of the following amino acid sequences: SEQ ID NO: 8 to 10.
• Another object of the present invention relates to a process for the hydrolysis of polysaccharides consisting of at least pure or branched mannan molecules linked together by a ⁇ bond (l-> 4), in which (1) a clone is cloned into a vector.
• DNA fragment coding for the enzyme according to the invention, said vector further comprising a sequence allowing autonomous replication or integration into a host cell, (2) a host cell is transformed by said vector, (3) then the transformed host cell is cultured under conditions suitable for the hydrolysis of such polysaccharides.
• the present invention therefore opens the possibility of using DNA fragments according to the invention to modify the production of polysaccharides consisting at least of pure or branched mannan molecules linked together by a ⁇ bond (l-> 4) in a host cell, in particular a coffee bean cell. It is thus possible to envisage expressing or overexpressing in a coffee bean cell the expression of the DNAs according to the invention, in order to produce such polysaccharides intended to modify the aroma and the structure of the coffee beans, for example.
• the present invention also makes it possible to have new means for identifying coffee genes involved in the hydrolysis of polysaccharides consisting of at least pure or branched mannan molecules linked together by a ⁇ bond (l-> 4).
• the present invention also provides new enzymes involved in the hydrolysis of such polysaccharides. These enzymes can thus be advantageously used to synthesize or modify in vitro such polysaccharides.
• the present invention also relates to a plant cell comprising, integrated into its genome or by means of a recombinant vector, a DNA fragment coding for at least one enzyme involved in the hydrolysis of polysaccharides consisting of at least pure mannan molecules or branched linked together by a ⁇ bond (l-> 4).
• this plant cell comprises a DNA fragment having the nucleotide sequence SEQ ID NO: 1 or a DNA fragment having a nucleic sequence homologous or which hybridizes to the nucleic sequence SEQ ID NO: 1 or a fragment d DNA comprising at least nucleotides 11 to 1294 of the nucleic sequence SEQ ID NO: 1.
• this plant cell is a coffee cell. It is possible in particular to choose, as coffee cells, cells derived from the plant of Coffea canephora var. robusta, Coffea arabica or any other species of the genus Coffea.
• the present invention also relates to any plant or any seed consisting of plant cells comprising, integrated into its genome or by means of a recombinant vector, a DNA fragment coding for at least one enzyme involved in the hydrolysis of polysaccharides constituted by fewer pure or branched mannan molecules linked together by a ⁇ bond (l-> 4).
• Any microorganism comprising, integrated into its genome or by means of a replicable plasmid, a DNA fragment according to the invention, so that it expresses at least one enzyme involved in the hydrolysis of polysaccharides consisting at least pure or branched mannan molecules linked together by a ⁇ bond (l-> 4), is also an object of the present invention.
• Another subject of the invention relates to any food, cosmetic or pharmaceutical composition comprising a DNA fragment according to the invention or a protein according to the invention.
• the present invention relates to a process for treating coffee beans, in which all or part of the protein according to the invention is used.
• the sediments After overexpression of the DNA fragment according to the invention in a microorganism, in a fungus or in an undifferentiated plant cell, the sediments can be treated with the more or less purified enzyme, so as to increase the extraction yields.
• the coffee liquor can also be treated, so as to reduce the sedimentation due to the mannan which gels.
• the total grain RNAs are extracted after 22 days of germination (days after soaking water - JAI 22).
• the grain is rapidly ground in liquid nitrogen and the powder obtained is resuspended in 8 ml of pH 8.0 buffer containing 100 mM Tris HC1, 0.1% w / v SDS and 0.5% v / v of ⁇ -mercaptoethanol, it is homogenized with a volume of phenol saturated with Tris HC1 100 mM pH 8.0, then centrifuged at 12,000 g for 10 min. at 4 ° C., so as to extract the aqueous phase which is centrifuged (i) once with an equivalent volume of phenol, (ii) twice with an equivalent volume of phenol: chloroform (1: 1) and (iii ) twice with an equivalent volume of chloroform.
• the total nucleic acids are then precipitated for 1 h at -20 ° C by adding to the aqueous phase 1/10 by volume of 3M sodium acetate, pH 5.2 and 2.5 volumes of ethanol.
• RNA is then taken up in 500 ⁇ l of H 2 0 and quantified by spectrophotometric assay at 260 nm. Their quality is analyzed by agarose gel electrophoresis in the presence of formaldehyde.
• the poly A + messenger RNAs are then purified from 500 ⁇ g of total RNA using the Oligotex-dT purification system.
• the bacteria which contain recombinant vectors are selected on boxes of LB medium (Luria-Bertani) containing 20 ⁇ g. ml "1 of ampicillin, 80 ⁇ g. ml " 1 of methicillin and in the presence of IPTG and X-Gal (Sambrook et al., 1989). They are then grown on petri dishes, to obtain approximately 300 clones per dish. These clones are transferred to a nylon filter and then treated according to the recommendations provided by Boehringer Mannheim (Boehringer Mannheim GmbH, Biochemica, Postfach 310120, Mannheim 31, DE).
• Plasmids from this cDNA library are also extracted from an overnight culture of these transformants, in the presence of 25 ml of LB medium containing 50 ⁇ g. ml "1 of ampicillin, and using the" QiaFilter Plasmid MidiKit "kit (Qiagen INC., USA).
• the synthesis of the second strand of the cDNA is then carried out by carrying out an RT (Reverse Transcription) -PCR reaction (US Patent 4,683,195 and US Patent 4,683,202) using the synthetic oligonucleotide MAN2, having the nucleic sequence SEQ ID NO: 3, and the synthetic oligonucleotide DT15, having the nucleic sequence SEQ ID NO: 4.
• RT Reverse Transcription
• the synthetic oligonucleotide MAN2 corresponds to the amino acid sequence located between amino acids 206 and 212 of the protein sequence of Lycopersicon esculentum (Bewley et al., Planta 203 r 454-459, 1997) which is also conserved within the endo- ⁇ -mannanase protein sequences of Trichoderma reesei (Stalbrand et al, GenBank Accession Number L25310, 1993) and Aspergillus aculeatus ( Christgau et al, Biochem Mol Biol Internat 917-925, 1994)
• the PCR reaction is carried out in the presence of 1 to 10 ng of first strand of cDNA, in a final volume of 50 ⁇ l containing 50 mM KC1, 10 mM Tris-HCl pH 8.8, 1.5 mM MgCl 2 , 0.1 mg. ml "1 gelatin, 0.2 mM of each dNTP, 0.25 ⁇ M of each oligonucleotide (MAN2 and DT15) and 3 units of Taq DNA polymerase (Stratagene, USA).
• the reaction mixture is covered with 50 ⁇ l of oil mineral and incubated for 35 cycles (94 ° C-30 s, 45 ° C-30 s, 72 ° C-3 min.) followed by a final extension at 72 ° C for 7 min.
• PCR fragment 50 ng of PCR fragment are added to a ligation mixture which comprises 10 mM KC1, 6 mM (NH 4 ) 2 SO 4 , 20 mM Tris-HCl pH 8, 0.1% Triton X-100, 2 mM MgCl 2 , 10 ⁇ g. ml "1 BSA, 10 ng of the cloning vector pPCR-Script SK (+), 5 units of the restriction enzyme Sfr1, 4 units of T4 DNA ligase and 5 M of rATP.
• This reaction is incubated for 60 min. 25 ° C. and is then used to transform the Escherichia coli XL2-Blue MRF 'strain (Stratagene, USA).
• the bacteria which contain recombinant vectors are selected, on LB medium dishes, containing 20 ⁇ g. Ml " of ampicillin , 80 ⁇ g. ml "1 of methicillin and in the presence of IPTG and X-Gal (Sambrook et al, 1989).
• This vector contains the PCR fragment obtained above, which is cloned at the Sfr1 site of the vector pPCR-Script (SK +).
• This cDNA was sequenced according to the “T7 sequencing kit” protocol (Amersham Pharmacia Biotech AB, SE-751 84 Uppsala, Sweden), in the presence of alpha- ( 35 S) -dATP.
• the transformants are transferred to a Nylon Hybond N + filter (Amersham International pic, Amersham place, Little Chalfont, Buckinghamshire HP7 9NA, UK) according to the supplier's recommendations and analyzed by molecular hybridization with the MAN1 probe (50 ng).
• This probe is obtained after digestion of the vector pMAN1 with the restriction enzyme Smai. It was purified on electrophoresis gel and labeled by primer extension at random with 50 ⁇ Ci of alpha- ( 32 P) -dCTP according to the protocol of the Megaprime kit (Amersham, UK). After hybridization, washing and autoradiography of the filters, a positive clone is detected which shelters the recombinant vector pMAN2, described in FIG. 2 below.
• This vector contains a DNA fragment of approximately 1000 bp which has been sequenced on both strands (Eurogentec Bel sa- Parc Scientifique du Sart Tilman - 4102 Seraing- Belgium). It actually comprises the last 1022 base pairs of the sequence SEQ ID NO: 1, but again constitutes only a partial cDNA of the endo- ⁇ -mannanase from coffee.
• a PCR reaction was carried out according to the conditions described above, but with the exception of the following parameters. 20 ng of the plasmid DNA library (22 JAI) were used, the synthetic oligonucleotide MAN60, corresponding to the sequence SEQ ID NO: 5 and the universal oligonucleotides ForM13 and RevM13, which correspond respectively to the sequences SEQ ID NO: 6 and SEQ ID NO: 7. These primers are each located approximately 100 bp on either side of the Sfr1 cloning site of the vector pPCR-Script Amp SK (+).
• the primer MAN60 is itself located between nucleotides 803 and 819 of the sequence SEQ ID NO: 1.
• an amplification fragment [MAN60 / ForM13] of approximately 900 bp was obtained which was digested with the restriction enzyme Smal, in order to eliminate the sequences of the plasmid pPCR-Script Amp SK (+).
• This digested DNA was ligated into the vector pPCR-Script Amp SK (+) to give the vector pMAN3 described in FIG. 3 below.
• Analysis of its sequence reveals that it corresponds to the 5 ′ end of the cDNA coding for coffee mannanase and that it is located between nucleotides 1 and 819 of the sequence SEQ ID NO: 1.
• This enriched library was amplified in Escherichia coli, then was screened using the MAN3 cDNA probe described above which was labeled by primer extension at random according to the protocol of the Megaprime kit (Amersham, UK). At the end of this screen, a positive clone was selected which contains a cDNA of approximately 1600 bp cloned in the vector pPCR Script Amp SK (+). This recombinant plasmid is named pMAN4, described in FIG. 4 below., And the cDNA it shelters has been sequenced. Its analysis in the Genbank database (release 106.0) (Genetics Computer Group Inc., University Research Park, 575 Science Drive, Madison, Wisconsin 53711 USA) showed that it corresponds to the entire sequence SEQ ID NO: 1 .
• the sequence SEQ ID NO: 1 contains an open reading phase of 428 codons, which begins with the ATG codon at position 11 and ends with a TGA codon (A at position 1294).
• the protein deduced from this complementary DNA has an approximate molecular weight of 48349 Da and has a very hydrophobic protein segment which corresponds to the first 30 amino acids of the sequence SEQ ID NO: 1.
• This protein sequence could correspond to a signal peptide type sequence . In this case, the molecular weight of the protein is expected to be less than or equal to 45,000 Da in its mature form.
• the first two are located in the potential signal peptide, in positions 8 and 11 of the sequence SEQ ID NO: 2, and are therefore assumed to be absent in the mature form of mannanase.
• the other two are located in the C-terminal position, in positions 389 and 412 of the sequence SEQ ID NO: 2.
• Test 2 Measurement of the endo- ⁇ -mannanase activity peak during germination
• Grains of the variety Coffea arabica var. caturra 2308 are harvested at the mature stage and treated as defined above during the isolation of the RNAs.
• the powder is homogenized at a rate of 1 g per 5 ml in an extraction buffer (phosphate-citrate 200/100 mM pH 5.0, metabisulfite Na 2 S 2 0 5 10 mM, EDTA 5 mM, protease inhibitor 'Complete' [cat. no 1836 145, Boehringer Mannheim, Mannheim, Germany] 1 tablet / 50 ml) for 20 min. at 4 ° C.
• the homogenate is then centrifuged at
• the reaction begins with the addition of the extract, and proceeds at 37 ° C with stirring.
• an aliquot of 400 ⁇ l of medium is taken every 15 min. for 1 h, heated to 100 ° C for 5 min. then centrifuged at 12000 g for 2 min.
• the optical density of the supernatant is measured at 590 nm and the specific activity is expressed in AU (optical absorption units) min "1 mg protein " 1 , after having determined the protein concentration in each extract by the method of Bradford (Bradford, Anal. Biochem. 22, 248-254, 1976).
• AU optical absorption units
• 1 1 9 endo- ⁇ -mannanase is 0.87 AU min " . Mg " x 10 " , which corresponds to an enrichment of the enzyme by a factor of 4 compared to the crude extract and a recovery of 10 AU min "1 of total activity, or 100%.
• hydrophobic interaction column Separation on hydrophobic interaction column
• the sample described above is then separated on a hydrophobic interaction column (Hiload HR 16/10 phenyl sepharose High performance, Amersham Pharmacia Biotech, Sweden).
• the column is previously equilibrated with an equilibration buffer (50 mM sodium phosphate, 400 mM ammonium sulfate, pH 7.0).
• the sample (1 ml) is then injected onto the column which is then washed with 5 column volumes of the equilibration buffer.
• a gradient from 0 to 99.5% water in 0.5 column volumes is applied followed by another from 99.5 to 100% in 5 column volumes.
• the activity is mainly concentrated in three fractions which are used to continue the purification.
• the purification yield of this step is 79% compared to the previous step.
• the specific activity is 27 AU min “1 , mg " 1 , ie an enrichment of approximately 137 times relative to the crude extract.
• the total activity recovered is approximately 9 AU min "1 x 10 " 2 or 90% of the initial activity.
• the three fractions described above are mixed, concentrated and changed buffer using Ultrafree tubes (Millipore, Bedford, MA, USA, centrifugation at 4000 g maximum).
• the volume recovered is 1 ml.
• This sample is injected into a Resource Q column (Amersham Pharmacia Biotech AB SE-751 84 Uppsala Sweden) anion exchange, previously equilibrated with 20 mM Tris / HCl buffer, pH 8.0. Elution is carried out with a linear gradient from 0 to 1 M NaCl in 20 column volumes.
• the total activity recovered is present exclusively in two fractions.
• the purification yield of this step is 58% compared to the previous step.
• the specific activity is 167 AU min "1 , mg " 1 , ie an enrichment of approximately 836 times relative to the crude extract.
• the total activity recovered is 0.9 AU min "1 , or approximately 9% of the initial activity.
• the two recovered fractions are concentrated by centrifugation at 4000 g at 100 1 using the tubes
• the fractions having the enzymatic activity at the outlet of the columns described above are analyzed during the purification by two-dimensional electrophoresis. To do this, the fractions are mixed and concentrated to 20 ⁇ l by centrifugation in the Ultrafree tubes (Millipore, USA) as described above. To this volume, 105 l of rehydration buffer (8 M urea, CHAPS 3% w / v, 0.8% v / v ampholines, DTT 1% w / v) are added and a non-linear 7 cm gel strip is rehydrated. (pH 3.0 to 10.0) (Immobiline Dry Strip, Amersham Pharmacia Biotech, Sweden), according to the manufacturer's instructions. The proteins are then separated according to their isoelectric point (pi) using, for example, the IPGphore system (Amersham
• the gel strip of the first dimension is balanced in a first solution (urea 6 M, glycerol 30% v / v, SDS 2% w / v, DTT 2%, Tris-HCl 50 mM, pH 8.0) for 5 min ., then is balanced in a second solution (6 M urea, 30% v / v glycerol, 2% w / v SDS, 2.5% w / v iodoacetamide, 50 mM Tris-HCl, pH 8.0) for 10 min.
• a first solution urea 6 M, glycerol 30% v / v, SDS 2% w / v, DTT 2%, Tris-HCl 50 mM, pH 8.0
• a second solution 6 M urea, 30% v / v glycerol, 2% w / v SDS, 2.5% w / v iodoacetamide, 50 mM Tri
• the gel strip is then loaded into a 10-20% acrylamide concentration gradient gel (dimensions 10 x 10 x 0.75 cm) with a single well, and covered with an agarose solution (1% w / v agarose , SDS 0.5% w / v, traces of bromophenol blue) previously heated to 90 ° C and maintained at 40 ° C.
• the gel is mounted in a vertical electrophoresis system and is subjected to a voltage of 170 V for 2 h. After migration, the proteins are stained with silver according to the method of Bjellqvist et al. (Electrophoresis, 14, 1357-1365, 1993).
• the profile of the mixture of the last three fractions thus obtained shows the presence of a single group of proteins which consists of a line of 5 proteins having the same approximate molecular weight of 48 kDa but having slight differences in pi.
• the proteins thus purified are analyzed by micro-sequencing of the amino acids. To do this, they are transferred to a PVDF membrane ('Problot', Perkin Elmer- Applied Biosystems, 850 Lincoln Center Drive, Foster City, CA 94404, USA) using, for example, a Trans-Blot transfer cell (Bio -Rad, 2000 Alfred Drive, Hercules, California 94547, USA).
• a transfer solution methanol 10% v / v, CAPS-NaOH 10 mM pH 11.0
• N-terminal sequencing of purified proteins and internal peptides is carried out with a Beckman automatic sequencer (Beckmann Instruments Inc., 250 Harbor Boulevard Box 3100, Fullerton, California, 92634 USA) according to the methods described in Teixeira et al. (Electrophoresis, 1 &, 1491-1497).
• SEQ ID NO: 8 An N-terminal sequence of 22 amino acids, called SEQ ID NO: 8, and two others relating to independent internal peptides of 10 (called SEQ ID NO: 9) and 17 (called SEQ ID NO: 10) amino acids. All the sequences obtained are unambiguous, and show that the 5 proteins, which make up the line described above, are isozynias of endo- ⁇ -mannanase which share an identical sequence in the regions concerned.
• the three sequences SEQ ID NO: 8 to 10 have a very strong homology with the sequence SEQ ID NO: 2. In particular, the sequence SEQ ID NO: 8 aligns with a strong homology against amino acids 35 to 50 of SEQ ID NO: 2.
• This region corresponds exactly to the N-terminal sequence of endo- ⁇ -mannanase in the coffee bean in its mature form, which begins just after a leader peptide corresponding to amino acids 1 to 34 of SEQ ID NO : 2.
• the internal sequences SEQ ID NO: 9 and SEQ ID NO: 10 align with very strong homology with the regions of SEQ ID NO: 2 corresponding respectively to amino acids 93 to 102 and 259 to 275. From the purification and micro-sequencing analysis described above, it can be concluded that the enzyme having endo- ⁇ -mannanase activity in the coffee seed being germinated shares the same sequence as the protein. described by the sequence SEQ ID NO: 2.
• Test 4 Expression of coffee mannananse in Escherichia coli
• This reaction is carried out in the presence of 50 ng of vector pMAN4, in a final volume of 100 ⁇ l containing 1.5 units of DNA polymerase Pwo (Boehringer Mannheim), 10 ⁇ l of buffer 10 x DNA polymerase Pwo (Boehringer Mannheim), 0.1 mM of each dNTP and 0.1 ⁇ M of each oligonucleotide (TAMl and TAM2).
• the reaction mixture is incubated for 30 cycles (94 ° C-30 s, 40 ° C-60 s, 72 ° C-2 min.) followeded by a final extension cycle at 72 ° C for 7 min.
• the use of the expression vector pQE31 makes it possible to introduce 6 histidines (6 His tag) in phase with the N-terminal end of coffee mannanase which then allows the purification of this recombinant protein after passage through a column of Ni-NTA resin containing Ni 2+ ions (Hochuli et al, J. Chromatography, ell, 177-184, 1987).
• the ligation mixture was used to transform the competent cells of the strain M15 [pREP4] ⁇ Escherichia coli according to the recommendations provided by Qiagen (USA) and the recombinant bacteria were selected on dishes of LB medium containing 25 ⁇ g. ml "1 of kanamycin and 100 ⁇ g. ml " 1 of ampicillin.
• the induction is carried out by the addition of IPTG (final concentration 1 mM) in the culture medium and the culture samples are taken every 30 min.
• the bacteria are lysed and the soluble proteins (Escherichia coli) are extracted under denaturing conditions and separated on a column of Ni-NTA resin following the protocol defined by QIAGEN (QIAexpress System).
• QIAGEN QIAexpress System

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• 1999
  • 1999-10-28 WO PCT/EP1999/008314 patent/WO2000028046A1/fr active Application Filing
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  • 1999-10-28 BR BR9915148-0A patent/BR9915148A/pt not_active Application Discontinuation
• 2001
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