Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-12-12
2003-01-28
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S183000, C435S200000, C435S207000, C435S252300, C435S252330, C435S320100, C536S023200, C536S023740
Reexamination Certificate
active
06511838
ABSTRACT:
The present invention relates to two novel genes coding for &bgr;-agarases and to their use for producing agar biodegradation enzymes.
It further relates to the
Cytophaga drobachiensis
strain from which these genes were isolated.
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 the agri-foodstuffs sector. Approximately 6000 tonnes of agars and 22,000 tonnes of carrageenans are extracted annually from marine red algae for this purpose. Agars are produced industrially from red algae of the genera Gelidium and Gracilaria. Carrageenans are widely extracted from the genera Chondrus, Gigartina and Euchema.
Agaro-colloids are polysaccharide complexes consisting mainly of agars and agaroids. Each agaro-colloid has a different content of each of the above compounds, so its gelling strength is different. Agar gel comprises a matrix of double-helix polymer chains held together by hydrogen bonds.
There are two types of enzyme capable of degrading agars: &agr;-agarases and &bgr;-agarases. &bgr;-Agarases act on the &bgr;-1,4 linkage and &agr;-agarases act on the &agr;-1,3 linkage.
Microorganisms which produce enzymes capable of hydrolyzing agars have already been isolated. This capacity to digest agar has been attributed to the genera Pseudomonas (MORRICE et al., Eur. J. Biochem. 137, 149-154, (1983)), Streptomyces (HODGSON and CHATER, J. Gen. Microbiol. 124, 339-348, (1981)), Cytophaga (VAN DER MEULEN and HARDER, J. Microbiol. 41, 431-447, (1975)) and Vibrio (SUGANO et al., Appl. Environ. Microbiol. 59, 1549-1554, (1993)). Several &bgr;-agarase genes have already been isolated. Thus BELAS et al. isolated the gene of an agarase from
Pseudomonas atlantica
(Appl. Environ. Microbiol. 54, 30-37, (1988)). BUTTNER et al. isolated an agarase from
Streptomyces coelicolor
and sequenced the corresponding gene (Mol. Gen. Genet. 209, 101-109, (1987)). SUGANO et al. cloned and sequenced two different agarase genes from Vibrio sp. JT0107, which they called agaA (Appl. Environ. Microbiol. 59, 3750-3756, (1993)) and agaB (Biochimica et Biophysica Acta 1 218, 105-108, (1994)).
The Applicant has now isolated, from the red alga
Delesseria sanguinea,
a bacterial strain which has agarase activity.
This strain was deposited in the DSMZ Collection (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures)) on May 8, 1998 under the number DSM 12170. It forms the first subject of the present invention.
Taxonomic investigation of this strain, performed by techniques well known to those skilled in the art, shows that it belongs to the genus Cytophaga (bacteria of the CFB or “Cytophaga/Flexibacter/Bacteroides” group). In fact, this strain develops by spreading and has yellow colonies encrusted in the agar, which is then liquefied. The bacterium is a Gram-negative bacterium and has the shape of a non-mobile rod of 0.3-0.4×3.0-8.0 &mgr;m×&mgr;m. When a drop of culture of the strain is inoculated at the center of an agar dish, the colony develops with concentric growth of the margin and this mobility is not inhibited by diethyl ether, which is an inhibitor of the flagellar apparatus. The strain is aerobic and has an oxidative metabolism. It produces flexirubin, which is a pigment rarely found in isolates of marine Cytophaga but present in non-marine Cytophaga. It is capable of assimilating various carbon sources and degrading several types of macromolecule such as agar, carrageenan, starch and gelatin.
The Applicant carried out an in-depth study to find out what species this strain belonged to. Thus it determined the percentage guanine and cytosine composition of the DNA of the strain of the invention and found that the values were between 43 and 49%. It also sequenced its 16S DNA by the method well known to those skilled in the art for finding out the taxonomic position of a strain (FOX et al., Int. J. Syst. Bacteriol. 22, 44-57, (1977)). The sequencing result shows that the strain of the invention is very similar to
Cytophaga uliginosa.
(There is a 99% similarity of sequence between the 16S DNA of
C. uliginosa
and that of the strain of the invention.) However, DNA/DNA hybridization between the two strains (45%) shows that they are different species.
Furthermore, the strain of the invention has similar morphological, biochemical and physiological characteristics to the strain
Pseudomonas drobachiensis
nov. comb. isolated by HUMM (Duke Univ. Mar. Stn. Bull. 3, 43-75, (1946)). It was therefore named
Cytophaga drobachiensis.
The Applicant also isolated two genes with &bgr;-agarase activity from
Cytophaga drobachiensis
DSM 12170.
Thus the present invention further relates to the novel agaA gene coding for a &bgr;-agarase, which has the DNA sequence SEQ ID No. 1.
It further relates to the novel agaB gene coding for a &bgr;-agarase, which has the DNA sequence SEQ ID No. 3.
These two genes code for two different &bgr;-agarases produced by
C. drobachiensis
DSM 12170, namely the &bgr;-agarases called proteins AgaA and AgaB.
The present invention further relates to the nucleic acid sequences, namely the genomic DNA sequences and the DNA or mRNA sequences, which comprise or consist of a concatenation of nucleotides coding for the protein AgaA or the protein AgaB or for any one of their peptide fragments as defined below.
The invention therefore relates to:
All the nucleic acid sequences coding for the protein AgaA in its entirety or for one or more of its peptide fragments. These sequences are preferably represented by:
a) the DNA sequence SEQ ID No. 1 coding for the protein AgaA, and its fragments coding for the peptide fragments of said protein;
b) the DNA sequences which hybridize under specific stringency conditions with the above sequence or one of its fragments;
c) the DNA sequences which, because of the degeneracy of the genetic code, are derived from one of the sequences a) and b) above and code for the protein AgaA or the fragments of said protein; and
d) the corresponding mRNA sequences.
All the nucleic acid sequences coding for the protein AgaB in its entirety or for one or more of its peptide fragments. These sequences are preferably represented by:
a) the DNA sequence SEQ ID No. 3 coding for the protein AgaB, and its fragments coding for the peptide fragments of said protein;
b) the DNA sequences which hybridize under specific stringency conditions with the above sequence or one of its fragments;
c) the DNA sequences which, because of the degeneracy of the genetic code, are derived from one of the sequences a) and b) above and code for the protein AgaB or the fragments of said protein; and
d) the corresponding mRNA sequences.
The present invention further relates to the nucleic acid sequence SEQ ID No. 5 coding for the specific peptide fragment AgaA′, which will be described below. This sequence corresponds to nucleic acids 223-1050 of SEQ ID No. 1.
The invention therefore further relates to the nucleic acid sequences coding for said peptide fragment AgaA′ and its peptide fragments, which are represented by:
a) the DNA sequence SEQ ID No. 5 coding for the peptide fragment AgaA′, and its fragments coding for the peptide fragments of said peptide fragment AgaA′;
b) the DNA sequences which hybridize under specific stringency conditions with the above sequence or one of its fragments;
c) the DNA sequences which, because of the degeneracy of the genetic code, are derived from one of the sequences a) and b) above and code for the peptide fragment AgaA′ or the fragments of said fragment; and
d) the corresponding mRNA sequences.
The nucleic acids according to the invention can be prepared by chemical synthesis or genetic engineering using the techniques well known to those skilled in the art and described for example in SAMBROOK et al. (“Molecular Cloning: a Laboratory Manual”, published by Cold Spring Harbor Press, N.Y., 1989).
For e
Barbeyron Tristan
Flament Didier
Kloareg Bernard
Potin Philippe
Yvin Jean-Claude
Laboratoires Goemar S.A.
Prouty Rebecca E.
Rao Manjunath N.
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