Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2001-11-19
2004-12-14
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
Reexamination Certificate
active
06830915
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to glycosyl hydrolase genes for the biotechnological production of oligosaccharides, especially sulfated oligo-carrageenans and more particularly oligo-iota-carrageenans and oligo-kappa-carrageenans, by the biodegradation of carrageenans.
The sulfated galactans of Rhodophyceae, such as agars and carrageenans, represent the major polysaccharides of Rhodophyceae and are very widely used as gelling agents or thickeners in various branches of activity, especially agri-foodstuffs. About 6000 tonnes of agars and 22,000 tonnes of carrageenans are extracted annually from red seaweeds for this purpose. Agars are commercially produced by red seaweeds of the genera
Gelidium
and
Gracilaria
. Carrageenans, on the other hand, are widely extracted from the genera
Chondrus, Gigartina
and
Eucheuma.
Carrageenans consist of repeat D-galactose units alternately bonded by &bgr;1→4 and &agr;1→3 linkages. Depending on the number and position of sulfate ester groups on the repeat disaccharide of the molecule, carrageenans are thus divided into several different types, namely: kappa-carrageenans, which possess one sulfate ester group, iota-carrageenans, which possess two sulfate ester groups, and lambda-carrageenans, which possess three sulfate ester groups.
The physicochemical properties and the uses of these polysaccharides as gelling agents are based on their capacity to undergo ball-helix conformational transitions as a function of the thermal and ionic environment [Kloareg et al., Oceanography and Marine Biology—An annual review 26: 259-315 (1988)].
Furthermore, carrageenans are structural analogs 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.
In particular, carrageenans are known:
(i)—for their action on the immune system, causing the secretion of interleukin or prostaglandins,
(ii)—for their antiviral action on the AIDS virus HIV1, the herpes virus HSV1 and the hepatitis A virus,
(iii)—as antagonists of the fixation of the growth factors of human cells,
(iv)—and also for their action on the proliferation of keratinocytes and their action on the contractility of fibroblasts.
Furthermore, oligocarrageenans act on the adherence, the division and the protein synthesis of human cell cultures, doubtless as structural analogs of the glycosylated part of the proteins of the extracellular matrix. In plants, oligocarrageenans very significantly elicit enzymatic activities which are markers of growth (amylase) or of the phenolic defense metabolism (laminarinase, phenyl-alanineammonium lyase).
Carrageenans are extracted from red seaweeds by conventional processes such as hot aqueous extraction, and oligocarrageenans are obtained from carrageenans by chemical hydrolysis or, preferably, by enzymatic hydrolysis.
The production of oligocarrageenans by enzymatic hydrolysis generally comprises the following steps:
1) production of a glycosyl hydrolase by the culture of a marine bacterium;
2) enzymatic hydrolysis of the carrageenan with the glycosyl hydrolase thus obtained; and
3) fractionation and purification of the oligocarrageenans obtained.
Microorganisms which produce enzymes capable of hydrolyzing iota- and kappa-carrageenans were isolated by Bellion et al. in 1982 [Can. J. Microbiol. 28: 874-80 (1982)]. Some are specific for &kgr;- or &igr;-carrageenan and others are capable of hydrolyzing both substrates. Another group of bacteria capable of degrading carrageenans was characterized by Sarwar et al. in 1983 [J. Gen. Appl. Microbiol. 29: 145-55 (1983)]. These yellow-orange bacteria are assigned to the
Cytophaga
group of bacteria and some of these bacteria have the property of hydrolyzing both agar and carrageenans.
Purification and characterisation of several &igr;-carrageenases and &kgr;-carrageenases, such as the &igr;-carrageenase and &kgr;-carrageenase of
Cytophaga drobachiensis,
the &igr;-carrageenase of
Alteromonas fortis
and the &kgr;-carrageenase of
Alteromonas carrageenovora,
were described in the thesis of P. Potin [“Recherche, production, purification et caractérisation de galactane-hydrolases pour la préparation des parois d'algues rouges”, (February 1992)]. A detailed study of the &kgr;-carrageenase of
Alteromonas carrageenovora
was described by Potin et al. [Eur. J. Biochem. 228, 971-975 (1995)].
The availability of specific enzymes and tools for obtaining oligocarrageenans by genetic engineering could markedly improve their production.
SUMMARY OF THE INVENTION
The Applicant has now found novel glycosyl hydrolase genes which make it possible specifically to obtain either oligo-iota-carrageenans or oligo-kappa-carrageenans.
Thus the present invention relates to novel 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 to 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].
The present invention further relates to 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% and 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.
In particular, 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].
The glycosyl hydrolase genes of the invention are obtained by a process which consists in selecting proteins 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 to 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,
and in sequencing the resulting genes by the conventional techniques well known to those skilled in the art.
The glycosyl hydrolase genes of the invention can also be obtained by a process which consists in selecting proteins having an HCA score with the kappa-carrageenase of
Alteromonas carrageenovora
which is greater than or equal to 75%, preferably greater than 80% and 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 resulting genes by the conventional techniques well known to those skilled in the art.
Finally, the present invention relates to the use of the above glycosyl hydrolase genes for obtaining, by genetic engineering, glycosyl hydrolases which are useful for the biotechnological production of oligocarrageenans.
The glycosyl hydrolases according to the invention are therefore characterized by the HCA score which they possess with a particular domain of the amino acid sequence of the iota-carrageenase of
Alteromonas fortis
or the kappa-carrageenase of
Alteromonas carrageenovora.
The HCA or “Hydrophobic Cluster Analysis” method is a method of analyzing the sequences of proteins represented as a two-dimensional structure, which has been described by Gaboriaud et al. [FEBS Letters 224, 149-155 (1987)].
It is known that the three-dimensiona
Barbeyron Tristan
Henrissat Bernard
Kloareg Bernard
Potin Philippe
Richard Christophe
Dennison Schultz Dougherty & MacDonald
Laboratoires Goemar S.A.
Patterson Jr. Charles L.
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