WO2005042735A1 - Carbohydrate-binding modules of a new family - Google Patents
Carbohydrate-binding modules of a new family Download PDFInfo
- Publication number
- WO2005042735A1 WO2005042735A1 PCT/DK2004/000734 DK2004000734W WO2005042735A1 WO 2005042735 A1 WO2005042735 A1 WO 2005042735A1 DK 2004000734 W DK2004000734 W DK 2004000734W WO 2005042735 A1 WO2005042735 A1 WO 2005042735A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- carbohydrate
- cbm
- seq
- polypeptide
- dna
- Prior art date
Links
Classifications
-
- 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)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
-
- 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)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
Definitions
- the present invention relates to non-catalytic carbohydrate-binding modules (CBM) be- longing to a new family of CBM's.
- CBM of the invention was found attached to a glycosyl hydrolase family 61 (GH61) polypeptide and was shown to have little homology with known CBM's indicating that it is the first known member of a new family of CBM's.
- GH61 glycosyl hydrolase family 61
- the present invention further relates to CBM's preferably exhibiting binding affinity for cellulose; to a method of producing such CBM's; and to methods for using such CBM's in the textile, detergent and cellulose fiber process- ing industries, for purification of polypeptides, immobilisation of active enzymes, baking, manufacturing of biofuel, modification of plant cell walls.
- a carbohydrate-binding module is defined as a contiguous amino acid sequence within a carbohydrate-active enzyme with a discrete fold having carbohydrate-binding activity.
- CBM's existing as modules within larger enzymes sets this class of carbohydrate- binding protein apart from other non-catalytic sugar binding proteins such as lectins and sugar transport proteins.
- CBM's were p reviously classified as cellulose-binding d omains (CBD's) based on the initial discovery of several modules that bound cellulose (Tomme et al. (1989) FEBS Lett. 243, 239-243; Gilkes et al. (1988) J. Biol. Chem.
- CBM's are of bacterial origin, and the known fungal carbohydrate-binding modules are mainly classified in the family CBM1. However, representatives of f ungal CBM's are also found in CBM13, CBM18, CBM19, CBM20 and CBM24. Until now, only the fungal carbohydrate-binding modules from CBM1 were known to bind to crystalline cellulose.
- the fungal CBM's from fami- lies C BM13, C BM18, C BM19, C BM20 a nd C BM24 h ave b een s hown to b ind t o s ubstrates such as chitin, starch and mutan.
- Endoglucanase I (gene egll) from Trichoderma reesei.
- Endoglucanase II (gene egl2) from Trichoderma reesei.
- Endoglucanase V (gene egl5) from Trichoderma reesei.
- Exocellobiohydrolase I (gene CBHI) from Humicola grisea, Neurospora crassa, Phanerochaete cht ⁇ sosporium, Trichoderma reesei, and Trichoderma viride.
- Exocellobiohydrolase II (gene CBHI I) from Trichoderma reesei.
- Exocellobiohydrolase 3 (gene cel3) from Agaricus bisporus.
- the CBD domain is found either at the N-terminal (Cbh-ll or egl2) or at the C-terminal extremity (Cbh-I, egll or egl5) of these enzymes.
- CBD domain There are four conserved cysteine residues in this type of CBD domain, all of which are involved in disulfide bonds. (Prosite, Swiss Institute of Bioinformatics).
- a DNA sequence encoding a CBD from a given organism can be obtained conventionally by using PCR techniques, and, also based on current knowledge; it is possible to find homologous sequences from other organisms.
- CBD's can be found by cloning cellulases, xylanases or other plant cell wall degrading enzymes and measure the binding to e.g. cellulose. If the enzyme activity is bound to Avicel under the standard conditions described below, it can be assumed that part of the gene codes for a binding domain.
- Examples of CBM-like polypeptides obtainable from plants are expansins. Expansins are not CBM's per se because they are not found encoded in the same amino acid sequence with an enzyme activity. However, it has been observed that isolated CBM domains can have expansin like activity on cellulose (Levy and Shoseyov, 2002 supra). Din et al.
- CBD CenA from Cellulomonas fimi endoglucanase A is capable of nonhydrolytic disruption activity of cellulose fibers resulting in small particle release. Furthermore, it was shown that CBD CenA could prevent the floccula- tion of microcrystalline bacterial cellulose (Gilkes et al. (1993) Int. J. Biol. Macromol. 15:347- 351). Similar phenomena were observed for other CBD's (Krull et al. (1988) Biotechnol. Bio- eng. 31 :321-327; Banka et al. (1998) World J. Microbiol. Biotechnol.
- CBM carbohydrate-binding module
- the novel CBM (called CBMX) was shown to have affinity for Avicel ® and had no observable homology (below 20%) to known CBM's. Also, none of the positions of the cysteine residues found on the CBM of the invention correspond to the positions of the well conserved cysteine residues in the family CBM1 described above. This indicates that the CBM of the invention is the first known member of a new family of CBM's. Apart from the fungal CBM's of family CBM1 which have binding affinity for cellulose, the CBM of the invention is the first known fungal CBM shown to have binding affinity for cellulose. The inventors have succeeded in cloning and expressing a CBM bound to a family GH61 enzyme.
- the inventors have expressed the domain only, without the GH61 enzyme and demonstrated that the CBM alone can bind cellulose, such as Avicel.
- Said CBM domain is encoded by the DNA sequence of positions 109-531 of SEQ ID NO:1 and has the amino acid sequence of positions 34-174 of SEQ ID NO:2.
- Positions 1-33 of SEQ ID NO:2 constitutes a signal peptide and an N-terminal region of the GH61 enzyme. Accordingly, the present invention relates to a CBM of a new family of CBM's which CBM is
- polypeptide produced by culturing a cell comprising the DNA sequence of positions 109- 531 of SEQ ID NO:1 under conditions wherein the DNA sequence is expressed;
- the invention provides an expression vector comprising a DNA segment which is e.g. a polynucleotide molecule of the invention; a cell comprising the DNA segment or the expression vector; and a method of producing a CBM polypeptide, which method comprises culturing the cell under conditions permitting the production of the CBM, and recovering the CBM from the culture.
- impurities such as homologous impurities can be removed from the recovered CBM by use of purification methods generally known in the art.
- the invention provides an isolated CBM polypeptide characterized in (i) being free from homologous impurities and (ii) being produced by the method described above.
- the novel CBM of the present invention is useful for washing, treatment of textile, purifi- cation of polypeptides, immobilisation of active enzymes, modification of cellulosic material, baking, manufacturing of biofuel, modification of plant cell walls.
- the present invention relates to non-catalytic carbohydrate-binding modules (CBM) ob- tainable from the fungus Pseudoplectania nigrella and belonging to a new family of fungal CBM's.
- CBM carbohydrate-binding modules
- the CBM of the invention was found in association with a protein belonging to family 61 of the glycosyl hydrolases.
- the CBM of the invention is encoded by the DNA sequence of positions 109-531 of SEQ ID NO:1 and has the amino acid sequence of positions 34-174 of SEQ ID NO:2.
- Said CBM preferably exhibits binding affinity for cellulose.
- the present invention relates to a method of producing such CBM's; and to methods for using such CBM's in washing applications, for treatment of textile, purification of polypeptides, immobilisation of active enzymes, modification of cellulosic material, baking, manufacturing of biofuel, modification of plant cell walls.
- the inventors have succeeded in cloning and expressing a CBM bound to a family GH61 enzyme.
- the inventors have expressed the domain only, without the GH61 enzyme and demonstrated that the CBM alone can bind cellulose. Accordingly, the invention relates to a CBM of a new family of CBM's which CBM is
- polypeptide produced by culturing a cell comprising the DNA sequence of positions 109- 531 of SEQ ID NO:1 under conditions wherein the DNA sequence is expressed;
- polypeptide having the amino acid sequence of positions 34-174 of SEQ ID NO:2 , or a polypeptide homologous to SEQ ID NO:2, which polypeptide has an amino acid sequence of at least 40% identity with positions 34-174 of SEQ ID NO:2, preferably at least 50% identity, more preferably at least 60% identity, more preferably at least 70% identity, more preferably at least 80%, more preferably at least 85%, more preferably at I east 90%, more preferably at least 95% identity, more preferably at least 97% identity, more preferably at least 98% identity, even more preferably at least 99% identity with positions 34-174 of SEQ ID NO:2;
- Hybridization Suitable experimental conditions for determining hybridization at low to very high stringency between a nucleotide probe and a homologous DNA or RNA sequence involves presoaking of the filter containing the DNA fragments or RNA to hybridize in 5 x SSC (Sodium chloride/Sodium citrate as described in Sambrook et al. ( 1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor, NY) for 10 min, and prehybridization of the filter in a solution of 5 x SSC, 5 x Denhardt's solution (Sambrook et al. 1989 supra), 0.5 % SDS and 100 ⁇ g/ml of denatured sonicated salmon sperm DNA (Sambrook et al.
- the filter is then washed twice for 30 minutes in 2 x SSC, 0.5 % SDS at at least 55°C (low stringency), more preferably at least 60°C (medium stringency), still more preferably at least 65°C (medium/high stringency), e ven m ore p referably a 1 1 east 70°C ( high s tringency), a nd e ven m ore p referably a t least 75°C (very high stringency).
- Nucleotide sequences may be aligned with the AlignX application of the Vector NTI Program Suite 7.0 (Informax, a subsidiary of Invitrogen Inc.) using the default settings, which employ a modified ClustalW algorithm (Thompson et al. (1994) Nuc. Acid Res. 22:4673-4680), the swgapdnamt score matrix, a gap opening penalty of 15 and a gap extension penalty of 6.66.
- Amino acid sequences may be aligned with the AlignX application of the Vector NTI
- Program Suite v8 (Informax, a subsidiary of Invitrogen Inc) using default settings, which employ a modified ClustalW algorithm (Thompson, et al. (1994) supra), the blosum62mt2 score matrix, a gap opening penalty of 10 and a gap extension penalty of 0.1.
- a Smith-Waterman search Smith and Waterman (1981) J. Mol. Biol. 147:195-197
- Two proteins registered some similarity at the amino acid level. The first was FIG2 of Saccharomyces cerevisiae (Swiss-Prot No. p25653).
- the Smith- Waterman score was 162, and showed 28.7% identity over a 143 base pair overlap.
- the CBM of the invention has six phenylalanine repeats at a spacing that would potentially put them on the same surface of a higher order structure such as a beta barrel or alpha helix.
- the three dimensional structures of representative members of CBM families 1-6, 9 and 15 have been resolved by x-ray crystallography and NMR and according to Levy and Shoseyov (Biotechnology Advances 20 (2002) 191-213), data from these structures indicate that CBD's from different families are structurally similar and that their cellulose binding capacity can be attributed, at least in part, to several aromatic amino acids that compose their hydrophobic surface.
- the present inventors therefore wish to point out several phenylalanine residues and their significance to the ability of the CBM of the invention to bind cellulose. Below are subregions of the CBM with the residues marked:
- VPNFTATDVPTFTATDIPTFTATDVPIFTKKPQQPS positions 64-99 of SEQ ID NO:2
- SVSFVAKPSAFIPKPSA positions 110-126 of SEQ ID NO:2
- the expressed CBM or CBM-containing polypeptide of the invention has a molecular weight (Mw) which is equal to or higher than about 15 kD in an unglycosylated form.
- Mw molecular weight
- the majority of the protein binding to Avicel appeared as a broad band of molecular weight 35-45 kDa, which is considerably higher than the 15 kDa of the protein part of the carbohydrate binding module.
- the high and heterogeneous molecular weight is probably due to heterogeneity in O- and N-glycosylation of the N-terminal part of the protein.
- CBMX For heterologously expressed CBMX in Aspergillus oryzae the size of CBMX can vary from 14 kDa to almost 70 kDa due to het- erologous glycosylation of the protein. Moreover, N-terminal sequencing of the 35-45 kDa band gave exclusively the sequence SFSSSGT (positions 47-53 of SEQ ID NO:9) indicating that heterogeneity in the N-terminal amino acid sequence is not present.
- the m olecular weight of the CBM of the invention in an unglycosylated form is equal to or below about 70 kD, more preferably equal to or below 50 kD, more preferably equal to or below about 40 kD or 30 kD, even more preferably equal to or below about 25 kD, even more preferably equal to or below about 20 kD, even more preferably equal to or be- low about 15 kD.
- CBD cellulose-binding domains
- a typical CBM will thus be one which occurs in a cellulase and which binds preferentially to cellulose and/or to poly- or oligosaccharide fragments thereof.
- Cellulose-binding (and other carbohydrate-binding) modules are polypeptide amino acid sequences which occur as integral parts of large polypeptides or proteins consisting of two or more polypeptide amino acid sequence regions, especially in hydrolytic enzymes
- hydrolases which typically comprise a catalytic domain containing the active site for substrate hydrolysis and a carbohydrate-binding domain for binding to the carbohydrate substrate in question.
- Such enzymes can comprise more than one catalytic domain and one, two or three carbohydrate-binding domains, and they may further comprise one or more polypeptide amino acid sequence regions linking the carbohydrate-binding domain(s) with the catalytic domain(s), a region of the latter type usually being denoted a "linker".
- a CBM is located either at the N or C terminal or is internal.
- a monomeric CBM typically consists of more than about 30 and less than about 250 amino acid residues.
- a CBM classified in Family I consists of
- a CBM classified in Family I la consists of 95-108 amino acid residues
- a CBM classified in Family VI consists of 85-92 amino acid residues.
- the molecular weight of a monomeric CBM will typically be in the range of from about 4kD to about 40kD, and usually below about 35kD.
- CBM's may be useful as a single domain polypeptide or as a dimer, a trimer, or a polymer; or as a part of a protein hybrid.
- hydrolytic enzymes comprising a carbohydrate-binding module are cellulases, xylanases, mannanases, arabinofuranosidases, acetylesterases, amylases, glucoamy-lases, mutanases and chitinases.
- CBM's have been shown to bind to carbohydrates such as cellulose, xylan, starch, chitin, mannan, beta-glucans, mutan and cyclodextrins.
- CBM's have been found in plants and algae, e.g. in the red alga Porphyra purpurea in the form of a non-hydrolytic polysaccharide-binding protein (see Tomme et al. (1996) Cellulose-Binding Domains, Classification and Properties in Enzymatic Degradation of Insoluble Carbohydrates, Saddler & Penner (Eds.), ACS Symposium Series, No. 618).
- the present invention thus relates, inter alia, to a process for removal or bleaching of soiling or stains present on cellulosic fabric or textile, wherein the fabric or textile is contacted in aqueous medium with a modified enzyme (enzyme hybrid) which comprises a catalytically (enzymatically) active amino acid sequence of a non-cellulolytic enzyme linked to an amino acid sequence comprising a carbohydrate-binding module, such as a CBD.
- a modified enzyme enzyme hybrid
- Soiling or stains which may be removed according to the present invention include those already mentioned above, i.e. soiling or stains originating from, for example, starch, proteins, fats, red wine, fruit (such as blackcurrant, cherry, strawberry or tomato, in particular tomato in ketchup or spaghetti sauce), vegetables (such as carrot or beetroot), tea, coffee, spices (such as curry or paprika), body fluids, grass, or ink (e.g. from ball-point pens or fountain pens).
- Other types of soiling or stains which are appropriate targets for removal or bleaching in accordance with the invention include sebum, soil (i.e. earth), clay, oil and paint.
- a process for removal or bleaching of soiling or stains present on cellulosic fabric is described in WO 97/28243.
- the process comprises con- tacting a fabric with an aqueous medium comprising a modified enzyme, which enzyme is a catalytically active amino acid sequence of a non-cellulolytic enzyme which is linked to an amino acid sequence comprising a cellulose-binding domain. It is an object of the present invention to use the CBM of SEQ ID NO:2 in a process for removal or bleaching of soiling or stains present on cellulosic fabric as described in WO 97/28243.
- cellulosic fabric is intended to indicate any type of fabric, in particular woven fabric, prepared from a cellulose-containing material, such as cotton, or from a cellulose-derived material (prepared, e.g., from wood pulp or from cotton).
- fabric is intended to include garments and other types of processed fabrics, and is used interchangeably with the term “textile”.
- cellulosic fabric manufactured from naturally occurring cellulosic fibre are cotton, ramie, jute and flax (linen) fabrics.
- cellulosic fabrics made from man-made cellulosic fibre are viscose (rayon) and lyocell (e.g.
- TencelTM fabric also of relevance in the context of the invention are all blends of cellulosic fibres (such as viscose, lyocell, cotton, ramie, jute or flax) with other fibres, e.g. with animal hair fibres such as wool, alpaca or camel hair, or with polymer fibres such as polyester, polyacrylic, polyamide or polyacetate fibres.
- cellulosic fibres such as viscose, lyocell, cotton, ramie, jute or flax
- other fibres e.g. with animal hair fibres such as wool, alpaca or camel hair
- polymer fibres such as polyester, polyacrylic, polyamide or polyacetate fibres.
- blended cellulosic fabric are viscose/cotton blends, lyocell/cotton blends (e.g.
- Enzyme classification numbers (EC numbers) referred to in the present patent application are in accordance with the Recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, Academic Press Inc., 1992.
- a modified enzyme (enzyme hybrid) for use in accordance with the invention comprises an enzymatically active amino acid sequence of a non-cellulolytic enzyme (i.e. a catalytically active amino acid sequence of an enzyme other than a cellulase) useful in relation to the cleaning of fabric or textile, typically the removal or bleaching of soiling or stains from fabrics or textiles in washing processes.
- enzymes selected from the group consisting of amylases (e.g.
- ⁇ -amylases EC 3.2.1.1
- proteases i.e. peptidases, EC 3.4
- lipases e.g. triacylglycerol lipases, EC 3.1.1.3
- oxidoreductases e.g. peroxidases, EC 1.11.1 , such as those classified under EC 1.11.1.7; or phenol-oxidizing oxidases, such as laccases, EC 1.10.3.2, or other enzymes classified under EC 1.10.3
- fused (linked) to an amino acid sequence comprising a cellulose-binding module fused (linked) to an amino acid sequence comprising a cellulose-binding module.
- the catalytically active amino acid sequence in question may comprise or consist of the whole, or substantially the whole of the full amino acid sequence of the mature enzyme in question, or it may consist of a portion of the full sequence which retains substantially the same enzymatic properties as the full sequence.
- Modified enzymes of the type in question, as well as detailed descriptions of the prepara- tion and purification thereof, are known in the art (see, e.g., WO 90/00609, WO 94/24158 and WO 95/16782).
- fusion protein may be prepared by transforming into a host cell a DNA construct comprising at least a fragment of DNA encoding the CBM ligated, with or without a linker, to a DNA sequence encoding the enzyme of i nterest, and growing the transformed h ost cell to express the fused gene.
- a DNA construct comprising at least a fragment of DNA encoding the CBM ligated, with or without a linker, to a DNA sequence encoding the enzyme of i nterest, and growing the transformed h ost cell to express the fused gene.
- CBM is an amino acid sequence comprising at least the carbohydrate- binding module (CBM) perse.
- MR the middle region; a linker
- MR may be a bond, or a linking group comprising from 1 to about 100 amino acid residues, in particular of from 2 to 40 amino acid residues, e.g. from 2 to 15 amino acid residues.
- MR may, in principle, alternatively be a non-amino-acid linker.
- X is an amino acid sequence comprising the above-mentioned, enzymatically active sequence of amino acid residues of a polypeptide encoded by a DNA sequence encoding the non-cellulolytic enzyme of interest.
- the moieties A and B are independently optional.
- a moiety A or B constitutes a terminal extension of a CBM or X moiety, and normally comprises one or more amino acid residues. It will thus, inter alia, be apparent from the above that a CBM in an enzyme hybrid of the type in question may be positioned C-terminally, N-terminally or internally in the enzyme hybrid.
- an X moiety in an enzyme hybrid of the type in question may be positioned N- terminally, C-terminally or internally in the enzyme hybrid.
- Enzyme hybrids of interest in the context of the invention include enzyme hybrids which comprise more than one CBM, e.g.
- two or more CBM's are linked directly to each other, or are separated from one another by means of spacer or linker sequences, consisting typically of a sequence of amino acid residues of appropriate length.
- Two CBM's in an enzyme hybrid of the type in question may, for example, also be separated from one another by means of an -MR-X- moiety as defined above.
- a very important issue in the construction of enzyme hybrids of the type in question is the stability towards proteolytic degradation.
- Cellulases (cellulase genes) useful for preparation of CBM's Techniques suitable for isolating a cellulase gene are well known in the art.
- the terms “cellulase” and “cellulolytic enzyme” refer to an enzyme which catalyses the degradation of cellulose to glucose, cellobiose, triose and/or other cello-oligosaccharides.
- Preferred cellulases i.e. cellulases comprising preferred CBM's
- microbial cellulases particularly bacterial or fungal cellulases.
- Endoglucanases notably endo- 1 ,4- ⁇ -glucanases (EC 3.2.1.4), particularly mono-component (recombinant) endo-1 ,4- ⁇ -gluc- anases, are a preferred class of cellulases.
- bacterial cellulases are cellulases derived from or producible by bac- teria from the group consisting of Pseudomonas, Bacillus, Cellulomonas, Clostridium, Microspora, Thermotoga, Caldocellum and Actinomycetes such as Streptomyces, Termomonospora and Acidothemus, in particular from the group consisting of Pseudomonas cellulolyticus, Bacillus lautus, Cellulomonas fimi, Clostridium thermocellum, Microspora bispora, Termomonospora fusca, Termomonospora cellulolyticum and Acidothemus cellulolyticus.
- the cellulase may be an acid, a neutral or an alkaline cellulase, i.e. exhibiting maximum cellulolytic activity in the acid, neutral or alkaline range, respectively.
- a useful cellulase is an acid cellulase, preferably a fungal acid cellulase, which is derived from or producible by fungi from the group of genera consisting of Trichoderma, Myrothecium, Aspergillus, Phanaerochaete, Neurospora, Neocallimastix and Botrytis.
- Another useful cellulase is a neutral or alkaline cellulase, preferably a fungal neutral or alkaline cellulase, which is derived from or producible by fungi from the group of genera consisting of Aspergillus, Penicillium, Myceliophthora, Humicola, Irpex, Fusarium, Stachybotrys, Scopu- lariopsis, Chaetomium, Mycogone, Verticillium, Myrothecium, Papulospora, Gliocladium, Cepha- losporium, Pseudoplectania nigrella and Acremonium.
- a preferred alkaline cellulase is one derived from or producible by fungi from the group of species consisting of Humicola insolens, Fusarium oxysporum, Myceliopthora thermophila, Penicillium janthinellum and Cephalosporium sp., preferably from the group of species consisting of Humicola insolens DSM 1800, Fusarium oxysporum DSM 2672, Myceliopthora thermophila CBS 117.65, and Cephalosporium sp. RYM-202.
- Other examples of useful cellulases are variants of parent cellulases of fungal or bacterial origin, e.g. variants of a parent cellulase derivable from a strain of a species within one of the fungal genera Humicola, Trichoderma or Fusarium.
- Amylases e.g. ⁇ - or ⁇ -amylases
- Amylases which are appropriate as the basis for enzyme hybrids of the types employed in the context of the present invention include those of bacterial or fungal origin. Chemically or g enetically modified m utants of s uch amylases are included in t his connection.
- Relevant ⁇ -amylases include, for example, ⁇ -amylases obtainable from Bacillus species, in particular a special strain of B. licheniformis, described in more detail in GB 1296839.
- amylases include Duramyl ® , Termamyl ® , Fungamyl ® and BAN ® (all available from Novozymes A/S, Bagsvaerd, Denmark), and RapidaseTM and MaxamylTM P (available from DSM, Holland).
- Other u seful a mylolytic enzymes a re C GTases ( cyclodextrin glucanotransferases, E C 2.4.1.19), e.g. those obtainable from species of Bacillus, Thermoanaerobactor or Thermoanaero- bacterium.
- proteases which are appropriate as the basis for enzyme hybrids of the types employed in the context of the present invention include those of animal, vegetable or microbial origin. Proteases of microbial origin are preferred. Chemically or genetically modified mutants of such proteases are included in this connection.
- the protease may be a serine protease, preferably an alkaline microbial protease or a trypsin-like protease.
- alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtil- isin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279).
- trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270.
- Relevant commercially available p rotease e nzymes i n include A lcalase ® , S avinase ® a nd Esperase ® (all available from Novozymes A/S, Bagsvaerd, Denmark), MaxataseTM, MaxacalTM, MaxapemTM and ProperaseTM (available from DSM, Holland), PurafectTM and PurafectTM OXP (available from Genencor International, USA), and OpticleanTM and OptimaseTM (available from by Solvay Enzymes).
- Lipolytic enzymes which are appropriate as the basis for enzyme hybrids of the types employed in the context of the present invention include those of bacterial or fungal origin. Chemically or genetically modified mutants of such lipases are included in this connection.
- useful lipases include a Humicola lanuginosa lipase, e.g. as described in EP 258 068 and EP 305 216; a Rhizomucor miehei lipase, e.g. as described in EP 238 023; a Candida lipase, such as a C. antarctica lipase, e.g. the C.
- antarctica lipase A or B described in EP 214 761 a Pseudomonas lipase, such as one of those described in EP 721 981 (e.g. a lipase ob- tainable from a Pseudomonas sp. SD705 strain having deposit accession number FERM BP-
- PCT/JP96/00426, in PCT/JP96/00454 e.g. a P. solanacearum lipase
- EP 571 982 or in WO 95/14783 e.g. a P. mendocina lipase
- a P. alcaligenes or P. pseudoalcaligenes lipase e.g. as described in EP 218 272
- a P. cepacia lipase e.g. as described in EP 331 376
- a P. stutz- eri lipase e.g. as disclosed in GB 1,372,034, or a P.
- fluorescens lipase a Bacillus lipase, e.g. a B. subtilis lipase (Dartois et al. (1993) Biochemica et Biophysica Acta 1131 :253-260), a B. stearo- thermophilus lipase (JP 64/744992) and a B. pumilus lipase (WO 91/16422).
- a number of cloned lipases may be useful, including the Penicillium cam- embertii I ipase d escribed b y Y amaguchi et a l.
- Suitable commercially available lipases include Lipolase ® and Lipolase Ultra ® (available from Novozymes A/S), M1 LipaseTM, LumafastTM and LipomaxTM (available from DSM, Holland) and Lipase P "Amano” (available from Amano Pharmaceutical Co. Ltd.).
- Oxidoreductases which are appropriate as the basis for enzyme hybrids of the types employed in the context of the present invention include peroxidases (EC 1.11.1) and oxidases, such as lac- cases (EC 1.10.3.2) and certain related enzymes.
- Peroxidases (EC 1.11.1) are enzymes acting on a peroxide (e.g. hydrogen peroxide) as acceptor.
- Very suitable peroxidases are those classified under EC 1.11.1.7, or any fragment derived there- from, exhibiting peroxidase activity. Synthetic or semisynthetic derivatives thereof (e.g. with por- phyrin ring systems, or microperoxidases, cf., for example, US 4,077,768, EP 537 381 , WO 91/05858 and WO 92/16634) may also be of value in the context of the invention.
- Very suitable peroxidases are peroxidases obtainable from plants (e.g. horseradish peroxidase or soy bean peroxidase) or from microorganisms, such as fungi or bacteria. In this re- spect, some preferred fungi include strains belonging to the subdivision Deuteromycotina, class
- Hyphomycetes e.g. Fusarium, Humicola, Tricoderma, Myrothecium, Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embellisia, Cladosporium or Dreschlera, in particular Fusarium oxysporum (DSM 2672), Humicola insolens, Trichoderma resii, Myrothecium verrucana (IFO 6113), Verticillum alboatrum, Verticillum dahlie, Arthromyces ramosus (FERM P-7754), Caldariomyces fumago, Ulocladium chartarum, Embellisia alii or Dreschlera halodes.
- DSM 2672 Fusarium oxysporum
- Humicola insolens Trichoderma resii
- Myrothecium verrucana IFO 6113
- Verticillum alboatrum Verticillum dahlie
- fungi include strains belonging to the subdivision Basidiomycotina, class Basidiomycetes, e.g. Coprinus, Phanerochaete, Coriolus or Trametes, in particular Coprinus cinereus f. microsporus (IFO 8371), Coprinus macrorhizus, Phanerochaete chrysospohum (e.g. NA-12) or Trametes versicolor (e.g. PR4 28-A). Further preferred fungi include strains belonging to the subdivision Zygomycotina, class Basidiomycetes, e.g. Coprinus, Phanerochaete, Coriolus or Trametes, in particular Coprinus cinereus f. microsporus (IFO 8371), Coprinus macrorhizus, Phanerochaete chrysospohum (e.g. NA-12) or Trametes versicolor (e.g. PR4 28-A). Further preferred fungi include strain
- Mycoraceae e.g. Rhizopus or Mucor, in particular Mucor hiemalis.
- Some preferred bacteria include strains of the order Actinomycetales, e.g. Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus (IFO 12382) or Streptoverticillum verticillium ssp. verticillium.
- Bacillus pumilus ATCC 12905
- Bacillus stearother- mophilus Rhodobacter sphaeroides
- Rhodomonas palust Streptococcus lactis
- Pseudomonas purrocinia ATCC 15958
- Pseudomonas fluorescens NRRL B-11
- Further preferred bacteria include strains belonging to Myxococcus, e.g. M. virescens.
- Other potential sources of useful particular peroxidases are listed in Saunders et al. (1964) Peroxidase, 41-43 London.
- the peroxidase may furthermore be one which is producible by a method comprising cultivating a host cell - transformed with a recombinant DNA vector which carries a DNA sequence encoding said peroxidase as well as DNA sequences encoding functions permitting the expression of the DNA sequence encoding the peroxidase - in a culture medium under conditions permitting the expression of the peroxidase, and recovering the peroxidase from the culture.
- a suitable recombinantly produced peroxidase is a peroxidase derived from a Coprinus sp., in particular C. macrorhizus or C. cinereus according to WO 92/16634, or a variant thereof, e.g. a variant as described in WO 94/12621.
- Preferred oxidases in the context of the present invention are oxidases classified under EC 1.10.3, w hich a re o xidases e mploying m olecular oxygen a s a cceptor (i.e. e nzymes c atalyzing oxidation reactions in which molecular oxygen functions as oxidizing agent).
- laccases EC 1.10.3.2
- examples of other useful oxidases in the context of the invention include the catechol oxidases (EC 1.10.3.1) and bilirubin oxidases (EC 1.3.3.5).
- laccases are obtainable from a variety of plant and microbial sources, notably from bacteria and fungi (including filamentous fungi and yeasts), and suitable examples of laccases are to found among those obtainable from fungi, including laccases obtainable from strains of Aspergillus, Neurospora (e.g. N. crassa), Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurot- us, Trametes (e.g. T. villosa or T. versicolor [some species/strains of Trametes being known by various names and/or having previously been classified within other genera; e.g.
- laccases in the context of the invention include laccase obtainable from species/strains of Trametes (e.g. T. villosa), Myceliophthora (e.g. M. thermophila), Schytalidium or Polyporus.
- pectinases such as pectate lyase (EC 4.2.2.2), pectin lyase (EC 4.2.2.10), rhamnogalacturonan lyase (EC not defined), endo-1 , 4-- galactanase (EC 3.2.1.89), xyloglucanase (EC not defined), xylanase (EC 3.2.1.8), arabinanase (EC 3.2.1.99), alpha-L-arabinofuranosidase (EC 3.2.1.55), Mannan endo-1 ,4-mannosidase (EC 3.2.1.78), beta-mannosidase (EC 3.2.1.25), beta-1 ,3-1 ,4-glucanase (EC 3.2.1.73), rhamnogalac- turonan hydrolase, exo-polygalacturonase (EC 3.2.1.67),
- Acetyl and methyl esterase en- zymes such as: rhamnogalacturonan methyl esterase, rhamnogalacturonan acetyl esterase, pectin methylesterase (EC 3.1.1.11), pectin acetylesterase (EC not defined), xylan methyl esterase, acetyl xylan esterase (EC 3.1.1.72), feruloyl esterase (EC 3.1.1.73), cinnamoyl esterase (EC 3.1.1.73).
- the CBM of the invention may be added to a detergent for washing textile, such as a laundry detergent or a detergent for washing hard surfaces, such as a dish washing detergent.
- a detergent composition comprising the CBM of the invention can further comprise one or more enzymes selected from the group consisting of p roteases, cellulases (endo-glucanases), beta-glucanases, hemicellulases, lipases, peroxidases, laccases, alpha-amylases, glucoamylases, cutinases, pectinases, reductases, oxidases, phenoloxidases, ligninases, pullulanases, pectate lyases, xyloglu- canases, xylanases, pectin acetyl esterases, polygalacturonases, rhamnogalacturonases, pectin lyases, other mannanases, pectin methyleste
- m ost common s izing agent is starch in native or m odified form.
- other polymeric substances for example poly-vinylalcohol (PVA), polyvinylpyrrolidone (PVP), polyacrylic acid (PAA) or derivatives of cellulose [e.g. carboxy-methylcellulose (CMC), hydroxyethylcellulose, hydroxypropyl-cellulose or methylcellulose] may also be abundant in the size. Small amounts of, e.g., fats or oils may also be added to the size as a lubricant.
- the threads of the fabric are not able to absorb water, finishing agents or other compositions (e.g. bleaching, dyeing or crease-proofing compositions) to a sufficient degree. Uniform and durable finishing of the fabric can thus be achieved only after removal of the size from the fabric; a process of removing size for this purpose is known as a "desizing" process.
- the desizing treatment may be carried out using a starch-degrading enzyme (e.g. an amylase).
- the desizing treatment may comprise the use of a lipolytic enzyme (a lipase).
- the desizing treatment may be carried out with a cellulolytic enzyme, either alone or in combination with other substances, optionally in combination with other enzymes, such as amylases and/or lipases. It is an object of the present invention to achieve improved enzyme performance under desizing conditions by modifying the enzyme so as to alter (increase) the affinity of the enzyme for cellulosic fabric, whereby the modified enzyme comes into closer contact with the sizing agent in question.
- the present invention thus relates, inter alia, to a process for desizing cellulosic fabric or textile, w herein t he fabric o r textile is treated (normally contacted i n aqueous medium) with a modified enzyme (enzyme hybrid) which comprises a catalytically (enzymatically) active amino acid sequence of an enzyme, in particular of a non-cellulolytic enzyme, linked to an amino acid sequence comprising a carbohydrate-binding module, such as the CBM of SEQ ID NO:2.
- a modified enzyme enzyme hybrid
- enzyme hybrid which comprises a catalytically (enzymatically) active amino acid sequence of an enzyme, in particular of a non-cellulolytic enzyme, linked to an amino acid sequence comprising a carbohydrate-binding module, such as the CBM of SEQ ID NO:2.
- the scouring process removes non-cellulosic material from the cotton fiber, especially the cuticle (mainly consisting of waxes) and primary cell wall (mainly consisting of pectin, protein and xyloglucan) before bleaching and dying of the textile.
- a proper wax removal is necessary for obtaining a high wettability, being a measure for obtaining a good dyeing.
- Removal of the primary cell wall improves wax removal and ensures a more even dyeing. Further this improves the whiteness in the bleaching process.
- the CBM's of family CBM1 are known to wedge into crystalline cellulose like ex- pansins and swollenins, and aid in the release of non cellulose components or contaminants from textile.
- T he d ye a ccessibility can be increased by treating t he textile with C BM's.
- T he CBM's of the invention have properties similar to the CBM's of family CBM1. Accordingly, the CBM's of the invention may be used to remove non-cellulosic material from the cotton fiber in the scouring process.
- CBM's that bind reversibly to carbohydrates are useful for separation and purification of target polypeptides.
- CBM's of family I bind reversibly to crystalline cellulose and are useful tags for affinity chromatography. It is an object of the present invention to achieve improved separation and purification of target polypeptides by use of CBM's as affinity tags as describes by Terpe (2003) Appl. Micro- biol. Biotechnol. 60:523-533 and US 5,670,623.
- CBM's bind irreversibly to cellulose and can be used for immobilization of molecules such as metallothioneins, phytochelatins or enzymes.
- Such an immobilization is useful in e.g. removal of heavy metal contaminations from the environment, wherein the heavy metal ions bind to polypeptides biosorbents such as metallothioneins or phytochelatins, and the CBM-biosorbent-heavy metal complex is irreversibly immobilized by the binding of the
- CBM to a carbohydrate material
- Xu et al. (2002) Biomacromolecules 3:462-465 It is an object of the present invention to achieve immobilization of molecules such as metallothioneins, phytochelatins or enzymes by use of the CBM of SEQ ID NO:2 as fusion proteins. Further, a method for removal contaminants such as heavy metals from the environment, by immobilization with CBM's it is an object of the present invention.
- a carbohydrate-binding domain conjugate such as a CBD conjugate, comprises at least two CBD's attached to a polysaccharide.
- the polysaccharide may be capable of binding to cellulose, and is conveniently locust bean gum.
- CBD conjugates are able to increase the strength of cellulosic material such as fabric by cross-linking fibres as described in GB 2376017 to Unilever. It is an object of the present invention to increase the strength and wear of the fabric by cross-linking fibres by use of the CBM of SEQ ID NO:2.
- the CBD conjugates may also be used as delivery vehicles to deposit materials on textile in any stage of the laundering process.
- This latter application can be achieved by coating the benefit agent, either directly by chemical means or indirectly via a compound associated with the benefit agent e.g. a capsule as described in GB 2376017 to Unilever.
- a compound associated with the benefit agent e.g. a capsule as described in GB 2376017 to Unilever.
- s uch b enefit a gents are s oftening a gents, finishing a gents, p rotecting agents, fragrances such as perfumes and bleaching agents.
- softening agents a re clays, cationic s urfactants o r silicon compounds.
- finishing agents and protecting agents are polymeric lubricants, soil repelling agents, soil release agents, photo-protective agents such as sunscreens, anti-static agents, dye-fixing agents, anti-bacterial agents and anti-fungal agents.
- T he fragrances or perfumes may be encapsulated, e.g. in latex or microcapsules or gelatin based coacervates. It is an object of the present invention to use of the CBM of SEQ ID NO:2 as a delivery vehicle to deposit materials on textile in the laundry process.
- CBM's for production of bioethanol Ethanol can be produced from agricultural waste or biomass (biofuel).
- biomass for example, corn stover, wood pulp and wheat straw
- crystalline cellulose is naturally resistant to enzymatic degradation because the cellulose fibrils are tightly packed together thus creating an accessibility problem for cellulose degrading enzymes.
- a number of methods for opening the structure of crys- talline cellulose in biomass are being investigated: acid pre-treatment with steam explosion is one well studied method (Bura et al. (2002) Appl Biochem Biotechnol. 98-100:59-72). Wet oxidation is another method described by Naito et al.
- CBD proteins have been shown to bind to newly synthesized cellulose fibres in plant cell walls, and this physico-mechanical interference uncouples cellulose synthesis by the subunits of the cellulose synthase enzyme complexes. This results in an increased rate of synthesis of the cel- lulose polymer, improved polymer qualities and enhanced biomass.
- the increased rate of cellulose synthesis in the cell wall leads to enhanced cellulose production, greater biomass at the plant level, improved fibre properties and may enhance resistance to biotic and abiotic stress.
- a CBD encoding gene can be inserted into hardwood forestry species and subsequent substantial volume increases with improvements in wood density and fibre properties can be dem- onstrated. These improvements will carry through to the finished paper, exhibiting enhanced tensile, tear and burst indices (US 6,184,440).
- the CBM of the invention may be part of a composition made for the specific application.
- Further components in such compositions comprise a carrier compound, and one or more enzymes selected from the group consisting of proteases, cellulases, beta-glucanases, hemicellulases, lipases, peroxidases, laccases, alpha-amylases, glucoamylases, cutinases, pectinases, reductases, oxidases, phenoloxidases, ligninases, pullu- lanases, pectate lyases, xyloglucanases, xylanases, pectin acetyl esterases, polygalacturonases, rhamnogalacturonases, pectin lyases, other mannanases, pectin methylesterases, cellobiohy- drolases, transglutaminases; or mixtures thereof.
- Nucleic acid constructs comprising nucleotide seguences
- the present invention relates to nucleic acid constructs comprising a nucleotide se- quence of the invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
- a nucleotide sequence encoding a CBM of the invention may be manipulated in a variety of ways to provide for expression of the CBM. Manipulation of the nucleotide sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleotide sequences utilizing recombinant DNA methods are well known in the art.
- the control sequence may be an appropriate promoter sequence, a nucleotide sequence which is recognized by a host cell for expression of the nucleotide sequence.
- T he promoter sequence contains transcriptional control sequences, which mediate the expression of the polypeptide.
- the promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention, especially in a bacterial host cell, are the promoters obtained from the E.
- Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis le- vansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha- amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al.
- promoters for directing the transcription of the nucleic acid con- structs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (WO 96/00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the
- yeast h ost u seful promoters a re o btained from the genes for S accharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomy- ces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.
- ENO-1 S accharomyces cerevisiae enolase
- GAL1 Saccharomyces cerevisiae galactokinase
- ADH2/GAP Saccharomy- ces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
- Saccharomyces cerevisiae 3-phosphoglycerate kinase Other useful promoters for yeast host cells
- the control sequence may also be a suitable transcription terminator sequence, a se- quence recognized by a host cell to terminate transcription.
- the terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the CBM. Any terminator which is functional in the host cell of choice may be used in the present invention.
- Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.
- Preferred terminators for yeast host cells are obtained from the genes for Saccharo- myces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al. (1992) supra.
- the control sequence may also be a suitable leader sequence, a non-translated region of an mRNA which is important for translation by the host cell.
- the leader sequence is operably linked to the 5' terminus of the nucleotide sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used in the present invention.
- Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
- Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydro- genase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
- the control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' terminus of the nucleotide sequence and which, when transcribed, is recog- nized by the host cell as a signal to add polyadenosine residues to transcribed mRNA.
- Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention.
- Preferred p olyadenylation s equences for filamentous fungal h ost cells a re o btained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Asper- gillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase.
- Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman (1995) Molecular Cellular Biology 15:5983-5990.
- the control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded
- the 5' end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted CBM.
- the 5' end of the coding sequence may contain a signal peptide coding region which is foreign to the coding sequence.
- the foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region.
- the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the CBM.
- any signal peptide coding region which directs the expressed CBM into the secretory pathway of a host cell of choice may be used in the present invention.
- the native signal peptide coding region of the CBM of the present invention is nucleotides 10 to 69 of SEQ ID NO:1 encoding amino acids 1 to 20 of SEQ ID NO:2.
- Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta- lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva (1993) Microbiological Reviews 57:109-137.
- Effective signal peptide coding regions for filamentous fungal host cells are the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic pro- teinase, Humicola insolens cellulase, Candida antarctica lipase and Humicola lanuginosa lipase.
- Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase.
- the control sequence may also be a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a CBM.
- the resultant polypeptide may be denoted a pro-CBM or propolypeptide.
- a propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
- the propeptide coding region may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila laccase (WO 95/33836). Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypep- tide and the signal peptide region is positioned next to the amino terminus of the propeptide region. In yeast, the ADH2 system or GAL1 system may be used.
- the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences.
- Other examples of regulatory sequences are those which allow for gene amplification. In eukaryotic systems, these include the dihydrofolate reductase gene which is amplified in the presence of meth- otrexate, and the metallothionein genes which are amplified with heavy metals. In these cases, the nucleotide sequence encoding the polypeptide would be operably linked with the regulatory sequence.
- Recombinant expression vector comprising nucleic acid construct
- the present invention also relates to recombinant expression vectors comprising the nucleic acid construct of the invention.
- the various nucleotide and control sequences described above may be joined together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleotide sequence encoding the polypeptide at such sites.
- the nucleotide sequence of the present invention may be expressed by inserting the nucleotide sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression.
- the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
- the recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleotide sequence.
- the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
- the vectors may be linear or closed circular plasmids.
- the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
- the vector may contain any means for assuring self-replication.
- the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
- a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
- the vectors of the present invention preferably contain one or more selectable markers which permit easy selection of transformed cells.
- a selectable marker is a gene the product of which p rovides for b iocide o r viral resistance, resistance to h eavy m etals, p rototrophy to auxotrophs, and the like.
- Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers which confer antibiotic resistance such as ampicillin, kana- mycin, chloramphenicol o r tetracycline resistance.
- S Preble m arkers for yeast h ost cells a re ADE2, HIS3, LEU2, LYS2, MET3, TRP1 , and URA3.
- Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine car- bamoyltransferase), bar (phosphinothricin acetyltransferase), hygB (hygromycin phosphotrans- ferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), as well as equivalents thereof.
- amdS acetamidase
- argB ornithine car- bamoyltransferase
- bar phosphinothricin acetyltransferase
- hygB hygromycin phosphotrans- ferase
- niaD nitrate reducta
- the vectors of the present invention preferably contain an element(s) that permits stable integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
- the vector may rely on the nucleotide sequence encoding the polypeptide or any other element of the vector for stable integration of the vector into the genome by homologous or nonhomolo- gous recombination.
- the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host cell.
- the additional nucleotide sequences enable the vector to be integrated into the host cell genome at a precise location(s) in the chromosome(s).
- the integrational elements should preferably contain a sufficient number of nucleo- tides, such as 100 to 1 ,500 base pairs, preferably 400 to 1 ,500 base pairs, and most preferably 800 to 1 ,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination.
- the integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell.
- the integrational elements may be non-encoding or encoding nucleotide sequences.
- the vector On t he other h and, the vector may b e i ntegrated i nto the g enome of the h ost cell by n on- homologous recombination.
- the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question.
- Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAM ⁇ l permitting replication in Bacillus.
- origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1 , ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.
- the origin of replication may be one having a mutation which makes it's functioning temperature-sensitive in the host cell (see, e.g., Ehrlich (1978) Proceedings of the National Academy of Sciences USA 75:1433). More than one copy of a nucleotide sequence of the present invention may be i n- serted into the host cell to increase production of the gene product.
- An increase in the copy number of the nucleotide sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleotide sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleotide sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
- the procedures used to ligate the elements described above to construct the recombinant expression vectors of the present i nvention a re well k nown to o ne s killed i n t he a rt (see e .g. Sambrook et al. (1989) supra).
- Recombinant host cell comprising nucleic acid construct
- the present invention also relates to recombinant a host cell comprising the nucleic acid construct of the invention, which are advantageously used in the recombinant production of the polypeptides.
- a vector comprising a nucleotide sequence of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier.
- Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagu- lans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus, or gram negative bacteria such as E.
- a Bacillus cell e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagu
- the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, or Bacillus subtilis cell.
- Bacillus cell is an alkalophilic Bacillus.
- the introduction of a vector into a bacterial host cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen (1979) Molecular General Genetics 168:111-115), using competent cells (see, e.g., Young and Spizizin (1961) Journal of Bacteriology 81 :823-829, or Dubnau and Davidoff-Abelson (1971) Journal of Molecular Biology 56:209-221), electroporation (see, e.g., Shigekawa and Dower (1988) B iotechniques 6:742- 751), or conjugation (see, e.g., Koehler and Thorne (1987) Journal of Bacteriology 169:5771- 5778).
- protoplast transformation see, e.g., Chang and Cohen (1979) Molecular General Genetics 168:111-115
- competent cells see, e.g., Young and Spizizin (1961) Journal of Bacteriology 81 :823-829, or
- the host cell may be a eukaryote, such as a mammalian, insect, plant, or fungal cell.
- the host cell is a fungal cell.
- "Fungi” as used herein includes the phyla A scomycota, B asidiomycota, C hytridiomycota, a nd Z ygomycota ( as d efined b y H awk- sworth et al. (1995) In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al.
- yeast as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore and Davenport, eds, (1980) Soc. App. Bacteriol. Symposium Series No. 9).
- the yeast host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.
- the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluy- veri, S accharomyces n orbensis o r S accharomyces o viformis c ell.
- the yeast host cell is a Kluyveromyces lactis cell.
- the yeast host cell is a Yarrowia lipolytica cell.
- the fungal host cell is a filamentous fungal cell.
- "Filamentous fungi" include all filamentous forms of the subdivision Eumycota and Oomy- cota (as defined by Hawksworth et al. (1995) supra). The filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obliga- tely aerobic.
- the filamentous fungal host cell is a cell of a species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, or Trichoderma.
- the filamentous fungal host cell is an Aspergillus awamori, Aspergillus foetid us, A spergillus j aponicus, A spergillus n idulans, A spergillus n iger o r A spergillus o ryzae cell.
- the filamentous fungal host cell is a Fusarium bac- tridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium ox- ysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sar- cochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum cell.
- Fusarium bac- tridioides Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusa
- the filamentous fungal parent cell is a Fusarium venenatum cell (Nirenberg sp. nov. such as the Fusarium venenatum deposited under Nos. CBS 458.93, CBS 127.95, CBS 128.95, CBS 148.95).
- the filamentous fungal host cell is a Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
- Fungal cells may be transformed by a process involving protoplast formation, trans- formation of the protoplasts, and regeneration of the cell wall in a manner known per se.
- Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and Yelton et a l. ( 1984) P roceedings of the N ational Academy of S ciences USA 8 1 :1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al. (1989) Gene 78:147-156 and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson and Simon, eds, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194:182-187, Academic Press, Inc., New York; Ito et al. (1983) Journal of Bacteriology 153:163; and Hinnen et al. (1978) Proceedings of the National Academy of Sciences USA 75:1920.
- the present invention also relates to methods for producing a CBM of the present invention comprising (a) cultivating a strain, which in its wild-type form is capable of producing the CBM; and (b) recovering the CBM.
- the strain is a fungus, more preferably of the genus Humicola, particularly Humicola insolens or Coprinus, such as Coprinus cinereus or
- Thielavia such as Thielavia terresths or Aspergillus such as Aspergillus oryzae.
- the present invention also relates to a method for producing a CBM polypeptide, the method comprising the steps of
- the cells are cultivated in a nutrient medium suitable for production of the CBM using methods known in the art.
- the cell may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated.
- the cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art.
- Suitable media are available from commercial suppliers or may be prepared according to pub- lished compositions (e.g., in catalogues of the American Type Culture Collection).
- the CBM can be recovered directly from the medium. If the CBM is not secreted, it can be recovered from cell lysates.
- the produced CBM may be detected using methods known in the art and modifications thereof that are specific for the CBM. These detection methods may include use of specific antibodies or determination of binding to a carbohydrate substrate, such as Avicel.
- the resulting CBM may be recovered by methods known in the art. For example, the CBM may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
- polypeptides of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g. ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g. Protein Purification, Janson and Ryden, eds (1989) VCH Publishers, New York).
- chromatography e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
- electrophoretic procedures e.g., preparative isoelectric focusing
- differential solubility e.g. ammonium sulfate precipitation
- SDS-PAGE or extraction
- a DNA sequence encoding a CBM from a given organism can be obtained conventionally by using PCR techniques, and, also based on current knowledge it is possible to find homologous sequences from other organisms.
- CBM's can be found by cloning carbohydrate degrading enzymes such as cellulases, xylanases or other plant cell wall degrading enzyme and measure the bind- ing to the target carbohydrate.
- carbohydrate degrading enzymes such as cellulases, xylanases or other plant cell wall degrading enzyme and measure the bind- ing to the target carbohydrate.
- carbohydrate degrading enzymes such as cellulases, xylanases or other plant cell wall degrading enzyme and measure the bind- ing to the target carbohydrate.
- carbohydrate degrading enzymes such as cellulases, xylanases or other plant cell wall degrading enzyme
- the enzyme is added in a ratio of 1 part cellulose binding domain to 150 parts Avicel ® . This is done on ice which gives optimum binding within 5 to 10 minutes.
- the Avicel ® can then be washed and applied directly to SDS-PAGE for visualization of the bound proteins (since the use of SDS and cooking will release the bound proteins).
- the slurry is packed into a column and washed.
- the bound protein is eluted, either in ionized water or in a high pH buffer such as triethylamine (pH 11.2; 1 % solution), where the pH eluted protein is quickly adjusted to neutral.
- the first procedure (A) uses inverse PCR to delete the enzymatic core of the family GH61 en- zyme obtained from Pseudoplectania nigrella. Ligation of the product resulted in a plasmid containing the native secretion signal of the GH61 enzyme, fused in frame to the DNA encoding the carbohydrate-binding module.
- the second method (B) pursued for recombinant overexpression of the CBM of the invention was to clone the DNA encoding the CBM domain into a vector containing a Candida lipase signal peptide.
- a - Inverse PCR Primers NP887U1 and NP887D1 were synthesized as 5' phosphorylated primers. Amplification of plasmid DNA encoding the full open reading frame (ORF) of the family GH61 enzyme was used as template. The OFR can be obtained from the deposited strain CBS 444.97 by use of primers NP887U1 and NP887D1. Approximately 100 nanograms of DNA were used as template in a PCR reaction with the two primers A and B.
- NP887D1 (SEQ ID NO:4) 5'-ACATCCTCCGGCACCTCCAATGACAAGGCCGTCG-3'
- the ligation was performed at 17 degrees Celsius overnight and then transformed into TOP10 Chemically competent cells according to the manufacturers instructions.
- the transformation was plated out on LB agar with 50mg/liter ampicillin. Eleven of the several hundred colonies that grew were miniprepped (Qiaspin ® columns, Qiagen Ltd.) and the DNA cut with EcoRI and Notl to liberate the insert. Eight out of the eleven plasmids had an insert of the correct size (ca. 700bp).
- the insert was sequenced for these plasmids containing inserts.
- the colonies were sequenced with vector primer PNA2I (5'-GTT TCC AAC TCA ATT TAC CTC-3' SEQ ID NO:5).
- pCBMX-K1 was chosen for a medium scale JetStar ® (GENOMED, Germany) plasmid preparation from 100 ⁇ l of LB ampicillin grown plasmid containing E. coli cells.
- the DNA sequence of the fusion construction of pCBMX-K1 , and the corresponding amino acid sequence, are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.
- Transformation of construct pCBMX-K1 into Aspergillus oryzae The DNA of SEQ ID NO:1 was transformed into Aspergillus oryzae strain JAL355 (disclosed in international patent application WO 01/98484A1 ). Transformants of SEQ ID NO:1 was re-isolated twice under selective and non-inducing conditions on Cove minimal plates (Cove (1966) Biochim. B iophys. Acta 133:51-56) with 1 M sucrose as a carbon source and 10mM nitrate.
- transformants were grown for 3 days and 4 days at 30 degrees Celsius in tubes with 10 ml YPM (2% peptone, 1 % yeast extract, 2% mal- tose). Supematants were run on NuPage ® 10% Bis-Tris SDS gels (Invitrogen, USA) as recommended by the manufacturer. All Aspergillus isolates grew well even when induced for the expression of the DNA of SEQ ID NO:1.
- the second method pursued for recombinant overexpression of the CBM domain of the invention was to clone the CBM domain into a specially prepared vector containing the necessary Aspergillus regulatory elements (promoter, terminator, etc.) a nd a signal peptide with signal cleavage site of a secreted lipase gene from Candida antarctica. Cloning of the PCR product consisting of the CBM domain into the vector allows for an in frame fusion of the CBM domain with the signal peptide.
- pDau109 is a derivative of pJAL721 , which is described in WO 03/008575.
- the plasmid pDau109 differs from pJaL721 in that the ampicillin resistance gene has been inserted into the pyrG selectable marker.
- the improvements made in pDaul 09 vector are first, the selection marker URA3 of E. coli that has been replaced by a URA3 gene disrupted by the insertion of the ampicillin resistance gene E. coli beta lactamase. This feature allows for facile selection for positive recombinant E.
- pDau109 has a Candida antarctica lipase (SWALL:LIPB_CANAR) signal sequence (amino acids 1-57 of SEQ ID NO:9) and cleavage site introduced after the fungal promoter in which a number of convenient cloning sites are available for in frame fusions of a supplied coding region with the C. antarctica secretion sig- nal.
- SWALL:LIPB_CANAR Candida antarctica lipase
- cleavage site introduced after the fungal promoter in which a number of convenient cloning sites are available for in frame fusions of a supplied coding region with the C. antarctica secretion sig- nal.
- pDau109 has 8 unique restriction sites that can be used to insert a cDNA
- Plasmid of pDau109 was prepared by medium scale Qiagen ® midi plasmid preparation (Qiagen) from 100mls of LB ampicillin grown plasmid containing E. coli cells.
- Plasmid NP887-1 encoding the GH61 coding region was used as PCR template.
- NEB T4 DNA ligase buffer
- the ligation was performed at 16 degrees Celsius overnight and then stored in -20 degree Celsius until used.
- Plasmid pCBMX-S1 was found to be error free and in the correct orientation and was therefore chosen for a medium scale Qiagen ® midi plasmid preparation (Qiagen) from 100 ⁇ l of LB ampicillin grown plasmid contain- ing E. coli cells.
- Qiagen medium scale Qiagen ® midi plasmid preparation
- the DNA sequence of the fusion construct pCBMX-S1 and the corresponding amino acid sequence, are shown in SEQ ID NO:8 and SEQ ID NO:9, respectively.
- the fusion construct pCBMX-S1 (SEQ ID NO:8) was transformed into Aspergillus oryzae strain BECh2, which was constructed as described in WO 00/39322 (BECh2 is derived from strain
- Transformation media AMDS media Agarose 20g
- AMDS media for re-isolation of transformants The same as above but without added CsCI and adding 100 ⁇ l triton-X100 per 1000ml media.
- Transformants of pCBMX-S1 were re-isolated twice on Cove sucrose media (Cove (1966) Bio- chim. Biophys. Acta 133:51-56) with 1 M sucrose as a carbon source and 10mM nitrate.
- pCBMX-S1 which contains the Candida antarctica lipase (SWALL:LIPB_CANAR) signal sequence and the P. nigrella CBM polypeptide of the invention (SEQ ID NO:8)
- 23 transformants were grown for 3 days and 4 days at 30 degrees Celsius in tubes with 10 ml YPG (2% peptone, 1% yeast extract, 2% glucose).
- FG4P 3% Soybean meal (SFK 102-2458), 1.5% Maltodextrin (Roquette), 0.5% Peptone bacto (Difco 0118), 1.5% KH2P04 (Merck 4873), 0.2mls/liter Pluronic ® PE 6100 (BASF).
- a heavy inoculum of several thousand spores was used for each and the shake flasks were agitated on an orbital shaker at 150 RPM at 30 degrees C. Aspergillus isolates grew well even when induced for the expression of the CBM polypeptide of the invention.
- SEQ ID NO:9 Purification of SEQ ID NO:9 from expression of SEQ ID NO:8 in Aspergillus.
- the Aspergillus oryzae strain described in Example 1 B expressing the CBM (CBMX) of the Pseudoplectania nigrella GH61 with Candida antarctica lipase signal peptide was grown in shake flasks. About 1 liter culture broth was sterile filtered and the filtrate loaded onto a column containing 50 g Avicel. Non-binding and weakly binding proteins were removed by washing with Milli-Q ® water. Proteins with affinity for Avicel were eluted with 0.1 M Tris, pH 11.5.
- EXAMPLE 3 Specificity of binding of purified CBMX
- the carbohydrate-binding domain with affinity for Avicel and purified as described in Example 2 (CBMX) was studied further.
- 50 ⁇ l purified CBMX was mixed with 500 ⁇ l 20 mM Tris, pH 7.5 containing varying amount of Avicel (0-100 mg/ml) in an Eppendorf tube. After 4 hours incubation at room temperature with agitation, the samples were centrifuged. 200 ⁇ l supernatant was transferred to the well of a microtiter plate (Costar, UV plate) and absorbance read at 280 nm on a microtiter plate reader (SpectraMax ® Plus, Molecular Devices). The results in Table 1 indicate that the large majority of the protein binds to the highest concentrations of Avicel.
- Table 1 Binding of CBMX to Avicel.
- A280 Absorbance at 280 nm of 200 ⁇ l supernatant in microtiter plate with absorbance of buffer and Avicel subtracted.
- Table 2 Binding of CBMX to PASC.
- A280 Absorbance at 280 nm of 200 ⁇ l supernatant in microtiter plate with absorbance of buffer subtracted.
- CBMX Affinity of CBMX for a number of soluble carbohydrates was tested in a competition assay by mixing 100 ⁇ l CBMX with both Avicel (400 ⁇ l 50 mg/ml in 20 mM Tris, pH 7.5) and the soluble carbohydrate (dissolved in 500 ⁇ l 20 mM Tris, pH 7.5). As references, samples without CBMX or soluble carbohydrate added were used. If CBMX has affinity for the soluble carbohydrate it should be able to keep CBMX in solution which can be measured as increase in absorbance at 280 nm compared to sample without soluble carbohydrate added.
- soluble carbohydrates were barley ⁇ -glucan (Megazyme, low viscosity), lichenan (Megazyme, Icelandic moss), CMC (carboxymethyl cellulose 7LF, Hercules, USA), Xyloglucan (Megazyme, amyloid, from tamarind seed), lupin galactan (Megazyme) and Locust bean gum (Sigma, G-0753).
- Table 3 Competition binding assay with CBMX, Avicel (20 mg/ml) and soluble carbohydrates.
- Concentration of carbohydrate Concentration of soluble carbohydrate during incubation with CBMX and Avicel.
- Difference in A280 Difference in absorbance at 280 nm between samples with and without CBMX added.
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Mycology (AREA)
- Detergent Compositions (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Saccharide Compounds (AREA)
- Chemical Or Physical Treatment Of Fibers (AREA)
- Peptides Or Proteins (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Laminated Bodies (AREA)
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE602004022967T DE602004022967D1 (en) | 2003-10-30 | 2004-10-26 | CARBOHYDRATE-BONDING MODULES |
EP04762952A EP1682655B1 (en) | 2003-10-30 | 2004-10-26 | Carbohydrate-binding modules |
US10/576,528 US8846340B2 (en) | 2003-10-30 | 2004-10-26 | Carbohydrate-binding modules of a new family |
AT04762952T ATE441705T1 (en) | 2003-10-30 | 2004-10-26 | CARBOHYDRATE BINDING MODULES |
DK04762952T DK1682655T3 (en) | 2003-10-30 | 2004-10-26 | Carbohydrate binding modules from a new family |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DKPA200301607 | 2003-10-30 | ||
DKPA200301607 | 2003-10-30 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2005042735A1 true WO2005042735A1 (en) | 2005-05-12 |
Family
ID=34530573
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/DK2004/000734 WO2005042735A1 (en) | 2003-10-30 | 2004-10-26 | Carbohydrate-binding modules of a new family |
Country Status (8)
Country | Link |
---|---|
US (2) | US8846340B2 (en) |
EP (1) | EP1682655B1 (en) |
CN (1) | CN1875098A (en) |
AT (1) | ATE441705T1 (en) |
DE (1) | DE602004022967D1 (en) |
DK (1) | DK1682655T3 (en) |
ES (1) | ES2332592T3 (en) |
WO (1) | WO2005042735A1 (en) |
Cited By (86)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011039319A1 (en) | 2009-09-30 | 2011-04-07 | Novozymes A/S | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
WO2011057140A1 (en) | 2009-11-06 | 2011-05-12 | Novozymes, Inc. | Compositions for saccharification of cellulosic material |
WO2011057083A1 (en) | 2009-11-06 | 2011-05-12 | Novozymes, Inc. | Polypeptides having xylanase activity and polynucleotides encoding same |
WO2011075677A2 (en) | 2009-12-18 | 2011-06-23 | Novozymes, Inc. | Methods for producing polypeptides in protease-deficient mutants of trichoderma |
WO2011080267A2 (en) | 2009-12-29 | 2011-07-07 | Novozymes A/S | Polypetides having detergency enhancing effect |
WO2011127802A1 (en) | 2010-04-14 | 2011-10-20 | Novozymes A/S | Polypeptides having glucoamylase activity and polynucleotides encoding same |
WO2012003379A1 (en) | 2010-06-30 | 2012-01-05 | Novozymes A/S | Polypeptides having beta-glucosidase activity and polynucleotides encoding same |
WO2012021395A1 (en) | 2010-08-12 | 2012-02-16 | Novozymes, Inc. | Compositions comprising a polypeptide having cellulolytic enhancing activity and a sulfur-containing compound and uses thereof |
WO2012030811A1 (en) | 2010-08-30 | 2012-03-08 | Novozymes A/S | Polypeptides having cellobiohydrolase activity and polynucleotides encoding same |
WO2012030849A1 (en) | 2010-08-30 | 2012-03-08 | Novozymes A/S | Polypeptides having xylanase activity and polynucleotides encoding same |
WO2012030858A2 (en) | 2010-08-30 | 2012-03-08 | Novozymes A/S | Polypeptides having hemicellulolytic activity and polynucleotides encoding same |
WO2012030845A2 (en) | 2010-08-30 | 2012-03-08 | Novozymes A/S | Polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same |
WO2012030799A1 (en) | 2010-08-30 | 2012-03-08 | Novozymes A/S | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
WO2012030844A1 (en) | 2010-08-30 | 2012-03-08 | Novozymes A/S | Polypeptides having endoglucanase activity and polynucleotides encoding same |
WO2012049181A1 (en) * | 2010-10-13 | 2012-04-19 | Novozymes A/S | Preparation of baked product from dough |
EP2468852A1 (en) | 2007-03-30 | 2012-06-27 | Novozymes A/S | Fungal peroxygenases and methods of application |
WO2012103293A1 (en) | 2011-01-26 | 2012-08-02 | Novozymes A/S | Polypeptides having cellobiohydrolase activity and polynucleotides encoding same |
WO2012103288A1 (en) | 2011-01-26 | 2012-08-02 | Novozymes A/S | Polypeptides having cellobiohydrolase activity and polynucleotides encoding same |
WO2012103322A1 (en) | 2011-01-26 | 2012-08-02 | Novozymes A/S | Polypeptides having endoglucanase activity and polynucleotides encoding same |
WO2012103300A2 (en) | 2011-01-26 | 2012-08-02 | Novozymes A/S | Polypeptides having cellobiohydrolase activity and polynucleotides encoding same |
WO2012122477A1 (en) | 2011-03-10 | 2012-09-13 | Novozymes A/S | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
WO2012122518A1 (en) * | 2011-03-09 | 2012-09-13 | Novozymes A/S | Methods of increasing the cellulolytic enhancing activity of a polypeptide |
WO2012135659A2 (en) | 2011-03-31 | 2012-10-04 | Novozymes A/S | Methods for enhancing the degradation or conversion of cellulosic material |
WO2012149192A1 (en) | 2011-04-28 | 2012-11-01 | Novozymes, Inc. | Polypeptides having endoglucanase activity and polynucleotides encoding same |
WO2013019780A2 (en) | 2011-08-04 | 2013-02-07 | Novozymes A/S | Polypeptides having endoglucanase activity and polynucleotides encoding same |
WO2013019827A2 (en) | 2011-08-04 | 2013-02-07 | Novozymes A/S | Polypeptides having xylanase activity and polynucleotides encoding same |
WO2013024021A1 (en) | 2011-08-15 | 2013-02-21 | Novozymes A/S | Polypeptides having cellulase activity and polynucleotides encoding same |
WO2013036526A1 (en) | 2011-09-06 | 2013-03-14 | Novozymes A/S | Glucoamylase variants and polynucleotides encoding same |
WO2013043910A1 (en) | 2011-09-20 | 2013-03-28 | Novozymes A/S | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
WO2013053801A1 (en) | 2011-10-11 | 2013-04-18 | Novozymes A/S | Glucoamylase variants and polynucleotides encoding same |
WO2013074956A2 (en) | 2011-11-18 | 2013-05-23 | Novozymes, Inc. | Polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same |
WO2013076253A1 (en) | 2011-11-25 | 2013-05-30 | Novozymes A/S | Polypeptides having lysozyme activity and polynucleotides encoding same |
WO2013079531A2 (en) | 2011-12-02 | 2013-06-06 | Novozymes A/S | Polypeptides having peroxygenase activity and polynucleotides encoding same |
WO2013082486A1 (en) | 2011-12-02 | 2013-06-06 | Novozymes A/S | Processes for producing fermentation products |
WO2013079533A1 (en) | 2011-12-02 | 2013-06-06 | Novozymes A/S | Polypeptides having peroxygenase activity and polynucleotides encoding same |
WO2013123871A1 (en) | 2012-02-20 | 2013-08-29 | Novozymes A/S | Polypeptides having endoglucanase activity and polynucleotides encoding same |
US20130302879A1 (en) * | 2010-12-30 | 2013-11-14 | Novozymes A/S | Processes for Treating Textile with Polypeptide Having Cellulolytic Enzyme Enhancing Activity |
WO2013167581A1 (en) | 2012-05-07 | 2013-11-14 | Novozymes A/S | Polypeptides having xanthan degrading activity and polynucleotides encoding same |
WO2014015256A2 (en) | 2012-07-20 | 2014-01-23 | Novozymes A/S | Enzymatic oxidation of 5-hydroxymethylfurfural and derivatives thereof |
WO2014037438A1 (en) | 2012-09-05 | 2014-03-13 | Novozymes A/S | Polypeptides having protease activity |
WO2014056920A2 (en) | 2012-10-12 | 2014-04-17 | Novozymes A/S | Polypeptides having peroxygenase activity |
WO2014056919A2 (en) | 2012-10-12 | 2014-04-17 | Novozymes A/S | Polypeptides having peroxygenase activity |
WO2014056917A2 (en) | 2012-10-12 | 2014-04-17 | Novozymes A/S | Polypeptides having peroxygenase activity |
WO2014056922A2 (en) | 2012-10-12 | 2014-04-17 | Novozymes A/S | Polypeptides having peroxygenase activity |
WO2014056921A2 (en) | 2012-10-12 | 2014-04-17 | Novozymes A/S | Polypeptides having peroxygenase activity |
WO2014056927A2 (en) | 2012-10-12 | 2014-04-17 | Novozymes A/S | Polypeptides having peroxygenase activity |
WO2014056916A2 (en) | 2012-10-12 | 2014-04-17 | Novozymes A/S | Polypeptides having peroxygenase activity |
WO2014058896A1 (en) | 2012-10-08 | 2014-04-17 | Novozymes A/S | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
WO2014066141A2 (en) | 2012-10-24 | 2014-05-01 | Novozymes A/S | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
WO2014085439A1 (en) | 2012-11-30 | 2014-06-05 | Novozymes A/S | Processes for producing fermentation products |
WO2014093835A1 (en) | 2012-12-14 | 2014-06-19 | Novozymes A/S | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
WO2014092832A2 (en) | 2012-09-19 | 2014-06-19 | Novozymes, Inc. | Methods for enhancing the degradation or conversion of cellulosic material |
WO2014099798A1 (en) | 2012-12-19 | 2014-06-26 | Novozymes A/S | Polypeptides having cellulolytic enhancinc activity and polynucleotides encoding same |
WO2014138672A1 (en) | 2013-03-08 | 2014-09-12 | Novozymes A/S | Cellobiohydrolase variants and polynucleotides encoding same |
WO2014147127A1 (en) | 2013-03-21 | 2014-09-25 | Novozymes A/S | Polypeptides with lipase activity and polynucleotides encoding same |
WO2014182990A1 (en) | 2013-05-10 | 2014-11-13 | Novozymes A/S | Polypeptides having xylanase activity and polynucleotides encoding same |
WO2015004102A1 (en) | 2013-07-09 | 2015-01-15 | Novozymes A/S | Polypeptides with lipase activity and polynucleotides encoding same |
WO2015035914A1 (en) | 2013-09-11 | 2015-03-19 | Novozymes A/S | Processes for producing fermentation products |
WO2015058700A1 (en) | 2013-10-25 | 2015-04-30 | Novozymes A/S | Polypeptides having endoglucanase activity and polynucleotides encoding same |
WO2016037096A1 (en) | 2014-09-05 | 2016-03-10 | Novozymes A/S | Carbohydrate binding module variants and polynucleotides encoding same |
WO2016096996A1 (en) | 2014-12-16 | 2016-06-23 | Novozymes A/S | Polypeptides having n-acetyl glucosamine oxidase activity |
WO2016138167A2 (en) | 2015-02-24 | 2016-09-01 | Novozymes A/S | Cellobiohydrolase variants and polynucleotides encoding same |
WO2016205127A1 (en) | 2015-06-18 | 2016-12-22 | Novozymes A/S | Polypeptides having trehalase activity and the use thereof in process of producing fermentation products |
WO2016207384A1 (en) | 2015-06-26 | 2016-12-29 | Novozymes A/S | Method for producing a coffee extract |
WO2017000922A1 (en) | 2015-07-02 | 2017-01-05 | Novozymes A/S | Animal feed compositions and uses thereof |
WO2017001703A1 (en) | 2015-07-02 | 2017-01-05 | Novozymes A/S | Methods of improving animal performance |
WO2017112540A1 (en) | 2015-12-22 | 2017-06-29 | Novozymes A/S | Processes for producing fermentation products |
WO2018002261A1 (en) | 2016-07-01 | 2018-01-04 | Novozymes A/S | Detergent compositions |
WO2018098381A1 (en) | 2016-11-23 | 2018-05-31 | Novozymes A/S | Improved yeast for ethanol production |
WO2018099965A1 (en) | 2016-11-30 | 2018-06-07 | Novozymes A/S | Method of baking |
WO2018127532A1 (en) | 2017-01-04 | 2018-07-12 | Novozymes A/S | Microbial lysozyme for use in the treatment of irritable bowel syndrome or inflammatory bowel disease |
WO2018150021A1 (en) | 2017-02-20 | 2018-08-23 | Novozymes A/S | Lipolytic enzyme for use in baking |
WO2018178061A1 (en) | 2017-03-31 | 2018-10-04 | Novozymes A/S | Polypeptides having rnase activity |
WO2019161227A1 (en) | 2018-02-15 | 2019-08-22 | Novozymes A/S | Improved yeast for ethanol production |
EP3553172A1 (en) | 2012-08-16 | 2019-10-16 | Novozymes A/S | Method for treating textile with endoglucanase |
WO2020053271A1 (en) | 2018-09-11 | 2020-03-19 | Dsm Ip Assets B.V. | Animal feed composition and use thereof |
WO2020053274A1 (en) | 2018-09-11 | 2020-03-19 | Dsm Ip Assets B.V. | Animal feed composition and use thereof |
WO2020053276A1 (en) | 2018-09-11 | 2020-03-19 | Dsm Ip Assets B.V. | Animal feed composition and use thereof |
WO2020053273A1 (en) | 2018-09-11 | 2020-03-19 | Dsm Ip Assets B.V. | Animal feed composition and use thereof |
WO2020053275A2 (en) | 2018-09-11 | 2020-03-19 | Dsm Ip Assets B.V. | Animal feed composition and use thereof |
WO2020123463A1 (en) | 2018-12-12 | 2020-06-18 | Novozymes A/S | Polypeptides having xylanase activity and polynucleotides encoding same |
CN111850019A (en) * | 2019-04-25 | 2020-10-30 | 新乡医学院 | Construction method of agarase fusion enzyme engineering strain |
WO2021064068A1 (en) * | 2019-10-03 | 2021-04-08 | Novozymes A/S | Polypeptides comprising at least two carbohydrate binding domains |
WO2022074170A1 (en) | 2020-10-07 | 2022-04-14 | Novozymes A/S | Enzymatic preservation of probiotics in animal feed |
WO2022079238A1 (en) | 2020-10-15 | 2022-04-21 | Dsm Ip Assets B.V. | Methods of modulating gastrointestinal metabolites |
WO2022194728A1 (en) | 2021-03-16 | 2022-09-22 | Dsm Ip Assets B.V. | Animal feed composition and use thereof |
Families Citing this family (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009002932A1 (en) * | 2007-06-22 | 2008-12-31 | Cornell Research Foundation, Inc. | The use of plant glycosyl hydrolases with carbohydrate binding modules to alter plant cell wall composition and structure, or enhance degradation |
US8586827B2 (en) * | 2009-09-30 | 2013-11-19 | Novozymes, Inc. | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
US9758802B2 (en) * | 2010-08-06 | 2017-09-12 | Novozymes A/S | Methods of degrading or hydrolyzing a polysaccharide |
WO2012087966A2 (en) | 2010-12-20 | 2012-06-28 | E. I. Du Pont De Nemours And Company | Targeted perhydrolases |
CA2868154A1 (en) | 2012-03-20 | 2013-09-26 | The Research Foundation For The State University Of New York | Flocculation of lignocellulosic hydrolyzates |
US9458440B2 (en) * | 2012-06-07 | 2016-10-04 | Roal Oy | Proteins for the treatment of cellulosic material |
US9850512B2 (en) | 2013-03-15 | 2017-12-26 | The Research Foundation For The State University Of New York | Hydrolysis of cellulosic fines in primary clarified sludge of paper mills and the addition of a surfactant to increase the yield |
CN103319607B (en) * | 2013-06-28 | 2015-05-27 | 中国科学院遗传与发育生物学研究所 | CBM fragment and application thereof in specific binding of plant-source crystallized cellulose |
CN103510180A (en) * | 2013-09-17 | 2014-01-15 | 上海婉静纺织科技有限公司 | Lyocell bamboo fiber and preparation method thereof |
US9951363B2 (en) | 2014-03-14 | 2018-04-24 | The Research Foundation for the State University of New York College of Environmental Science and Forestry | Enzymatic hydrolysis of old corrugated cardboard (OCC) fines from recycled linerboard mill waste rejects |
US20210009927A1 (en) * | 2018-04-17 | 2021-01-14 | Novozymes A/S | Polypeptides Comprising Carbohydrate Binding Activity in Detergent Compositions And Their use in Reducing Wrinkles in Textile or Fabrics |
CN113307885B (en) * | 2021-05-26 | 2022-09-27 | 江南大学 | Fusion protein with improved product specificity and application thereof in preparation of linear chain maltopentaose |
CN114368833B (en) * | 2021-12-27 | 2024-07-16 | 江鹰 | Denitrification and dephosphorization composite filler and purifying device containing same |
Citations (38)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB1296839A (en) | 1969-05-29 | 1972-11-22 | ||
GB1372034A (en) | 1970-12-31 | 1974-10-30 | Unilever Ltd | Detergent compositions |
US4077768A (en) | 1975-06-20 | 1978-03-07 | The Procter & Gamble Company | Inhibiting dye transfer in washing or bleaching |
EP0214761A2 (en) | 1985-08-07 | 1987-03-18 | Novo Nordisk A/S | An enzymatic detergent additive, a detergent, and a washing method |
EP0218272A1 (en) | 1985-08-09 | 1987-04-15 | Gist-Brocades N.V. | Novel lipolytic enzymes and their use in detergent compositions |
EP0238023A2 (en) | 1986-03-17 | 1987-09-23 | Novo Nordisk A/S | Process for the production of protein products in Aspergillus oryzae and a promoter for use in Aspergillus |
EP0258068A2 (en) | 1986-08-29 | 1988-03-02 | Novo Nordisk A/S | Enzymatic detergent additive |
WO1988009367A1 (en) | 1987-05-29 | 1988-12-01 | Genencor, Inc. | Cutinase cleaning composition |
EP0305216A1 (en) | 1987-08-28 | 1989-03-01 | Novo Nordisk A/S | Recombinant Humicola lipase and process for the production of recombinant humicola lipases |
JPS6474992A (en) | 1987-09-16 | 1989-03-20 | Fuji Oil Co Ltd | Dna sequence, plasmid and production of lipase |
WO1989006279A1 (en) | 1988-01-07 | 1989-07-13 | Novo-Nordisk A/S | Mutated subtilisin genes |
WO1989006270A1 (en) | 1988-01-07 | 1989-07-13 | Novo-Nordisk A/S | Enzymatic detergent |
EP0331376A2 (en) | 1988-02-28 | 1989-09-06 | Amano Pharmaceutical Co., Ltd. | Recombinant DNA, bacterium of the genus pseudomonas containing it, and process for preparing lipase by using it |
WO1990000609A1 (en) | 1988-07-08 | 1990-01-25 | The University Of British Columbia | Cellulose binding fusion proteins |
WO1990009446A1 (en) | 1989-02-17 | 1990-08-23 | Plant Genetic Systems N.V. | Cutinase |
JPH02238885A (en) | 1989-03-13 | 1990-09-21 | Oji Paper Co Ltd | Phenol oxidase gene recombination dna, microorganism transformed with same recombinant dna, culture mixture thereof and production of phenol oxidase |
WO1991005858A1 (en) | 1989-10-13 | 1991-05-02 | Novo Nordisk A/S | A microperoxidase preparation containing a hemopeptide as an active component |
WO1991016422A1 (en) | 1990-04-14 | 1991-10-31 | Kali-Chemie Aktiengesellschaft | Alkaline bacillus lipases, coding dna sequences therefor and bacilli which produce these lipases |
WO1992001046A1 (en) | 1990-07-06 | 1992-01-23 | Valtion Teknillinen Tutkimuskeskus | Laccase production by recombinant organisms |
WO1992016634A1 (en) | 1991-03-22 | 1992-10-01 | Novo Nordisk A/S | A process for producing heme proteins |
EP0537381A1 (en) | 1991-10-14 | 1993-04-21 | The Procter & Gamble Company | Detergent compositions inhibiting dye transfer in washing |
EP0571982A1 (en) | 1992-05-27 | 1993-12-01 | Showa Denko Kabushiki Kaisha | Alkaline lipase, method for producing the same, microorganism producing the same and detergent composition containing alkaline lipase |
WO1994012621A1 (en) | 1992-12-01 | 1994-06-09 | Novo Nordisk | Enhancement of enzyme reactions |
WO1994024158A1 (en) | 1993-04-14 | 1994-10-27 | The Regents Of The University Of California | Cellulose binding domain |
WO1995014783A1 (en) | 1993-11-24 | 1995-06-01 | Showa Denko K.K. | Lipase gene and variant lipase |
WO1995016782A1 (en) | 1993-12-17 | 1995-06-22 | Genencor International, Inc. | Novel cellulase enzymes and systems for their expression |
WO1995033836A1 (en) | 1994-06-03 | 1995-12-14 | Novo Nordisk Biotech, Inc. | Phosphonyldipeptides useful in the treatment of cardiovascular diseases |
EP0721981A1 (en) | 1993-08-30 | 1996-07-17 | Showa Denko Kabushiki Kaisha | Novel lipase, microorganism producing the lipase, process for producing the lipase, and use of the lipase |
WO1996027002A1 (en) | 1995-02-27 | 1996-09-06 | Novo Nordisk A/S | Novel lipase gene and process for the production of lipase with the use of the same |
WO1996027659A1 (en) | 1995-03-06 | 1996-09-12 | Novo Nordisk A/S | Novel lipase, process for producing the same, and microorganism producing the same |
WO1997028243A1 (en) | 1996-01-29 | 1997-08-07 | Novo Nordisk A/S | Process for removal or bleaching of soiling or stains from cellulosic fabric |
WO1998012300A1 (en) | 1996-09-19 | 1998-03-26 | Novo Nordisk A/S | Novel host cells and methods of producing proteins |
WO1998016112A1 (en) | 1996-10-11 | 1998-04-23 | Novo Nordisk A/S | Use of a carbohydrate binding domain in baking |
WO1999027082A1 (en) | 1997-11-21 | 1999-06-03 | Novo Nordisk A/S | Protease variants and compositions |
US6184440B1 (en) | 1997-07-27 | 2001-02-06 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Transgenic plants of altered morphology |
WO2003000941A2 (en) * | 2001-06-26 | 2003-01-03 | Novozymes A/S | Polypeptides having cellobiohydrolase i activity and polynucleotides encoding same |
WO2003008575A2 (en) | 2001-07-20 | 2003-01-30 | Novozymes A/S | Dna sequences for regulating transcription |
WO2003044049A1 (en) * | 2001-11-20 | 2003-05-30 | Novozymes A/S | Antimicrobial polypeptides from pseudoplectania nigrella |
-
2004
- 2004-10-26 ES ES04762952T patent/ES2332592T3/en active Active
- 2004-10-26 DE DE602004022967T patent/DE602004022967D1/en active Active
- 2004-10-26 EP EP04762952A patent/EP1682655B1/en not_active Not-in-force
- 2004-10-26 WO PCT/DK2004/000734 patent/WO2005042735A1/en active Application Filing
- 2004-10-26 US US10/576,528 patent/US8846340B2/en not_active Expired - Fee Related
- 2004-10-26 DK DK04762952T patent/DK1682655T3/en active
- 2004-10-26 AT AT04762952T patent/ATE441705T1/en not_active IP Right Cessation
- 2004-10-26 CN CNA2004800325968A patent/CN1875098A/en active Pending
-
2014
- 2014-09-29 US US14/500,564 patent/US20150024465A1/en not_active Abandoned
Patent Citations (39)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB1296839A (en) | 1969-05-29 | 1972-11-22 | ||
GB1372034A (en) | 1970-12-31 | 1974-10-30 | Unilever Ltd | Detergent compositions |
US4077768A (en) | 1975-06-20 | 1978-03-07 | The Procter & Gamble Company | Inhibiting dye transfer in washing or bleaching |
EP0214761A2 (en) | 1985-08-07 | 1987-03-18 | Novo Nordisk A/S | An enzymatic detergent additive, a detergent, and a washing method |
EP0218272A1 (en) | 1985-08-09 | 1987-04-15 | Gist-Brocades N.V. | Novel lipolytic enzymes and their use in detergent compositions |
EP0238023A2 (en) | 1986-03-17 | 1987-09-23 | Novo Nordisk A/S | Process for the production of protein products in Aspergillus oryzae and a promoter for use in Aspergillus |
EP0258068A2 (en) | 1986-08-29 | 1988-03-02 | Novo Nordisk A/S | Enzymatic detergent additive |
WO1988009367A1 (en) | 1987-05-29 | 1988-12-01 | Genencor, Inc. | Cutinase cleaning composition |
EP0305216A1 (en) | 1987-08-28 | 1989-03-01 | Novo Nordisk A/S | Recombinant Humicola lipase and process for the production of recombinant humicola lipases |
JPS6474992A (en) | 1987-09-16 | 1989-03-20 | Fuji Oil Co Ltd | Dna sequence, plasmid and production of lipase |
WO1989006279A1 (en) | 1988-01-07 | 1989-07-13 | Novo-Nordisk A/S | Mutated subtilisin genes |
WO1989006270A1 (en) | 1988-01-07 | 1989-07-13 | Novo-Nordisk A/S | Enzymatic detergent |
EP0331376A2 (en) | 1988-02-28 | 1989-09-06 | Amano Pharmaceutical Co., Ltd. | Recombinant DNA, bacterium of the genus pseudomonas containing it, and process for preparing lipase by using it |
WO1990000609A1 (en) | 1988-07-08 | 1990-01-25 | The University Of British Columbia | Cellulose binding fusion proteins |
WO1990009446A1 (en) | 1989-02-17 | 1990-08-23 | Plant Genetic Systems N.V. | Cutinase |
JPH02238885A (en) | 1989-03-13 | 1990-09-21 | Oji Paper Co Ltd | Phenol oxidase gene recombination dna, microorganism transformed with same recombinant dna, culture mixture thereof and production of phenol oxidase |
WO1991005858A1 (en) | 1989-10-13 | 1991-05-02 | Novo Nordisk A/S | A microperoxidase preparation containing a hemopeptide as an active component |
WO1991016422A1 (en) | 1990-04-14 | 1991-10-31 | Kali-Chemie Aktiengesellschaft | Alkaline bacillus lipases, coding dna sequences therefor and bacilli which produce these lipases |
WO1992001046A1 (en) | 1990-07-06 | 1992-01-23 | Valtion Teknillinen Tutkimuskeskus | Laccase production by recombinant organisms |
WO1992016634A1 (en) | 1991-03-22 | 1992-10-01 | Novo Nordisk A/S | A process for producing heme proteins |
EP0537381A1 (en) | 1991-10-14 | 1993-04-21 | The Procter & Gamble Company | Detergent compositions inhibiting dye transfer in washing |
EP0571982A1 (en) | 1992-05-27 | 1993-12-01 | Showa Denko Kabushiki Kaisha | Alkaline lipase, method for producing the same, microorganism producing the same and detergent composition containing alkaline lipase |
WO1994012621A1 (en) | 1992-12-01 | 1994-06-09 | Novo Nordisk | Enhancement of enzyme reactions |
US5670623A (en) | 1993-04-14 | 1997-09-23 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Methods of use of cellulose binding domain proteins |
WO1994024158A1 (en) | 1993-04-14 | 1994-10-27 | The Regents Of The University Of California | Cellulose binding domain |
EP0721981A1 (en) | 1993-08-30 | 1996-07-17 | Showa Denko Kabushiki Kaisha | Novel lipase, microorganism producing the lipase, process for producing the lipase, and use of the lipase |
WO1995014783A1 (en) | 1993-11-24 | 1995-06-01 | Showa Denko K.K. | Lipase gene and variant lipase |
WO1995016782A1 (en) | 1993-12-17 | 1995-06-22 | Genencor International, Inc. | Novel cellulase enzymes and systems for their expression |
WO1995033836A1 (en) | 1994-06-03 | 1995-12-14 | Novo Nordisk Biotech, Inc. | Phosphonyldipeptides useful in the treatment of cardiovascular diseases |
WO1996027002A1 (en) | 1995-02-27 | 1996-09-06 | Novo Nordisk A/S | Novel lipase gene and process for the production of lipase with the use of the same |
WO1996027659A1 (en) | 1995-03-06 | 1996-09-12 | Novo Nordisk A/S | Novel lipase, process for producing the same, and microorganism producing the same |
WO1997028243A1 (en) | 1996-01-29 | 1997-08-07 | Novo Nordisk A/S | Process for removal or bleaching of soiling or stains from cellulosic fabric |
WO1998012300A1 (en) | 1996-09-19 | 1998-03-26 | Novo Nordisk A/S | Novel host cells and methods of producing proteins |
WO1998016112A1 (en) | 1996-10-11 | 1998-04-23 | Novo Nordisk A/S | Use of a carbohydrate binding domain in baking |
US6184440B1 (en) | 1997-07-27 | 2001-02-06 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Transgenic plants of altered morphology |
WO1999027082A1 (en) | 1997-11-21 | 1999-06-03 | Novo Nordisk A/S | Protease variants and compositions |
WO2003000941A2 (en) * | 2001-06-26 | 2003-01-03 | Novozymes A/S | Polypeptides having cellobiohydrolase i activity and polynucleotides encoding same |
WO2003008575A2 (en) | 2001-07-20 | 2003-01-30 | Novozymes A/S | Dna sequences for regulating transcription |
WO2003044049A1 (en) * | 2001-11-20 | 2003-05-30 | Novozymes A/S | Antimicrobial polypeptides from pseudoplectania nigrella |
Non-Patent Citations (43)
Title |
---|
"App. Bacteriol. Symposium Series No. 9", 1980, article "Biology and Activities of Yeast" |
"Biology and Activities of Yeast", 1980, SOC. APP. BACTERIOL. SYMPOSIUM SERIES NO. 9 |
"Current Protocols in Molecular Biology", 1995, JOHN WILEY AND SONS |
"International Union of Biochemistry and Molecular Biology", 1992, ACADEMIC PRESS INC. |
"Molecular Biological Methods for Bacillus", 1990, JOHN WILEY AND SONS |
"Nomenclature Committee of the International Union of Biochemistry and Molecular Biology", 1992, ACADEMIC PRESS INC., article "Recommendations (1992" |
"Protein Purification", 1989, VCH PUBLISHERS |
"Useful proteins from recombinant bacteria", SCIENTIFIC AMERICAN, vol. 242, 1980, pages 74 - 94 |
"Useful proteins from recombinant bacteria", vol. 242, 1980, SCIENTIFIC AMERICAN, pages: 74 - 94 |
BANKA ET AL., WORLD J. MICROBIOL. BIOTECHNOL., vol. 14, 1998, pages 551 - 558 |
BLUME J E ET AL: "A Dictyostelium discoideum cellulase is a member of a spore germination-specific gene family.", THE JOURNAL OF BIOLOGICAL CHEMISTRY. 15 AUG 1991, vol. 266, no. 23, 15 August 1991 (1991-08-15), pages 15432 - 15437, XP002314160, ISSN: 0021-9258 * |
BOURNE Y ET AL: "Glycoside hydrolases and glycosyltransferases: families and functional modules.", CURRENT OPINION IN STRUCTURAL BIOLOGY. OCT 2001, vol. 11, no. 5, October 2001 (2001-10-01), pages 593 - 600, XP002314159, ISSN: 0959-440X * |
BURA ET AL., APPL BIOCHEM BIOTECHNOL., vol. 98-100, 2002, pages 59 - 72 |
CHANG; COHEN, MOLECULAR GENERAL GENETICS, vol. 168, 1979, pages 111 - 115 |
COUTINHO P M ET AL: "Carbohydrate-active enzymes: An integrated database approach", ROYAL SOCIETY OF CHEMISTRY SPECIAL PUBLICATION. RECENT ADVANCES IN CARBOHYDRATE BIOENGINEERING, 1999, CAMBRIDGE, UK, pages 3 - 12, XP008041544, ISSN: 0-85404-774-3 * |
COVE, BIOCHIM. BIOPHYS. ACTA, vol. 133, 1966, pages 51 - 56 |
DIN ET AL., BIO/TECHNOLOGY, vol. 9, 1991, pages 1096 - 1099 |
DUBNAU; DAVIDOFF-ABELSON, JOURNAL OF MOLECULAR BIOLOGY, vol. 56, 1971, pages 209 - 221 |
EHRLICH, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA, vol. 75, 1978, pages 1433 |
GAO ET AL., ACTA BIOCHIM. BIOPHYS. SIN., vol. 33, 2001, pages 13 - 18 |
GILKES ET AL., INT. J. BIOL. MACROMOL., vol. 15, 1993, pages 347 - 351 |
GILKES ET AL., J. BIOL. CHEM., vol. 263, 1988, pages 10401 - 10407 |
GUO; SHERMAN, MOLECULAR CELLULAR BIOLOGY, vol. 15, 1995, pages 5983 - 5990 |
HASS ET AL., GENE, vol. 109, 1991, pages 117 - 113 |
HAWKSWORTH ET AL.: "Ainsworth and Bisby's Dictionary of The Fungi", 1995, CAB INTERNATIONAL, UNIVERSITY PRESS |
ITO ET AL., JOURNAL OF BACTERIOLOGY, vol. 153, 1983, pages 163 |
KOEHLER; THORNE, JOURNAL OF BACTERIOLOGY, vol. 169, 1987, pages 5771 - 5778 |
KRULL ET AL., BIOTECHNOL. BIO- ENG., vol. 31, 1988, pages 321 - 327 |
LEVY I ET AL: "Cellulose-binding domains - Biotechnological applications", BIOTECHNOLOGY ADVANCES, ELSEVIER PUBLISHING, BARKING, GB, vol. 20, no. 3-4, November 2002 (2002-11-01), pages 191 - 213, XP004392778, ISSN: 0734-9750 * |
LEVY; SHOSEYOV, BIOTECHNOLOGY ADVANCES, vol. 20, 2002, pages 191 - 213 |
MALARDIER ET AL., GENE, vol. 78, 1989, pages 147 - 156 |
NAITO ET AL., JOURNAL OF CHEMICAL ENGINEERING OF JAPAN, vol. 34, no. 12, 2001, pages 1545 - 1548 |
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LAB. |
SIMONEN; PALVA, MICROBIOLOGICAL REVIEWS, vol. 57, 1993, pages 109 - 137 |
SMITH; WATERMAN, J. MOL. BIOL., vol. 147, 1981, pages 195 - 197 |
TEPLYAKOV ET AL., PROTEIN ENGINEERING, vol. 5, 1992, pages 413 - 420 |
TERPE, APPL. MICROBIOL. BIOTECHNOL., vol. 60, 2003, pages 523 - 533 |
THOMPSON ET AL., NUC. ACID RES., vol. 22, 1994, pages 4673 - 4680 |
TOMME ET AL.: "Cellulose-Binding Domains, Classification and Properties in Enzymatic Degradation of Insoluble Carbohydrates", 1996, ACS SYMPOSIUM SERIES |
TOMME ET AL.: "Enzymatic Degradation of Insoluble Polysaccharides", 1995, AMERICAN CHEMICAL SOCIETY, pages: 142 - 163 |
VILLA-KAMAROFF ET AL., PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA, vol. 75, 1978, pages 3727 - 3731 |
XU ET AL., BIOMACROMOLECULES, vol. 3, 2002, pages 462 - 465 |
YAMAGUCHI ET AL., GENE, vol. 103, 1991, pages 61 - 67 |
Cited By (111)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2468852A1 (en) | 2007-03-30 | 2012-06-27 | Novozymes A/S | Fungal peroxygenases and methods of application |
EP2471911A2 (en) | 2007-03-30 | 2012-07-04 | Novozymes A/S | Fungal peroxygenases and methods of application |
WO2011039319A1 (en) | 2009-09-30 | 2011-04-07 | Novozymes A/S | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
WO2011057140A1 (en) | 2009-11-06 | 2011-05-12 | Novozymes, Inc. | Compositions for saccharification of cellulosic material |
WO2011057083A1 (en) | 2009-11-06 | 2011-05-12 | Novozymes, Inc. | Polypeptides having xylanase activity and polynucleotides encoding same |
EP3222716A1 (en) | 2009-11-06 | 2017-09-27 | Novozymes, Inc. | Composition for saccharification of cellulosic material |
EP3550016A1 (en) | 2009-11-06 | 2019-10-09 | Novozymes, Inc. | Composition for saccharification of cellulosic material |
WO2011075677A2 (en) | 2009-12-18 | 2011-06-23 | Novozymes, Inc. | Methods for producing polypeptides in protease-deficient mutants of trichoderma |
EP3000871A1 (en) | 2009-12-18 | 2016-03-30 | Novozymes, Inc. | Methods for producing polypeptides in protease-deficient mutants of trichoderma |
WO2011080267A2 (en) | 2009-12-29 | 2011-07-07 | Novozymes A/S | Polypetides having detergency enhancing effect |
WO2011127802A1 (en) | 2010-04-14 | 2011-10-20 | Novozymes A/S | Polypeptides having glucoamylase activity and polynucleotides encoding same |
WO2012003379A1 (en) | 2010-06-30 | 2012-01-05 | Novozymes A/S | Polypeptides having beta-glucosidase activity and polynucleotides encoding same |
WO2012021394A1 (en) | 2010-08-12 | 2012-02-16 | Novozymes, Inc. | Compositions comprising a polypeptide having cellulolytic enhancing activity and a quinone compound and uses thereof |
WO2012021395A1 (en) | 2010-08-12 | 2012-02-16 | Novozymes, Inc. | Compositions comprising a polypeptide having cellulolytic enhancing activity and a sulfur-containing compound and uses thereof |
WO2012021399A1 (en) | 2010-08-12 | 2012-02-16 | Novozymes, Inc. | Compositions comprising a polypeptide having cellulolytic enhancing activity and a nitrogen-containing compound and uses thereof |
WO2012021400A1 (en) | 2010-08-12 | 2012-02-16 | Novozymes, Inc. | Compositions comprising a polypeptide having cellulolytic enhancing activity and a heterocyclic compound and uses thereof |
WO2012030799A1 (en) | 2010-08-30 | 2012-03-08 | Novozymes A/S | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
WO2012030811A1 (en) | 2010-08-30 | 2012-03-08 | Novozymes A/S | Polypeptides having cellobiohydrolase activity and polynucleotides encoding same |
WO2012030844A1 (en) | 2010-08-30 | 2012-03-08 | Novozymes A/S | Polypeptides having endoglucanase activity and polynucleotides encoding same |
EP2735611A2 (en) | 2010-08-30 | 2014-05-28 | Novozymes A/S | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
WO2012030849A1 (en) | 2010-08-30 | 2012-03-08 | Novozymes A/S | Polypeptides having xylanase activity and polynucleotides encoding same |
WO2012030845A2 (en) | 2010-08-30 | 2012-03-08 | Novozymes A/S | Polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same |
EP3470514A1 (en) | 2010-08-30 | 2019-04-17 | Novozymes A/S | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
WO2012030858A2 (en) | 2010-08-30 | 2012-03-08 | Novozymes A/S | Polypeptides having hemicellulolytic activity and polynucleotides encoding same |
WO2012049181A1 (en) * | 2010-10-13 | 2012-04-19 | Novozymes A/S | Preparation of baked product from dough |
US20130302879A1 (en) * | 2010-12-30 | 2013-11-14 | Novozymes A/S | Processes for Treating Textile with Polypeptide Having Cellulolytic Enzyme Enhancing Activity |
WO2012103322A1 (en) | 2011-01-26 | 2012-08-02 | Novozymes A/S | Polypeptides having endoglucanase activity and polynucleotides encoding same |
WO2012103293A1 (en) | 2011-01-26 | 2012-08-02 | Novozymes A/S | Polypeptides having cellobiohydrolase activity and polynucleotides encoding same |
WO2012103288A1 (en) | 2011-01-26 | 2012-08-02 | Novozymes A/S | Polypeptides having cellobiohydrolase activity and polynucleotides encoding same |
WO2012103300A2 (en) | 2011-01-26 | 2012-08-02 | Novozymes A/S | Polypeptides having cellobiohydrolase activity and polynucleotides encoding same |
EP3235903A1 (en) | 2011-01-26 | 2017-10-25 | Novozymes A/S | Polypeptides having cellobiohydrolase activity and polynucleotides encoding same |
US9677060B2 (en) | 2011-03-09 | 2017-06-13 | Novozymes A/S | Methods of increasing the cellulolytic enhancing activity of a polypeptide |
EP3339442A1 (en) * | 2011-03-09 | 2018-06-27 | Novozymes A/S | Methods of increasing the cellulolytic enhancing activity of a polypeptide |
WO2012122518A1 (en) * | 2011-03-09 | 2012-09-13 | Novozymes A/S | Methods of increasing the cellulolytic enhancing activity of a polypeptide |
US9150842B2 (en) | 2011-03-09 | 2015-10-06 | Novozymes A/S | Methods of increasing the cellulolytic enhancing activity of a polypeptide |
WO2012122477A1 (en) | 2011-03-10 | 2012-09-13 | Novozymes A/S | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
WO2012135659A2 (en) | 2011-03-31 | 2012-10-04 | Novozymes A/S | Methods for enhancing the degradation or conversion of cellulosic material |
WO2012149192A1 (en) | 2011-04-28 | 2012-11-01 | Novozymes, Inc. | Polypeptides having endoglucanase activity and polynucleotides encoding same |
EP3091073A2 (en) | 2011-08-04 | 2016-11-09 | Novozymes Inc. | Polypeptides having xylanase activity and polynucleotides encoding same |
WO2013019827A2 (en) | 2011-08-04 | 2013-02-07 | Novozymes A/S | Polypeptides having xylanase activity and polynucleotides encoding same |
WO2013019780A2 (en) | 2011-08-04 | 2013-02-07 | Novozymes A/S | Polypeptides having endoglucanase activity and polynucleotides encoding same |
WO2013024021A1 (en) | 2011-08-15 | 2013-02-21 | Novozymes A/S | Polypeptides having cellulase activity and polynucleotides encoding same |
WO2013036526A1 (en) | 2011-09-06 | 2013-03-14 | Novozymes A/S | Glucoamylase variants and polynucleotides encoding same |
WO2013043910A1 (en) | 2011-09-20 | 2013-03-28 | Novozymes A/S | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
WO2013053801A1 (en) | 2011-10-11 | 2013-04-18 | Novozymes A/S | Glucoamylase variants and polynucleotides encoding same |
WO2013074956A2 (en) | 2011-11-18 | 2013-05-23 | Novozymes, Inc. | Polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same |
EP3382017A1 (en) | 2011-11-18 | 2018-10-03 | Novozymes A/S | Polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same |
EP3409769A1 (en) | 2011-11-18 | 2018-12-05 | Novozymes A/S | Polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same |
WO2013076253A1 (en) | 2011-11-25 | 2013-05-30 | Novozymes A/S | Polypeptides having lysozyme activity and polynucleotides encoding same |
WO2013076259A2 (en) | 2011-11-25 | 2013-05-30 | Novozymes A/S | Polypeptides having lysozyme activity and polynucleotides encoding same |
WO2013079531A2 (en) | 2011-12-02 | 2013-06-06 | Novozymes A/S | Polypeptides having peroxygenase activity and polynucleotides encoding same |
WO2013082486A1 (en) | 2011-12-02 | 2013-06-06 | Novozymes A/S | Processes for producing fermentation products |
WO2013079533A1 (en) | 2011-12-02 | 2013-06-06 | Novozymes A/S | Polypeptides having peroxygenase activity and polynucleotides encoding same |
WO2013123871A1 (en) | 2012-02-20 | 2013-08-29 | Novozymes A/S | Polypeptides having endoglucanase activity and polynucleotides encoding same |
WO2013167581A1 (en) | 2012-05-07 | 2013-11-14 | Novozymes A/S | Polypeptides having xanthan degrading activity and polynucleotides encoding same |
WO2014015256A2 (en) | 2012-07-20 | 2014-01-23 | Novozymes A/S | Enzymatic oxidation of 5-hydroxymethylfurfural and derivatives thereof |
EP3553172A1 (en) | 2012-08-16 | 2019-10-16 | Novozymes A/S | Method for treating textile with endoglucanase |
WO2014037438A1 (en) | 2012-09-05 | 2014-03-13 | Novozymes A/S | Polypeptides having protease activity |
WO2014092832A2 (en) | 2012-09-19 | 2014-06-19 | Novozymes, Inc. | Methods for enhancing the degradation or conversion of cellulosic material |
EP3586610A1 (en) | 2012-10-08 | 2020-01-01 | Novozymes A/S | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
WO2014058896A1 (en) | 2012-10-08 | 2014-04-17 | Novozymes A/S | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
WO2014056919A2 (en) | 2012-10-12 | 2014-04-17 | Novozymes A/S | Polypeptides having peroxygenase activity |
WO2014056920A2 (en) | 2012-10-12 | 2014-04-17 | Novozymes A/S | Polypeptides having peroxygenase activity |
WO2014056916A2 (en) | 2012-10-12 | 2014-04-17 | Novozymes A/S | Polypeptides having peroxygenase activity |
WO2014056927A2 (en) | 2012-10-12 | 2014-04-17 | Novozymes A/S | Polypeptides having peroxygenase activity |
WO2014056921A2 (en) | 2012-10-12 | 2014-04-17 | Novozymes A/S | Polypeptides having peroxygenase activity |
WO2014056922A2 (en) | 2012-10-12 | 2014-04-17 | Novozymes A/S | Polypeptides having peroxygenase activity |
WO2014056917A2 (en) | 2012-10-12 | 2014-04-17 | Novozymes A/S | Polypeptides having peroxygenase activity |
WO2014066141A2 (en) | 2012-10-24 | 2014-05-01 | Novozymes A/S | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
WO2014085439A1 (en) | 2012-11-30 | 2014-06-05 | Novozymes A/S | Processes for producing fermentation products |
WO2014093835A1 (en) | 2012-12-14 | 2014-06-19 | Novozymes A/S | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
WO2014099798A1 (en) | 2012-12-19 | 2014-06-26 | Novozymes A/S | Polypeptides having cellulolytic enhancinc activity and polynucleotides encoding same |
WO2014138672A1 (en) | 2013-03-08 | 2014-09-12 | Novozymes A/S | Cellobiohydrolase variants and polynucleotides encoding same |
WO2014147127A1 (en) | 2013-03-21 | 2014-09-25 | Novozymes A/S | Polypeptides with lipase activity and polynucleotides encoding same |
WO2014182990A1 (en) | 2013-05-10 | 2014-11-13 | Novozymes A/S | Polypeptides having xylanase activity and polynucleotides encoding same |
WO2015004102A1 (en) | 2013-07-09 | 2015-01-15 | Novozymes A/S | Polypeptides with lipase activity and polynucleotides encoding same |
WO2015035914A1 (en) | 2013-09-11 | 2015-03-19 | Novozymes A/S | Processes for producing fermentation products |
EP3712274A1 (en) | 2013-09-11 | 2020-09-23 | Novozymes A/S | Processes for producing fermentation products |
WO2015058700A1 (en) | 2013-10-25 | 2015-04-30 | Novozymes A/S | Polypeptides having endoglucanase activity and polynucleotides encoding same |
EP3594335A1 (en) | 2014-09-05 | 2020-01-15 | Novozymes A/S | Carbohydrate binding module variants and polynucleotides encoding same |
WO2016037096A1 (en) | 2014-09-05 | 2016-03-10 | Novozymes A/S | Carbohydrate binding module variants and polynucleotides encoding same |
EP4406964A2 (en) | 2014-09-05 | 2024-07-31 | Novozymes A/S | Carbohydrate binding module variants and polynucleotides encoding same |
WO2016096996A1 (en) | 2014-12-16 | 2016-06-23 | Novozymes A/S | Polypeptides having n-acetyl glucosamine oxidase activity |
EP3739045A2 (en) | 2015-02-24 | 2020-11-18 | Novozymes A/S | Cellobiohydrolase variants and polynucleotides encoding same |
WO2016138167A2 (en) | 2015-02-24 | 2016-09-01 | Novozymes A/S | Cellobiohydrolase variants and polynucleotides encoding same |
WO2016205127A1 (en) | 2015-06-18 | 2016-12-22 | Novozymes A/S | Polypeptides having trehalase activity and the use thereof in process of producing fermentation products |
WO2016207384A1 (en) | 2015-06-26 | 2016-12-29 | Novozymes A/S | Method for producing a coffee extract |
EP4032409A1 (en) | 2015-07-02 | 2022-07-27 | Novozymes A/S | Animal feed comprising a feed additive for improving animal performance |
WO2017001703A1 (en) | 2015-07-02 | 2017-01-05 | Novozymes A/S | Methods of improving animal performance |
WO2017000922A1 (en) | 2015-07-02 | 2017-01-05 | Novozymes A/S | Animal feed compositions and uses thereof |
WO2017112540A1 (en) | 2015-12-22 | 2017-06-29 | Novozymes A/S | Processes for producing fermentation products |
WO2018002261A1 (en) | 2016-07-01 | 2018-01-04 | Novozymes A/S | Detergent compositions |
WO2018098381A1 (en) | 2016-11-23 | 2018-05-31 | Novozymes A/S | Improved yeast for ethanol production |
WO2018099965A1 (en) | 2016-11-30 | 2018-06-07 | Novozymes A/S | Method of baking |
WO2018127532A1 (en) | 2017-01-04 | 2018-07-12 | Novozymes A/S | Microbial lysozyme for use in the treatment of irritable bowel syndrome or inflammatory bowel disease |
EP4052722A1 (en) | 2017-01-04 | 2022-09-07 | Novozymes A/S | Compositions for human treatment |
WO2018150021A1 (en) | 2017-02-20 | 2018-08-23 | Novozymes A/S | Lipolytic enzyme for use in baking |
WO2018178061A1 (en) | 2017-03-31 | 2018-10-04 | Novozymes A/S | Polypeptides having rnase activity |
WO2019161227A1 (en) | 2018-02-15 | 2019-08-22 | Novozymes A/S | Improved yeast for ethanol production |
WO2020053275A2 (en) | 2018-09-11 | 2020-03-19 | Dsm Ip Assets B.V. | Animal feed composition and use thereof |
WO2020053273A1 (en) | 2018-09-11 | 2020-03-19 | Dsm Ip Assets B.V. | Animal feed composition and use thereof |
WO2020053276A1 (en) | 2018-09-11 | 2020-03-19 | Dsm Ip Assets B.V. | Animal feed composition and use thereof |
WO2020053274A1 (en) | 2018-09-11 | 2020-03-19 | Dsm Ip Assets B.V. | Animal feed composition and use thereof |
WO2020053271A1 (en) | 2018-09-11 | 2020-03-19 | Dsm Ip Assets B.V. | Animal feed composition and use thereof |
WO2020123463A1 (en) | 2018-12-12 | 2020-06-18 | Novozymes A/S | Polypeptides having xylanase activity and polynucleotides encoding same |
CN111850019A (en) * | 2019-04-25 | 2020-10-30 | 新乡医学院 | Construction method of agarase fusion enzyme engineering strain |
WO2021064068A1 (en) * | 2019-10-03 | 2021-04-08 | Novozymes A/S | Polypeptides comprising at least two carbohydrate binding domains |
WO2022074170A1 (en) | 2020-10-07 | 2022-04-14 | Novozymes A/S | Enzymatic preservation of probiotics in animal feed |
WO2022074163A2 (en) | 2020-10-07 | 2022-04-14 | Novozymes A/S | Enzymatic feed preservation |
WO2022079238A1 (en) | 2020-10-15 | 2022-04-21 | Dsm Ip Assets B.V. | Methods of modulating gastrointestinal metabolites |
WO2022194728A1 (en) | 2021-03-16 | 2022-09-22 | Dsm Ip Assets B.V. | Animal feed composition and use thereof |
Also Published As
Publication number | Publication date |
---|---|
DK1682655T3 (en) | 2010-01-11 |
US20150024465A1 (en) | 2015-01-22 |
US20070072185A1 (en) | 2007-03-29 |
EP1682655B1 (en) | 2009-09-02 |
ES2332592T3 (en) | 2010-02-09 |
EP1682655A1 (en) | 2006-07-26 |
US8846340B2 (en) | 2014-09-30 |
CN1875098A (en) | 2006-12-06 |
DE602004022967D1 (en) | 2009-10-15 |
ATE441705T1 (en) | 2009-09-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP1682655B1 (en) | Carbohydrate-binding modules | |
US6015783A (en) | Process for removal or bleaching of soiling or stains from cellulosic fabric | |
EP2046819B1 (en) | Methods of increasing secretion of polypeptides having biological activity | |
KR100423670B1 (en) | Alkaline cellulase and method for producing same | |
CA2158357C (en) | Purification and molecular cloning of eg iii cellulase | |
EP0877799A1 (en) | Process for desizing cellulosic fabric | |
US8309338B2 (en) | Polypeptides having endoglucanase activity and polynucleotides encoding same | |
CA2405954C (en) | Novel expression-regulating sequences and expression products in the field of filamentous fungi | |
US7981654B2 (en) | Cellulases and their uses | |
WO2015040159A2 (en) | Polypeptides having mannanase activity and polynucleotides encoding same | |
Wang et al. | A unique endoglucanase-encoding gene cloned from the phytopathogenic fungus Macrophomina phaseolina | |
US10927358B2 (en) | Endoglucanase compositions and methods | |
US20010031490A1 (en) | Laccase mutants | |
US11987824B2 (en) | Additional endoglucanase variants and methods | |
US10808234B2 (en) | Variant amylase enzyme compositions and methods | |
US20220170001A1 (en) | Amylase Enzymes | |
US20220162576A1 (en) | Amylase enzymes | |
Yano et al. | Cloning and expression of chitinase A gene from Streptomyces cyaneus SP-27: the enzyme participates in protoplast formation of Schizophyllum commune | |
CN118256469A (en) | Lysophospholipase mutant and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 200480032596.8 Country of ref document: CN |
|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2004762952 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2007072185 Country of ref document: US Ref document number: 10576528 Country of ref document: US |
|
WWP | Wipo information: published in national office |
Ref document number: 2004762952 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 10576528 Country of ref document: US |