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  • 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)
• • 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)
  • C12Y302/01083—Kappa-carrageenase (3.2.1.83)
• • 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)
  • C12Y302/01157—Iota-carrageenase (3.2.1.157)

Definitions

• the present invention relates to glycosyl hydrolase genes for the biotechnological production of oligosaccharides, in particular of sulfated oligo-carrageenans, more particularly of oligo-iota-carrageenans and of oligo-kappa-carrageenans by biodegradation of carrageenans.
• Rhodophyceae such as agars and carrageenans
• agars and carrageenans represent the major polysaccharides of Rhodophyceae and are very widely used as gelling agents or thickeners in various branches of activity, in particular the food industry.
• About 6,000 tonnes of agars and 22,000 tonnes of carrageenans are extracted annually from marine red algae for this purpose.
• Agars are commercially produced by red algae of the genera Gelidium and Gracilaria.
• Carrageenans are largely extracted from the genera Chondrus, Gigartina and Eucheuma.
• the carrageenans consist of the repetition of D-galactose units alternately linked by ⁇ l-> 4 and ⁇ l-> 3 bonds.
• ester-sulphate groups on the molecule repeat dissacharide, several types of carrageenans are distinguished, namely: kappa-carrageenans which have an ester-sulphate group, iota-carrageenans which have two groups ester-sulfate and lambda-carrageenans which have three ester-sulfate groups.
• carrageenans are structural analogues of the sulfated polysaccharides of the animal extracellular matrix (heparin, chondroitin, keratan, dermatan) and they exhibit biological activities which are related to certain functions of these glycosaminoglycans.
• carrageenans are known:
• HSV1 and hepatitis A as antagonists of the binding of human cell growth factors and also,
• oligocarraghénanes act on the adhesion, the division and the protein synthesis of cultures of human cells, undoubtedly as structural analogues of the glycosylated part of the proteins of the extracellular matrix. In plants, oligocarraghenans very significantly elicit enzymatic activities that mark growth (amylase) or defense phenolic metabolism (laminarinase, phenylalanine-ammonium lyase).
• the carrageenans are extracted from red seaweed by conventional methods, such as hot aqueous extraction and the oligocarraghenans are obtained from carrageenans by chemical hydrolysis or preferably by enzymatic hydrolysis.
• the production of oligo-carrageenans by enzymatic hydrolysis generally includes the steps of:
• Microorganisms producing enzymes capable of hydrolyzing iota- and kappa-carrageenans have been isolated by Bellion et al. in 1982 [Can. J. Microbiol. 28: 874-80, (1982)]. Some are specific for K- OR i-carrageenan and others are capable of hydrolyzing the two substrates. Another group of bacteria capable of degrading carrageenans has been characterized by
• oligo-carrageenans could be improved considerably if we had specific enzymes and tools to obtain them by genetic engineering.
• the Applicant has now found new glycosyl hydrolase genes, which make it possible to specifically obtain either oligo-iota-carrageenans or oligo-kappa-carrageenans.
• the present invention relates to new genes which code for glycosyl hydrolases having an HCA score with the iota-carrageenase of Alteromonas fortis which is greater than or equal to 65%, preferably greater than or equal to 70% and advantageously greater than or equal at 75%, over the domain extending between amino acids 164 and 311 of the sequence [SEQ ID No. 2] of the iota-carrageenase of Alteromonas fortis.
• the present invention relates more particularly to the nucleic acid sequence [SED ID No. 1] which codes for an iota-carrageenase as defined above, the amino acid sequence of which is the sequence [SEQ ID No. 2].
• a subject of the present invention is also the genes which code for glycosyl hydrolases having an HCA score with the kappa-carrageenase of Alteromonas carrageenovora which is greater than or equal to 75%, preferably greater than 80%, advantageously greater than 85%, on the domain extending between amino acids 117 and 262 of the sequence [SEQ ID No. 6] of Alteromonas carrageenovora kappa-carrageenase.
• the invention relates to the nucleic acid sequence [SEQ ID No. 7] which codes for a kappa-carrageenase having a score as defined above, the amino acid sequence of which is the sequence [SEQ ID No. 8].
• glycosyl hydrolase genes of the invention are obtained by the process which consists in selecting proteins which have an HCA score with the iota-carrageenase of Alteromonas fortis which is greater than or equal to 65%, preferably greater than or equal to 70 % and advantageously greater than or equal to 75%, over the domain extending between amino acids 164 and 311 of the sequence [SEQ ID NO: 1]
• glycosyl hydrolase genes of the invention can also be obtained by the method which consists in selecting proteins which have an HCA score with Alteromonas carrageenovora kappa-carrageenase which is greater than or equal to 75%, preferably greater than 80% , advantageously greater than 85%, over the domain extending between amino acids 117 and 262 of the sequence [SEQ ID No. 6] of the kappa-carrageenase of Alteromonas carrageenovora and in sequencing the genes thus obtained according to conventional techniques well known to those skilled in the art.
• the present invention relates to the use of the above glycosyl hydrolase genes for obtaining glycosyl hydrolases by genetic engineering, which are useful for the biotechnological production of oligo-carrageenans.
• the glysosyle hydrolases according to the invention are therefore characterized by the HCA score which they have with a particular domain of the amino acid sequence of the iota-carrageenase of Alteromonas fortis or of the kappa-carrageenase of Alteromonas carrageenovora.
• the HCA method from the English "Hydrophobic Cluster Analysis” is a method for analyzing the sequences of proteins represented in two-dimensional structure, which has been described by Gaboriaud et al. [FEBS Letters 224 149-155 (1987)].
• the two-dimensional representation used in the HCA method is an ⁇ helix where, by computer processing, the amino acids are arranged at the rate of 3.6 residues per revolution.
• the propeller is cut in the longitudinal direction.
• the diagram is duplicated.
• the method uses a code which recognizes only two states: the hydrophobic state and the hydrophilic state.
• Amino acids recognized as being hydrophobic are identified and grouped into characteristic geometric figures. The use of these two states makes it possible to get rid of the tolerance that the two- and three-dimensional structures show vis-à-vis the variability of the primary sequences. In addition, this representation makes it possible to quickly observe interactions over a short or medium distance since the first amino acid and the second amino acid neighboring a given residue are located on a segment of 17 amino acids. Finally, no "window" of predefined length is used, unlike analysis methods based on the primary or secondary structures of proteins.
• the fundamental characteristic of the ⁇ helix representation is that, for a given globular protein or only one domain of this protein, the distribution of the hydrophobic residues on the diagram is not random.
• Hydrophobic residues form clusters of varied geometry and size.
• the hydrophilic and hydrophobic faces of the amphiphilic propellers are very recognizable.
• a horizontal diamond cluster corresponds to the hydrophobic face of an ⁇ helix
• the internal helices appear as large horizontal hydrophobic clusters
• the ⁇ strands as fairly short and vertical hydrophobic clusters.
• the method makes it possible to identify the hydrophobic residues forming the heart of the globular proteins and to locate the elements of secondary structure that are the ⁇ helices and the ⁇ strands, independently of any knowledge of the secondary structure of the protein studied.
• HCA score 2CR / (RC ⁇ + RC 2 ) x 100% where - RCi and RC2 represent respectively the number of hydrophobic residues in the cluster of protein 1 (cluster 1) and the cluster of protein 2 (cluster 2).
• the amino acids are represented by their standard code with a single letter with the exception of proline (P), glycine (G), serine (S), threonine (T).
• Proline introduces strong constraints into the polypeptide chain and is systematically considered as an interruption in the clusters.
• proline residues stop or deform the propellers and the sheets.
• Glycine has a very high conformational flexibility due to the absence of side chain in this amino acid.
• Serine and threonine are normally hydrophilic, but they can also be encountered in hydrophobic environments, such as ⁇ -helices, in which their hydroxyl group loses their hydrophilic nature due to the hydrogen bond formed with the carbonyl group of the main chain. . Within hydrophobic ⁇ sheets, threonine is sometimes capable of replacing hydrophobic residues, thanks to the methyl group of its side chain.
• proteins which have an HCA score with the iota-carrageenase of Alteromonas fortis greater than or equal to 65% on the domain extending between amino acids 164- 311 of said iota- carrageenan are glycosyl hydrolase type enzymes and more particularly iota-carrageenases suitable for the production of oligo-iota-carrageenans from carrageenans.
• Proteins which have an HCA score greater than or equal to 70%, preferably greater than or equal to 75%, with the domain 164-311 above, are particularly preferred for the purposes of the invention.
• a particular example of a glycosyl hydrolase obtained with a gene according to the invention is the protein having the amino acid sequence [SEQ ID No. 2] extracted from Alteromonas fortis.
• glycosyl hydrolase obtained with a gene according to the invention is the protein having the amino acid sequence [SEQ ID No. 4] extracted from Cytophaga drobachiensis.
• the proteins which have an HCA score with the kappa-carrageenase of Alteromonas carrageenovora greater than or equal to 75% on the domain extending between amino acids 117 and 262 of said kappa-carrageenan are glycosyl hydrolase type enzymes, and more particularly kappa-carrageenases suitable for the production of oligo-kappa-carrageenans from carrageenans.
• Proteins which have an HCA score greater than or equal to 80, preferably greater than or equal to 85%, with the domain 117-262 above, are particularly preferred for the purposes of the invention.
• the above proteins are advantageously extracted from marine bacteria.
• glycosyl hydrolase obtained with a gene according to the invention is the protein having the amino acid sequence [SEQ ID No. 6] extracted from Alteromonas carrageenovora.
• Another particular example of a glycosyl hydrolase obtained with a gene according to the invention is the protein having the amino acid sequence [SEQ ID No. 8] extracted from Cytophaga drobachiensis.
• the genes coding for the glycosyl hydrolases according to the invention can be obtained by sequencing the genetic heritage of the bacteria which produce the glycosyl hydrolases, as defined above, according to the conventional methods well known to those skilled in the art. .
• the invention also relates to the expression vectors which carry, with the means for their expression, the nucleic sequences according to the invention.
• These expression vectors can be used to transform prokaryotic microorganisms, in particular Escherichia coli or eukaryotic cells, such as yeasts or fungi.
• prokaryotic microorganisms in particular Escherichia coli or eukaryotic cells, such as yeasts or fungi.
• FIGS. 1 to 4 respectively represent:
• Fig. 1 Alignment according to maximum similarity according to the method of Needleman and Wunsch, [J. Mol. Biol. 48, 443-453 (1970)] of the amino acid sequence of the iota-carrageenan Alteromonas fortis (upper part) and the iota-carrageenan of G drobachiensis (lower part).
• Fig. 2 The HCA profiles of the amino acid sequences of the iota-carrageenases of Cytophaga drobachiensis and Alteromonas fortis.
• Fig. 3 Alignment according to maximum similarity according to the method of Needleman and Wunsch 1970, J. Mol. Biol. 48, 443-453 of the amino acid sequence of the kappa-carrageenase of Alteromonas carrageenovora (upper part) and Cytophaga drobachiensis (lower part).
• Fig. 4 The HCA profiles of the amino acid sequences of the kappa-carrageenases of Cytophaga drobachiensis and Alteromonas fortis.
• the special abbreviations or symbols used in the examples below for amino acids are as follows:
• Threonine I - I Serine:
• Genomic DNA libraries of C. drobachiensis and A. fortis are generated by C. drobachiensis and A. fortis.
• Luria-Bertani medium LB medium
• Culture media agar or not were added with ampicillin (50 / g / ml) or tetracycline (15 ⁇ g / ml), from stock solutions prepared in 50% ethanol (to avoid solidification at the storage temperature, -20 ° C.) except for the non-recombinant DH5 ⁇ strain.
• the expression vector used is the plasmid pAT153 described in
• This plasmid contains two antibiotic resistance genes: a tetracycline resistance gene and a gene which codes for a ⁇ -lactamase, enzyme of the cytoplasmic membrane which degrades ampicillin.
• the total DNA of C. drobachiensis and the total DNA of A. fortis were prepared according to the method described by Barbeyron et al. [J. Bacteriol. 160, 586-590 (1984)].
• the genomic DNAs of C. drobachiensis and A. fortis were cut with the restriction endonucleases, respectively Nd ⁇ ll and Sau3Al. Indeed, in the case of C. drobachiensis, the restriction endonuclease Ndell was used preferentially because the DNA of this bacterium is methylated on the residue C of the sequence GATC.
• Plasmid DNA was isolated from the above five clones by the alkaline lysis method [Nucleic Acid. Res 1: 1513 (1979)]. The sizes and the mapping of the inserts showing i-carrageenase activity were determined by agarose gel electrophoresis after simple and double digestion of their plasmids using various restriction enzymes.
• the DNA fragments were extracted from the agarose by the glass wool method. All the plasmids obtained contain an identical 3.3 kb PVMII fragment.
• the DNA of the clones pIP1 and pIP2 showed inserts of 10.45 kb and 4.125 kb, respectively having a common fragment of 3kb. These clones showed positive i-carrageenase activity. Different fragments were subcloned and spread as described above. However, none of the subclones obtained was found to be i-carrageenase positive.
• SECTION 2 Determination of the nucleotide sequences of the genes coding for the i-carrageenases of Cytophaga drobachiensis and Alteromonas fortis
• This nucleotide sequence is composed of 1,837 bp.
• the translation of the six reading frames revealed only one open phase called cgiA.
• the potential initiation codon is located 333 bp beyond the 5'P end of the sequence.
• the deduced protein sequence [SEQ ID No. 4] from the cgiA sequence is composed of 391 amino acids, which corresponds to a theoretical molecular mass of 53.4 kDa.
• the hydropathic profile of this protein shows a hydrophobic region covering the first 24 amino acids.
• the presence of a positively charged amino acid (Lys) followed by a hydrophobic body, then a polar segment of six amino acids suggests that this domain could be a signal peptide.
• the analyzes carried out according to the method of Von Heijne J. Mol. Biol. 184: 99-105 (1985)]
• the signal-peptidase would cut between valine (Val 24 ) and threonine (Thr ⁇ 5).
• the mature protein devoid of its signal peptide would have a theoretical molecular mass of 50.7 kDa.
• the identity of the cgL gene was confirmed by the determination of amino acids on the NH 2 side of the partially purified protein.
• the sequence obtained conforms to that deduced from the nucleotide sequence.
• the first amino acid is located 14 residues from the NH 2 end generated by the signal-peptidase.
• the presence of the two prolines which follow the amino acids determined by microsequencing having slightly disturbed the order of appearance of the N-terminal residues, the sequence of an internal oligopeptide, purified by HPLC after cleavage with trypsin, has been established.
• the NH2ATYKCOOH sequence obtained is located towards the C-terminal end of the iotase (residues 396 to 399).
• the plasmids pIHP15 and pIHPX17 subcloned from pIP1 and pIP2 were used to determine the nucleotide sequence of the gene responsible for the i-carrageenan activity of Alteromonas fortis SEQ ID No 1.
• the 2085 bp fragment contains a single phase open reading of 1,473 bp called cgiA.
• the sequence located upstream of the initiator codon (ATG ⁇ H) is not coding.
• the protein sequence deduced from the sequence of the A. i-carrageenase gene. fortis [SEQ ID No. 2] consists of 491 amino acids, which corresponds to a theoretical molecular mass of 54.802 kDa.
• the N-terminal portion of this protein a large hydrophobicity suggesting that this field could be a signal peptide
• the hypothetical cleavage site would be between glycine (Gly26) e t alanine (Ala27) .
• the mature protein devoid of its signal peptide would have a theoretical molecular mass of 51.95 kDa, which corresponds to a value similar to the molecular mass obtained with the protein purified by SDS-PAGE of 57 kDa.
• HCA between the two proteins is 82% over a domain of 293 amino acids and reaches 90.5% in the case of said domain 164-311 (Fig. 2).
• Alteromonas carrageenovora ATCC 43555 was obtained from the American Type Culture Collection. The A. carrageenovora and C. drobachiensis strains were cultivated under conditions identical to those mentioned in section 1 of Example I. Similarly, genomic libraries were produced using the Escherichia coli DH5 strain and the plasmid vector. pAT153. 1. Cloning from Alteromonas carrageenovora
• the plasmids pKA1 to pKA4 were purified from the four independent clones and mapped using the restriction endonucleases BamHI, Oral, EcoRl, Hindlli, MM, Pstl, Pvull, Sali, Sspl, Xbal and Xhol. In each plasmid, the presence of a Dral- fragment was noted
• This common fragment which is the entire insert of the plasmid pKA3, was entirely sequenced from the plasmid pKA3.
• E. coli called pKCl to pKC5 were able to dig a hole in the substrate.
• the plasmids isolated and purified from said clones were mapped with restriction endonucleases.
• the plasmids pKCl, pKCEll and pKCN ⁇ were used to determine the nucleotic sequence of the kappa-carrageenase gene.
• SECTION 2 Determination of the sequences of the genes coding for the kappa-carrageenases of Alteromonas carrageenovora and
• the translation product of the cg 4 gene corresponds to a protein of 397 amino acids for a theoretical molecular mass of 44,212 Da (SEQ ID NO:
• the 4,425 bp pKCl insert contains a single 1,635 bp open reading frame called cgkA (SEQ ID NO: 7).
• the protein translated from the kappa-carrageenase gene is a protein comprising 545 amino acids with a molecular mass of 61.466 kDa [SEQ ID N ° 8]
• the hydropathic profile of this protein shows a highly hydrophobic domain at the N-terminus, suggesting that it is a signal peptide.
• the site of cleavage of the signal peptidase must be between threonine and serine at the respective positions 35 and 36, with codon ATG 75 as the initiating codon.
• the K-carrageenan of C. drobachiensis is similar to 36.1% over the entire alignment in linear sequences with the K-carrageenan of Alteromonas carrageenovora.
• HCA analysis shows an HCA score between the two proteins of 75.4% on said domain of 145 amino acids (Fig. 4).
• the HCA analysis also shows that these two proteins belong to the family 16 of glycosyl hydrolases which includes endoxyglucan transferases (XET), laminarinases, lichenases and agarases.
• XET endoxyglucan transferases
• the HCA score of the two kappa-carrageenases with XETs is 67.5%, 67.6% with laminarinases, 73.7% with lichenases and 71.5% with agarases.
• NAME LABORATOIRES GOEMAR S.A.
• AAGCTTTCCG ATTCTATCAT CGAAGTCATA GGAGTGGGTA AACAAAAAAG CATGAAACTA 60 GCTTTTTAAA ATACAGACTT TCAATATAGG TCGCACACAA TATTAACGAA TAAATAAGCA 120 AATCATATAC ATAATCATTG CTTTAAATAT GTTTTAATAC AGATATAAAC ATAGTATGTT 180 TGTGTTTTTG GTATCTATCG GAGTGAAAAC ATG CGC TTA TAT TTT AGA AAG TTG 234
• GGC AAA AAC CAC CGA CTA TTT GAA GTT GGC GTA AAC AAT ATT GTA AGA 618 Gly Lys Asn His Arg Leu Phe Glu Val Gly Val Asn Asn Ile Val Arg 125 130 135
• GCT AAA AAA GGG GGG GTA AGG GAT ATT TTT GCC ACA AAG ATC AAG AAT 1169 Ala Lys Lys Gly Gly Val Arg Asp Ile Phe Ala Thr Lys Ile Lys Asn 265 270 275
• GGT AAA GTG ACC ATA GAT GAT GTA ACC GCC ATC GGT TGT GCA TAT GCC 1265 Gly Lys Val Thr Ile Asp Asp Val Thr Ala Ile Gly Cys Ala Tyr Ala 300 305 310
• GCA AGT GCC GAC GCT TTC AAG AAC TAT ATT GAA GGT ATT CTA GGA GCT 1361 Ala Ser Ala Asp Ala Phe Lys Asn Tyr Ile Glu Gly Ile Leu Gly Ala 330 335 340
• GCA AAT TAC CCA CTT TAT TAT ACA TCG
• GGT GTC GCT AAA TCC
• AGA GCT 1097 Ala Asn Tyr Pro Leu Tyr Tyr Thr Ser Gly Val Ala Lys Ser Arg Ala 270 275 280 285
• CTTCCTTCCC TTTGGGAACC TATGGTACAG ACTTGCCTTT TTTAAACCGG TTACTTCAGC 360
• GTA GCC AAT GGG GAA ACG
• GTA TAC AGT GAA ATA
• GAT GTA GTT GAA CTA 1372
• Val Ala Asn Gly Glu Thr Val Tyr Ser Glu Ile Asp Val Val Glu Leu 155 160 165

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