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WO1998001569A1 - Cellulases - Google Patents

Cellulases Download PDF

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Publication number
WO1998001569A1
WO1998001569A1 PCT/NL1997/000400 NL9700400W WO9801569A1 WO 1998001569 A1 WO1998001569 A1 WO 1998001569A1 NL 9700400 W NL9700400 W NL 9700400W WO 9801569 A1 WO9801569 A1 WO 9801569A1
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WIPO (PCT)
Prior art keywords
ser
ala
val
gly
leu
Prior art date
Application number
PCT/NL1997/000400
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English (en)
Inventor
Arjen Schots
Jacob Bakker
Johannes Helder
Frederik Gommers
Wilhelmus Johannes Stiekema
Jan Roosien
Aska Goverse
Alexander Schouten
Geert Smant
Jan Marinus DE BOER
Jacobus Petrus Wilhelmus Stokkermans
Pierre Abad
Marie Noëlle Françoise ROSSO
Original Assignee
Rijkslandbouwuniversiteit Wageningen
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from EP96201890A external-priority patent/EP0818538A1/fr
Application filed by Rijkslandbouwuniversiteit Wageningen filed Critical Rijkslandbouwuniversiteit Wageningen
Publication of WO1998001569A1 publication Critical patent/WO1998001569A1/fr

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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)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase

Definitions

  • the invention relates to ccllulases ot .1 novel class, to their am o acid sequences, and to nucleotides encoding them.
  • the invention also concerns the use of these enzymes in agriculture and in industry and the use ot peptides 01 other compounds inhibiting these enzymes in the protection of crops against plagues Background
  • Plant parasitic ncmatodcs are an important threat to agriculture because they feed on roots of host plants and reduce the possibilities of the plant to take up nutrients from the soil.
  • sedentary parasites have swollen females
  • migratory cndoparasitcs craw l in the root
  • se i- endoparasites 3.
  • ectoparasites 5. epidermal and root hair feeders
  • Plant parasitic ncmatodcs comprise
  • Sedentary ncmatodcs include a. Cyst ncmatodcs (Hcterode ⁇ dae tamih ) b. Root knot ncmatodcs (Meloidogynidac tamil v) c. genus Natobbus
  • cyst and root knot ncmatodcs are ot most economic importance
  • Cyst ncmatodcs are ncmatodcs of the Hcterode ⁇ dae tamih
  • Thev represent an important group of pest organisms in agriculture
  • Subfamilies are the Hcterode ⁇ nae, Meloidodertnae (one genus one species) and Atalodcrinae (4 genera 12 species)
  • the Heterode ⁇ nac are divided into 85 species in 7 genera three of w hich comprise economically important species Hcicrodcta s species including H sdiaduu (sugar beet), H avenae (oat) H btfencsuci (grass) H ui ift'i m (cabbage ) // (soybean) H goettingwna (pea) H o ⁇ (ncc) H SUL CIUI ⁇ (sugarcane) H t/ ifoln (clover sugar beet) and H ze c (ma
  • Root-knot ncmatodcs include the ceonomicalh important genus Meloidogvnc Meloidogvnc species are pol phagous Thc ⁇ produce c onspicuous knots or gall-like sw ellings on roots Thc ⁇ attack most ig ⁇ culturalh important plants such as vegetables, cotton straw erries orchard trees garden and ornamental plants Losses can be severe and there can be several generations (the number depending on conditions) per year Species in temperate zones comprise M ap/a (northern root-knot nematode).
  • M chitwoodi and M naasi In (sub)trop ⁇ eal zones M nu M jcivcinn. ii and M ⁇ enaria occur Sedentary plant parasitic ncmatodcs feed from their host b ⁇ transforming root cells into multinuclcate feeding cells
  • this feeding site consists of a large svncytium w ich results from a fusion of ad ⁇ cent root cells
  • the successful formation and exploitation of these feeding cells involves a complex interaction between nematodc and host plant, in w hich nematodc secretions ot both polypcptide and polysaccha ⁇ de nature arc considered to play an important role
  • Feeding cells of root-knot ncmatodcs arc so-called giant cells Juveniles of Meloidogvnc migrate interccllularly in the root to the region of c ell differentiation and locate cells in the vascular c ⁇ Under of the stele
  • the multinucleate giant cells become the permanent feeding site toi the parasite throughout its life-cycle Giant cell formation is one of the most complex responses elicited in plant tissue by any parasite Cells parasitised by juveniles undergo repeated division of their nuclei without cell division.
  • ncmatodcs have three large csophageal gland ce ls one dorsal and tw o subventral Thev inject secretions from these csophageal (also referred to herein as salivary) glands via their sn lct into the cells of their host plant
  • salivary also referred to herein as salivary
  • these saliva proteins are presumabK inv olv ed in feeding site induction and they are also necessary for feeding itself .is follo from the foi ation of mtraccllular feeding tubes
  • the invention is based on the iscov c ⁇ of ccilulolvtic enzymes produced by nematodes in their salivary glands
  • Thev probably play a similar role in root parasitism by endoparasitic nematodes
  • these enzvmes mav be multifunctional and may therefore play a role in the induction of feeding cells w hich are formed b ⁇ species belonging to the plant parasitic nematodes
  • the inv ention pertains to cellulotvtic peptides and parts thereof and their use as w ell as to nucleotides encoding them
  • the invention pertains to nov el peptides having cellulasc (cndo-fi- 1 4-glucanasc) activity and exhibiting at least 40 r f amino acid identity in the primary structure w ith the amino acid sequence of the celluloh tie subventral csophageal proteins (u>//s) of Heleroderci ghetnes and/or Globoderci lostoducnsis w hich are show n in SEO ID NO 's 1-4 or with the majority sequence show n in Fig 1
  • amino acid homology is meant here amino acid identity in the p ⁇ marv structure
  • Ammo acid similarity is usually higher than the figures given for identity
  • Said amino acid identity in the primary structure is higher for partial sequences
  • the amino acid identity ith the sequence 19- 318 of SEQ ID No 1 (29-330 of SEQ ID No 2 20-324 of SEQ ID No s 3 and 4) is at least 50%, especially at least 60
  • the peptides arc characterised bv comprising a constitus of at least 8 contiguous amino acids of the amino acid sequence sho n in one of SEQ ID NO 's 1-4 or of the ammo acid sequence encoded b ⁇ the nucleotide sequence sho n in one of SEQ ID NO 's 1 -7
  • said series comprises at least 10 especially at least 12 or even at least 15 contiguous amino acids of the amino acid sequence show n in one of SEQ ID NO 's 1 -4
  • the peptide according to the inv ention preferably corresponds to a nematodal peptide or part thereof especially to a peptide of a sedentary nematodc
  • the c ⁇ st and root- knot nematodes ⁇ TC most important and thus their c luloh tic enzvmes arc a preferred group according to the present invention Parts of the peptides especially refer to immunogenic parts capable of inducing antibodies i
  • the invention furthermore rel ites to a nucleotide sequence encoding a peptide having cellulasc activity and exhibiting at least 40' ⁇ ammo aci homolog) w ith the amino acid sequence show n in one of SEQ ID NO s 1 -4 or hy bridising under stringent conditions with a nucleotide sequence sho n in SEQ I D NO s 1 — 1 or 5-7 or a part thereof having at least 15.
  • the coding sequences may contain mutations (insertions deletions or both) which serve to modify the structure and/or the activity of the expression product
  • the minimum identity and/or the hybridisation characteristic as defined above should preferably be maintained
  • the nucleotide sequence may also correspond to regulating or signal sequences of nematodal ccllulases
  • the nucleotide sequence may comprise substantially the encoding and/or regulating sequences of the cellulasc
  • the nucleotide sequence mav be used as a primer or probe in detecting nematodal cellulasc encoding sequences.
  • Also part of the invention are expression vec tors and plasmids containing the nucleotide sequences described above under the control of a homologous or hctcrologous promoter. Furthermore, the invention is concerned w ith the use of these sequences for the production of ccllulases by a different host under the control of its own. or hctcrologous regulatory sequences.
  • the expression vectors and host cells may contain multiple copies of the cellulase-encoding sequences (altered or not w ith respect to SEO ID NO's 1 -4 or 5-7) and also of other genes. Host organisms may be homologous production strains or alternatively hetero- logous hosts.
  • Suitable host organisms include fungi y easts bacteria and plants Examples are A pergdlus species. Ti id xk'i inci species Dae din s spec ies klnw romvc cs species and Sacchcironivc cs species For food applic ations of the ce llulasc the host organism is preferably food-grade
  • Examples of ow n control regions and hctcrologous regulatory regions include fungal constitutive and/or inducible promoters such as the p ruvatc kinase promoter (pkiA) and the glyceraldehyde-3-phosphatc dchvdrogenasc (gpd) promoters
  • Examples of strong yeast promoters are alcohol dehydrogenasc 3-phos ⁇ hogl vcerate kinase and t ⁇ ose phosphate lsomcrase promoters.
  • Examples of bac terial promoters arc ⁇
  • the host cell may advantageously express or ov erexprcss other relevant proteins, including enzvmes. in particular other glycanolvtic enzy mes such as xvlanascs. amvlases. glucanascs, -glucosidascs and/or other enzvmes such as oxidoreductascs such as hexose oxidase, u-glucuronidase. lipases. cstcrascs and/or proteases
  • the corresponding genes may be under the control of homologous control regions or under the control region of the cellulasc gene.
  • the invention is also concerned w nh antibodies directed at the ccllulolvtic enzymes described above or to immunogenie parts thereof
  • These antibodies or one or more of the variable parts thereof can be used in protecting crops against the action of parasitic plant nematodes, especially sedentary ncmatodcs cither as such or as an antibody-encoding insert in a transgenic plant
  • SEQ ID NO's 1 and 2 depict the genes HG-c/nA (about 1 kb) and UG-engl (about 1.5 kb) respectively encoding fi-1 .4-endoglucan ⁇ scs of H icrodera glvcincs with deduced amino acid sequence; these listings show the complete protein including the signal sequences.
  • SEQ ID NO's 3 and 4 depict the genes GR-en ⁇ ;2 and GR-eng I respectively encoding ⁇ -1.4-endoglucanascs of Globoderci lostocluensis w ith deduced amino acid sequence.
  • SEQ ID NO's 5, 6 and 7 depict the partial nucleotide sequences of the corresponding genes respectively encoding — 1,4— endoglucanases of Meloidogvnc incognita
  • GR-ENG2 amino acids 1 - 19 signal sequence amino acids 20-324 catalytic domain, amino acids 325-386 linker sequence This protein has no cellulose binding domain, HG-ENG1 ammo acids 1 -28 signal sequence amino acids 29-330 catalytic domain, am o acids 331 -380 linker sequence 381 — 176 cellulose binding domain HG-ENG2 1-18 signal sequence amino acids 19-318 catalytic domain This protein has no linker sequence and no cellulose binding domain
  • HG-ENG 1 having a homology of 80' " ? w ith the catalytic domain of HG-ENG2 shows homologies w ith the follo ing proteins Endoglucanase Z precursor (cndo- 1 4- -glucanasc cellulasc) from Ft wima du wanihemi, 35.6% in 385 amino acids overlap (AC P07103)
  • Endoglucanase precursor (endo-1 4-
  • Endoglucanase IV (cndo-1.4-
  • Endoglucanase CELA precursor endo- 1.4-(Aglucanase; cellulasc) from Sircptonivces lividans; 35.4% in 297 amino acids overlap (AC P27035)
  • Endoglucanase A precursor endo-1.4- i-gluca ⁇ asc; cellulasc; EGA) from Butyrivtbrio fibrisolvcns; 36.5% in 277 amino acids overlap (AC P22541 )
  • Endoglucanase A (endo- 1.4-
  • Figure 2 shows an alignment of the partial nucleotide sequences of the
  • Figures 3-5 show the putative partial amino acid sequences of M. incognita endoglucanases as derived from SEQ ID NO's 5-7. respectively, in the boxed areas.
  • Figure 6 shows an alignment of the seven nematodal cellulases of SEQ ID No's 4-1 (GR-ENG1. GR-ENG2, HG-ENGl . HG-ENG2) and of figures 3-5 (MI-ENG1 , MI-ENG2. MI-ENG3) with the Erwinia chrysanihemi cellulasc (GunZ-Erwch). Identical matches are shaded. Also the majority sequence is mentioned.
  • the criteria for peptides of the invention (minimum homology and minimum identical sequence length) as defined above, can also be used with regard to the sequences of Figure 6 including the majority sequence.
  • the enzymes may show multifunctionality including e.g. ccllobiohydrolase,
  • the cellulases according to the invention can be used in enzyme preparations.
  • the invention is also concerned with such enzyme preparations.
  • Such preparation may further contain other enzvmes, such as other glucanases. xylanascs. amvlascs. pascs. proteinases. oxulorcductascs etc
  • the cellulase assists in removing cellulose residues and also facilitates drainage in paper making and deinking of paper (see WO 94/00578 and WO 96/195 9).
  • sample buffer 240 mM Tris-Cl (pH 6.8), 40% glycerol, 8% SDS, 0.1 % bromophenol blue. 20 % 2-mcrcaptoethanol
  • sample was heated for 3 min at 100 °C and centrituged tor 5 mm. at 14.000 rpm ( 16.000 x g).
  • the overlaying supernatant was used in the preparativ e purification. Purification of proteins
  • a 10% SDS-polyacrylamide gel was poured in a preparative elcctrophoresis system (Prep Cell, Bio-Rad, Richmond. Ca, USA) and overlaid w ith a 4% stacking gel according to the manufacturer.
  • the 28 mm diameter gel tube was used.
  • Standard SDS- PAGE buffers were used (Lacmmli. 1971 ).
  • the follow mg running conditions were applied: 40 mA constant current (about 2X0 V). After 2.00 h ciution ys s started at 1 ml/min and collected in a 50 ml tube. After approximately 2:35 h the bromophenol blue migrated into the ciution chamber, w hereafter the traction collector as started and 2 ml per fraction was collected.
  • Oligonucleotidcs were designed based on the N-tcrmini and used for the cloning of the genes encoding these proteins as described in examples 1 and 2
  • RNA Extraction Kit Pharmacia Biotech. SE
  • PolyA RNA was purified using mRNA Purification Kit (Pharmacia Biotech. SE).
  • a cDNA library containing 2.5 * 10 6 primary rccombinants w ith an average insert size of 1500 bascpairs was constructed by Invitrogen (San Diego CA USA) This library in cloning vector pcDNAII was used as target DNA for amplification of svp encoding sequences using primers: 5'- CTTCCGTGTCTTCCTCCTCCATG -3' v s as 5' primer 5'- GGGTCG ACGCGCAACCAC 1 Ti Tl TATCATCATC - 3'. and
  • the rccombinant fusion proteins w ere purified from the E. co lysate ith affinity chromatography on amylose resin (New England Biolabs). and tested on Western blot for detection with a cocktail of svp31 , svp39, and svp49 specific monoclonal antibodies (MGR).
  • the rccombinant proteins w ere recognized as fusion proteins, and migrated in the SDS-PAGE betw een 80 and 95 kDa. w ith approx. 10 kDa difference in molecular mass.
  • the fusion proteins w ere incubated 17 h at 30 °C in a cup- plate assay including 0.2% carbo.xy methyl cellulose in 0.5% agarosc (see example 4).
  • Carboxymcthylccllulose ( CMC). 0 4% in 50 mM K-phosphaie/cit ⁇ c acid buffer pH 5.2 (PCA buffer) w as mixed with an equal volume of 1 % agarosc (Seakem) in PCA buffer and a layer of 4 mm w as poured in a 10 cm pct ⁇ dish ( Matcos et al. 1992). Holes were punched out able to contain 4 ⁇ [. From a homogenate of second stage larvae of G. rosioduensis in 50 mM Na-phosphate buffer pH 7.2 containing ⁇ b ⁇ vj ⁇ protein. 3 ⁇ l was added to a hole.
  • homogenates were prepared in 50 mM Na-phosphate buffer pH 7.2 and subjected to elcctrophoresis on a 10% polvacrylamidc gel containing 20 ⁇ g per ml bovine serum albumin. Samples were prepared with a sample buffer having a final SDS concentration of 0.4% while reducing agent was omitted.
  • J2 homogenates 1 to 5 ⁇ g of protein and 15 j of 20-fold concentrated salivary gland secretions (standard pore water (Schouten & VandcrBrugge, 1989) w herein the juveniles were incubated overnight after hatching in potato root diffusatc) were loaded on two gels without having been boiled.
  • MGR48 eight bands are visible in the molecular w eight range of 30 to 50 kD.
  • w ere found corresponding to apparent molecular weights of 32. 49 and 60 kD.
  • These proteins w ere not recognised bv MGR48
  • the hetcrologously expressed proteins MalE:./GR-ENGl and MalE /GR-ENG2 sho ed cellulasc activity and could be detected on a Western blot w ith the cocktail ot MGRs 46-60.
  • Example 6 Testing inhibiting capacity of anti-cellulase monoclonal antibodies.
  • ORGANISM Heterodera glycines
  • AAG AAT GTG ATC ATT CTC CGC ACT CCC AAA TGG TCT CAA GAT GTT GAC 636 Lys Asn Val He He Leu Gly Thr Pro Lys Trp Ser Gin Asp Val Asp 180 185 190
  • ORGANISM He erodera glycmes
  • AAG GTC ATT GCT GCC ATC CGA GCT ATT GAC AAG AAA AAT GTG ATC ATT 576
  • GGC ACC AAA TTG GTC GGC TCC AAC GGA CAA CCC GTT CAG CTG ATC GGA 144 Gly Thr Lys Leu Val Gly Ser Asn Gly Gin Pro Val Gin Leu He Gly 35 40 45
  • GCT TAC AAT CTG GCG GTT GCC GTG ATT GAG GCC GCC ATC TCC CAG GGC 336 Ala Tyr Asn Leu AJa Val Ala Val He Glu Ala Ala He Ser Gin Gly 100 105 110
  • GCT GCC AAA AAA TCG CCT CCG GCC AAA CCT GCC GCT GCC AAA AAG CCC 1104 Ala Ala Lys Lys Ser Pro Pro Ala Lys Pro Ala Ala Ala Lys Lys Pro 355 360 365
  • AAC GCT CTG ACT GCC ACG CCT CCC CCA TAC GGG CAA TTG TCC CTT TCC 96 Asn Ala Leu Thr Ala Thr Pro Pro Pro Tyr Gly Gin Leu Ser Val Ser 20 25 30
  • GGC ACC AAA TTG GTC GGC TCC AGT GGA CAA CCC GTT CAG CTG ATC GGA 144 Gly Thr Lys Leu Val Gly Ser Ser Gly Gin Pro Val Gin Leu He Gly 35 40 5
  • GCT TAC AAT CAG GCG GTT GCC GTC ATC GAG GCA GCC ATC TCC CAG GGC 336 Ala Tyr Asn Gin Ala Val Ala Val He Glu Ala Ala He Ser Gin Gly
  • CAA GAT GTG GAT ATT GCT TCG CAG AAT CCG ATC AAA GAG TAC AAA AAT 624 Gin Asp Val Asp He Ala Ser Gin Asn Pro He Lys Glu Tyr Lys Asn 195 200 205
  • TCT GGC AAT TCT GCC GCC ACA ACG ACC ACC AAA AAG CCC CCA TCT AAT 1056 Ser Gly Asn Ser Ala Ala Thr Thr Thr Lys Lys Pro Pro Ser Asn 340 345 350
  • TCT GGT CAA ACC ACT AAC CAA AAG CCT TCG TCT TCG GCG GGG TCC AGT 1104 Ser Gly Gin Thr Thr Asn Gin Lys Pro Ser Ser Ser Ala Gly Ser Ser 355 360 365
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

L'invention concerne des peptides possédant une activité de cellulase et montrant une identité d'au moins 40 % d'acides aminés, dans la structure primaire, avec la séquence d'acides aminés montrée dans SEQ ID NO 1, 2, 3 et 4, ou comprenant une série d'au moins huit acides aminés contigus de la séquence d'acides aminés montrée dans SEQ ID NO 1-7 ou dérivée de celle-ci. Ce peptide correspond à un peptide de nématode, notamment d'un nématode sédentaire (tel qu'un nématode à kyste ou un nématode cécidogène), ou à une partie de celui-ci. Des anticorps dirigés contre de tels peptides constituent des agents précieux de protection des cultures. En outre, on décrit des séquences nucléotidiques codant ces peptides et des systèmes d'expression comprenant au moins une telle séquence nucléotidique. Ces peptides enzymatiques sont utiles dans la protection de plantes contre des nématodes parasites, et dans l'industrie alimentaire, dans celle des boissons, du papier et de l'habillement, ou dans le traitement des déchets.
PCT/NL1997/000400 1996-07-08 1997-07-08 Cellulases WO1998001569A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP96201890.9 1996-07-08
EP96201890A EP0818538A1 (fr) 1996-07-08 1996-07-08 Cellulases nouvelles
EP97200136 1997-01-17
EP97200136.6 1997-01-17

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WO1998001569A1 true WO1998001569A1 (fr) 1998-01-15

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6812018B2 (en) 2000-06-15 2004-11-02 Prokaria Ltd. Thermostable cellulase
US7576261B2 (en) * 2004-10-13 2009-08-18 University Of Georgia Research Foundation, Inc. Nematode resistant transgenic plants

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1996022372A2 (fr) * 1995-01-17 1996-07-25 Landbouwuniversiteit Wageningen Anticorps utilisables dans la lutte contre les nematodes kystiques, et plantes transgeniques les exprimant

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Publication number Priority date Publication date Assignee Title
WO1996022372A2 (fr) * 1995-01-17 1996-07-25 Landbouwuniversiteit Wageningen Anticorps utilisables dans la lutte contre les nematodes kystiques, et plantes transgeniques les exprimant

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DE BOER JM ET AL: "Secretory granule proteins from the subventral esophageal glands of the potato cyst nematode identified by monoclonal antibodies to a protein fraction from second-stage juveniles.", MOL PLANT MICROBE INTERACT, JAN 1996, 9 (1) P39-46, UNITED STATES, XP000570632 *
GOVERSE A ET AL: "Monoclonal antibodies to the esophageal glands and stylet secretions of Heterodera glycines", JOURNAL OF NEMATOLOGY, 26 (3). 1994. 251-259., XP002002336 *
HALL J ET AL: "The non-catalytic cellulose-binding domain of a novel cellulase from pseudomonas fluoresces subsp. cellulosa is important for the efficient hydrolysis of Avicel", BIOCHEM. JOURNAL, vol. 309, 1995, pages 749 - 756, XP000612732 *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6812018B2 (en) 2000-06-15 2004-11-02 Prokaria Ltd. Thermostable cellulase
US7576261B2 (en) * 2004-10-13 2009-08-18 University Of Georgia Research Foundation, Inc. Nematode resistant transgenic plants
AU2005295796B2 (en) * 2004-10-13 2010-07-22 University Of Georgia Research Foundation, Inc. Nematode resistant transgenic plants
US7915479B2 (en) 2004-10-13 2011-03-29 University Of Georgia Research Foundation, Inc. Nematode resistant transgenic plants

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