Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-07-02
2001-03-27
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S183000, C435S195000, C435S200000, C536S023200
Reexamination Certificate
active
06207436
ABSTRACT:
The present invention relates to an endo-&bgr;-1,4-glucanase preparation derived from a Saccharothrix strain, an isolated polynucleotide molecule encoding an endo-&bgr;-1,4-glucanase, an isolated enzyme produced by recombinant techniques, and use of the preparation or the enzyme in the detergent, paper and pulp, oil drilling, oil extraction, wine and juice, food ingredients, animal feed and textile industries.
BACKGROUND OF THE INVENTION
Cellulose is a polymer of glucose linked by &bgr;-1,4-glucosidic bonds. Cellulose chains form numerous intra- and intermolecular hydrogen bonds, which result in the formation of insoluble cellulose microfibrils. Microbial hydrolysis of cellulose to glucose involves the following three major classes of cellulases: (i) endoglucanases (EC 3.2.1.4) which cleave &bgr;-1,4-glucosidic links randomly throughout cellulose molecules; (ii) cellobiohydrolases (EC 3.2.1.91) which digest cellulose from the nonreducing end, releasing cellobiose; and (iii) &bgr;-glucosidases (EC 3.2.1.21) which hydrolyse cellobiose and low-molecular-mass cellodextrins to release glucose.
Cellulases are produced by many microorganisms and are often present in multiple forms. Recognition of the economic significance of the enzymatic degradation of cellulose has promoted an extensive search for microbial cellulases which can be used industrially. As a result, the enzymatic properties and the primary structures of a large number of cellulase have been investigated. On the basis of the results of a hydrophobic cluster analysis of the amino acid sequence of the catalytic domain, these cellulases have been placed into different families of glycosyl hydrolases; fungal and bacterial glycosyl hydrolases have been grouped into 35 families (Henrissat et. al. (1991), (1993)). Most cellulases consist of a cellulose-binding domain (CBD) and a catlytic domain (CAD) separated by a linker which may be rich in proline and hydroxy amino residues. Another classification of cellulases has been established on the basis of the similarity of their CBDs (Gilkes et al. (1991)) giving five families of glycosyl hydrolases (I-V).
Cellulases are synthesized by a large number of microorganisms which include fungi, actinomycetes, myxobacteria and true bacteria but also by plants. Especially endo-&bgr;-1,4-glucanases of a wide variety of specificities have been identified. Many bacterial endoglucanases have been described (Henrissat (1993); Gilbert et al.,(1993)).
According to the NCBI Taxonomy Browser (Internet address: http://www.ncbi.nlm.nih.gov/Taxonomy/tax.html), the taxonomic family Pseudonocardiaceae belongs to the order of Actinomycetes under the class of gram-positive bacteria (Firmicutes) and covers the genera Actinokineospora, Actinopolyspora, Amycolata, Amycolatopsis, Kibdelosporangium, Pseudonocardia, Saccharomonospora, Saccharopolyspora, Saccharothrix, and Thermocrispum. According to Taxonomy of bacteria and Archaea refers to the Ribosomal Database Project (B. L. Maidak et al. (1996)
Nucleic Acids Res.
24:82-85), the Gram-Positive Phylum, High mol % G+C Subdivision comprises Streptomyces group (e.g.
Kitasatosporia setae, Streptomyces albogriseolus, Streptomyces lividans, Streptomyces celluflavus, Streptomyces flavogriseus
and
Streptomyces griseus
), Arthrobacter group (e.g.
Cellulomonas fimi, Cellulomonas gelida, Cellulomonas flavigena
and
Cellulomonas uda
), and Sachharopolyspora group (e.g.
Thermomonospora curvata, Thermomonospora mesouviformis micromonospora inositola, Saccharothix australiensis, Saccharothrix longispora, Saccharothrix mutabilis, Amycolatopsis mediterranei
and
Nocardiopsis albus
).
A very important industrial use of cellulolytic enzymes is the use for treatment of cellulosic textile or fabric, e.g. as ingredients in detergent compositions or fabric softener compositions, for bio-polishing of new fabric (garment finishing), and for obtaining a “stone-washed” look of cellulose-containing fabric, especially denim, and several methods for such treatment have been suggested, e.g. in GB-A-1 368 599, EP-A-0 307 564 and EP-A-0 435 876, WO 91/17243, WO 91/10732, WO 91/17244, PCT/DK95/000108 and PCT/DK95/00132. Another important industrial use of cellulolytic enzymes is the use for treatment of paper pulp, e.g. for improving the drainage or for deinking of recycled paper.
There is an ever existing need for providing novel cellulase enzymes or enzyme preparations which may be used for applications where cellulase, preferably an endo-&bgr;-1,4-glucanase, activity is desirable.
The object of the present invention is to provide novel enzymes and enzyme compositions having substantial cellulolytic activity under slightly acidic to alkaline conditions and improved performance in paper pulp processing, textile treatment, laundry processes, extraction processes or in animal feed; preferably are such novel well-performing endoglucanases producible or produced by using recombinant techniques in high yields.
SUMMARY OF THE INVENTION
It has now been found that a variety of strains belonging to the genus Saccharothrix produces an enzyme having substantial cellulolytic activity, i.e. an endo-&bgr;-1,4-glucanase which is endogenous to Saccharothrix, and the inventors have succeeded in cloning and expressing a DNA sequence encoding such an enzyme.
Accordingly, in its first aspect the present invention relates to an enzyme preparation comprising an enzyme having endo-&bgr;-1,4-glucanase activity which is obtainable from or endogeneous to a strain belonging to the genus Saccharothrix, preferably belonging to a strain selected from the species
Saccharothrix australiensis, Saccharothrix texasensis, Saccharothrix waywayandensis, Saccharothrix cryophilis, Saccharothrix flava, Saccharothrix coeruleofusca, Saccharothrix longispora, Saccharothrix mutabilis
ssp.
capreolus, Saccharothrix aerocolonigenes, Saccharothrix mutabilis
ssp.
mutabilis, Saccharothrix syringae,
Saccharothrix sp., and in its second aspect the invention relates to an isolated polynucleotide molecule, preferably a DNA molecule, encoding an enzyme or enzyme core (ie the catalytically active domain of the enzyme) exhibiting endo-&bgr;-1,4-glucanase activity selected from the group consisting of (a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID NO:1 from nucleotide 676 to nucleotide 1470, (b) polynucleotide molecules that encode a polypeptide that is at least 80% identical to the amino acid sequence of SEQ ID NO:2 from amino acid residue 226 to amino acid residue 490, and (c) degenerate nucleotide sequences of (a) or (b); preferably a polynucleotide molecule capable of hybridizing to a denatured double-stranded DNA probe under medium stringency conditions, wherein the probe is selected from the group consisting of DNA probes comprising the sequence shown in positions 676-1470 of SEQ ID NO:1 and DNA probes comprising a subsequence of positions 676-1470 of SEQ ID NO:1 having a length of at least about 100 base pairs.
A plasmid pSJ1678 comprising a DNA sequence encoding the endoglucanase of the invention has been transformed into a strain of the
Escherichia coli
which was deposited by the inventors according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Federal Republic of Germany, on Mar. 17, 1997 under the deposition number DSM 11476.
In its third, fourth and fifth aspect the invention provides an expression vector comprising a DNA segment which is eg a polynucleotide molecule of the invention; a cell comprising the DNA segment or the expression vector; and a method of producing an enzyme exhibiting cellulolytic activity, which method comprises culturing the cell under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.
In yet another aspect the invention provides an isolated enzyme exhibiting cellulolytic activity, characterized in (i) being free from homologou
Bjørnvad Mads Eskelund
Hatakeyama Mariko
Nielsen Jack Bech
Schulein Martin
Laubiris, Esq. Elisa J.
Novonordisk A/S
Prouty Rebecca E.
Rao Manjunath N.
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