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
  • 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 present invention relates to enzymes with endoglucanase activity, a method of producing the enzymes, and an enzyme preparation containing an enzyme of the invention.
• Endoglucanases constitute a group of hydrolases, which catalyse endo hydrolysis of 1,4- ⁇ -D- glycosidic linkages in cellulose, cellulose derivatives (such as carboxy methyl cellulose and hydroxy ethyl cellulose) lichenin, ⁇ -1,4 bonds in mixed ⁇ -1,3 glucans such as cereal ⁇ - D-glucans or xyloglucans and other plant material containing cellulosic parts.
• the authorized name is endo-1, 4- ⁇ -D-glucan 4- glucano hydrolase, but the abbreviated term endoglucanase is used in the present specification. Reference can be made to R.F.
• Endoglucanases have been found to be produced by various types of organisms such as plants and microorganisms, and endoglucanases of a wide variety of specificities have been identified. For instance, xyloglucan specific endoglucanases have been identified in various plants, cf the disclosure of Fry et al. (1992), Nishitani and Tominaga (1992), Hayashi et at. (1984), McDougall and Fry (1991), and WO 93/17101. All of these enzymes have been found to have transferase activity (as defined e.g. by Fry et al., 1992 and Nishitani et al., 1992) and are not, accordingly, classified as a real endoglucanase. Hitherto, xyloglucan specific endoglucanases have not been identified in microorganisms.
• Microbial endoglucanases have been described by Beldman et al., 1985 (Trichoderma viride) and in WO 93/20193 (Aspergillus aculeatus) , the latter reference being published only after the priority dates of the present invention. Furthermore, Sharma et al., 1991, Ooi, et al., 1990, and Gilkes et al. 1991 describe microbial endoglucanases.
• Endoglucanases may advantagously be used for the degradation of cellulose components present in plant cell walls and bacterial polysaccharides. Endoglucanases having a high xyloglucan- degrading activity may be of particular use for degradations of cell wall material having a high xyloglucan content, for instance in the wine and fruit industry, for pectin-extraction and for removal of hemicelluloses from textile fibres.
• the object of the invention is to provide novel endoglucanases exhibiting useful substrate specificities and a method for producing the endoglucanases in a better yield and higher purity than hitherto possible.
• a further object is to provide novel products, wherein the proportion of the endo- ⁇ -1,4- glucanase is either increased or decreased relative to the proportion in the original product.
• the endoglucanases and the novel products of the invention may be used for the degradation of plant cell wall tissue.
• the present invention relates to an enzyme exhibiting endoglucanase activity, which enzyme is encoded by a DNA sequence comprising at least one of the following partial sequences (a) ATTCATTTGTG GACAGTGGAC (SEQ ID No: 1)
• the term "endoglucanase activity" is intended to indicate a capability of hydrolysing 1,4- ⁇ -D- glycosidic linkages present in any cellulosic material, such as cellulose, cellulose derivatives, lichenin, /3-D-glucan, or xyloglucan.
• the endoglucanase activity may be determined in accordance with methods known in the art, examples of which are described in the Materials and Methods and Examples section herein.
• One unit of endoglucanase activity e.g.
• CMCU, AVIU, XGU or BGU is defined as the production of 1 ⁇ mol reducing sugar/min from a glucan substrate, the glucan substrate being, e.g., CMC (CMCU), acid swollen Avicell (AVIU), xyloglucan (XGU) or cereal ⁇ -glucan (BGU).
• CMCU CMC
• AVIU acid swollen Avicell
• XGU xyloglucan
• BGU cereal ⁇ -glucan
• the reducing sugars are determined as described in the Materials and Methods section herein.
• the specific activity of an endoglucanase towards a substrate is defined as units/mg of protein.
• the invention relates to an enzyme exhibiting endoglucanase activity, which enzyme i) is encoded by a DNA sequence comprising or included in at least one of the following partial sequences AAAGATTCATTTGTGGACAGTGGACGTTGATCGCACATTGAACCAACCCCAGCCGACCGATT- GTCCTTCCTTACCTCACCATCATTTAACATCTTTTCACCATGAAGCTTTCCCTTCTCTCCC TTGCCACCCTGGCTTCCGCTGCCAGCCTCCAGCGCCGCACACTTCTGCGGTCAGTGGGATA CCGCCACCGCCGGTGACTTCACCCTGTACAACGACCTTTGGGGCGAGACGGCCGGCACCGG CTCCCAGTGCACTGGAGTCGACTCCTACAGCGGCGACACCATCGCTTGTCACACCAGCAGG TCCTGGTCGGAGTAGCAGCAGCGTCAAGAGCTATGCCAACG (SEQ ID No: 17) or
• ii) is immunologically reactive with an antibody raised against a highly purified endoglucanase encoded by the DNA sequence defined in i) and derived from Aspergillus aculeatus , CBS 101.43, and/or iii) is specific for xyloglucan.
• the term "specific for xyloglucan” is intended to indicate that the enzyme exhibits a high activity on a xyloglucan substrate and only low, if any, activity on other cellulose-containing substrates such as carboxymethyl cellulose, cellulose, or other glucans.
• the specificity of an endoglucanase towards xyloglucan may be defined as a relative activity determined as the release of reducing sugars at optimal conditions obtained by incubation of the enzyme with xyloglucan and the other substrate to be tested, respectively.
• the specificity may be defined as the xyloglucan to ⁇ -glucan activity (XGU/BGU) or xyloglucan to carboxy methyl cellulose activity (XGU/CMCU) or xyloglucan to acid swollen Avicell activity (XGU/AVIU).
• the enzyme defined by properties i)-iii) above is referred to as of endoglucanase type II or (for short Endoglucanase II or EG II).
• the term "derived from” is intended not only to indicate an endoglucanase produced by strain CBS 101.43, but also an endoglucanase encoded by a DNA sequence isolated from strain CBS 101.43 and produced in a host organism transformed with said DNA sequence.
• the term "homologue” is intended to indicate a polypeptide encoded by DNA which hybridizes to the same probe as the DNA coding for an endoglucanase enzyme of the invention under certain specified conditions (such as presoaking in 5xSSC and prehybridizing for 1 h at ⁇ 40°C in a solution of 5xSSC, 5xDenhardt's solution, and 50 ⁇ g of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with 50 ⁇ Ci 32-P-dCTP labelled probe for 18 h at ⁇ 40°C and washing three times in 2xSSC, 0.2% SDS at 40°C for 30 minutes).
• certain specified conditions such as presoaking in 5xSSC and prehybridizing for 1 h at ⁇ 40°C in a solution of 5xSSC, 5xDenhardt's solution, and 50 ⁇ g of denatured sonicated calf thymus DNA, followed by hybrid
• the term is intended to refer to a DNA sequence which is at least 70% homologous to any of the sequences shown above encoding an endoglucanase of the invention, such as at least 75%, at least 80%, at least 85%, at least 90% or even at least 95% with any of the sequences shown above.
• the term is intended to include modifications of any of the DNA sequences shown above, such as nucleotide substitutions which do not give rise to another amino acid sequence of the polypeptide encoded by the sequence, but which correspond to the codon usage of the host organism into which a DNA construct comprising any of the DNA sequences is introduced or nucleotide substitutions which do give rise to a different amino acid sequence and therefore, possibly, a different protein structure which might give rise to an endoglucanase mutant with different properties than the native enzyme.
• Other examples of possible modifications are insertion of one or more nucleotides into the sequence, addition of one or more nucleotides at either end of the sequence, or deletion of one or more nucleotides at either end or within the sequence.
• the invention relates to an enzyme exhibiting endoglucanase activity, which enzyme iv) is encoded by a DNA sequence comprising or included in the following partial sequence GACAGTAGCAATCCAGCATTAGCATCAGCCGCTTTGTACACCATGAAGTTCACCGTATTGGCA CTGCTTCTCTCCCAGGTGTGGGCGGCCCCTCAGGCAAACGCTCCTCCAATTTTCTCTGGCTT GGTAGTAATGAGTCTGGCGCAGAGTTTGGCCAGGCCAACATCCCCGGTGTTCTGGG (SEQ ID No: 19) or a sequence homologous thereto encoding a polypeptide with endoglucanase activity, and/or v) is immunologically reactive with an antibody raised against a highly purified endoglucanase encoded by the DNA sequence defined in iv) and derived from Aspergrillus aculeatus, CBS 101.43.
• the enzyme defined by properties iv) and v) is referred to as of Endoglucanase type IV (or for short Endoglucanase IV or EG IV).
• the present invention relates to an enzyme preparation useful for the degradation of plant cell wall components, said preparation being enriched in an enzyme exhibiting endoglucanase activity as described above, as well to various uses of said enzyme preparation.
• Endoglucanase type II of the present invention has been found to have a surprisingly high specificity for xyloglucan and a very high specific activity towards this substrate.
• Endoglucanase type II of the present invention is preferably one which has a XGU/BGU, XGU/CMU and/or XGU/AVIU ratio (as defined above) of more than 50, such as 75, 90 or 100.
• the endoglucanase type II is preferably substantially devoid of activity towards ⁇ -glucan and/or exhibits at the most 3% such as at the most 2% or about 1% activity towards carboxymethyl cellulose and/or Avicell when the activity towards xyloglucan is 100%.
• endoglucanase type II of the invention is preferably substantially devoid of transferase activity, an activity which has been observed for most xyloglucan specific endoglucanases of plant origin.
• an endoglucanase of type II of the present invention may be obtained from the fungal species A. aculeatus .
• Microbial endoglucanases with endoglucanase II type specificity has not previously been described.
• Xyloglucan specific endoglucanases from plants have been described, but these enzymes have transferase activity and therefore must be considered inferior to microbial xyloglucan specific endoglucanases whenever extensive degradation of xyloglucan is desirable.
• An additional advantage of a microbial enzyme is that it, in general, may be produced in higher amounts in a microbial host, than enzymes of other origins.
• endoglucanase of type IV exhibits only low activity towards xyloglucan.
• endoglucanase type IV may be substantially devoid of xyloglucan degrading activity.
• endoglucanase of type IV exhibits a surprisingly high specificity and specific activity towards ⁇ -glucan.
• An enzyme of the invention may be isolated by a general method involving - cloning, in suitable vectors, a DNA library from Aspergillus spp.,
• Example 1 A more detailed description of this screening method is given in Example 1 below.
• the DNA sequence coding for the enzyme may for instance be isolated by screening a cDNA library of Aspergillus aculeatus, e.g strain CBS 101.43, publicly available from Centraalbureau voor Schimmelcultures, and selecting for clones expressing the appropriate enzyme activity (i.e. endoglucanase activity as defined by the ability of the enzyme to hydrolyse 0-1,3 and/or ⁇ - 1,4 bonds between two glucose molecules in polymers containing glucose (e.g. cellulose, cereal ⁇ -glucans or xyloglucans).
• the appropriate DNA sequence may then be isolated from the clone by standard procedures, e.g. as described in Example 1.
• a DNA sequence coding for a homologous enzyme may be derived by similarly screening a cDNA library of another microorganism, in particular a fungus, such as a strain of Aspergillus , in particular A. aculeatus or A. niger, a strain of Trichoderma , in particular T. harzianum , T. reesie , a strain of Fusarium , in particular F . oxysporum or a strain of Humicola .
• a fungus such as a strain of Aspergillus , in particular A. aculeatus or A. niger, a strain of Trichoderma , in particular T. harzianum , T. reesie , a strain of Fusarium , in particular F . oxysporum or a strain of Humicola .
• the DNA coding for an endoglucanase of the invention may, in accordance with well-known procedures, conveniently be isolated from DNA from any of the above mentioned organisms by use of oligonucleotide probes, such as 20mer probes, prepared on the basis of a DNA sequence disclosed herein.
• oligonucleotide probes such as 20mer probes
• a suitable oligonucleotide probe may, e.g., be prepared on the basis of any of the partial nucleotide sequences a)-p) listed above.
• the DNA sequence may subsequently be inserted into a recombinant expression vector.
• This may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
• 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.
• the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
• the DNA sequence encoding the endoglucanase should be operably connected to a suitable promoter and terminator sequence.
• the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
• the procedures used to ligate the DNA sequences coding for the endoglucanase, the promoter and the terminator, respectively, and to insert them into suitable vectors are well known to persons skilled in the art (cf., for instance, Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, NY, 1989).
• the host cell which is transformed with the DNA sequence encoding the enzyme of the invention is preferably a eukaryotic cell, in particular a fungal cell such as a yeast or filamentous fungal cell.
• the cell may belong to a species of Aspergillus , most preferably Aspergillus oryzae or Aspergillus niger.
• Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se.
• Aspergillus as a host microorganism is described in EP 238 023 (of Novo Nordisk A/S), the contents of which are hereby incorporated by reference.
• the host cell may also be a yeast cell, e.g.
• the present invention relates to a method of producing an enzyme according to the invention, wherein a suitable host cell transformed with a DNA sequence encoding the enzyme is cultured under conditions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture.
• the medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells in question.
• the expressed endoglucanase may conveniently be secreted into the culture medium and may be recovered therefrom by well-known procedures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
• the thus purified endoglucanase may be employed for immunization of animals for the production of antibodies. More specifically, antiserum against the endoglucanase may be raised by immunizing rabbits (or other rodents) according to the procedure described by N. Axelsen et al. in: A Manual of Quantitative Immunoelectrophoresis, Blackwell Scientific Publications, 1973, Chapter 23, or A. Johnstone and R. Thorpe, Immunochemistry in Practice, Blackwell Scientific Publications, 1982 (more specifically pp. 27-31). Purified immunoglobulins may be obtained from the antisera, for example by salt precipitation ((NH 4 ) 2 SO 4 ), followed by dialysis and ion exchange chromatography, e.g.
• the endoglucanases according to the invention may be produced essentially free from other plant cell wall degrading enzymes. This makes it possible to use the enzymes alone or together with other enzymes, such as galactanases and xylanases, to give the optimal combination of enzymes for a particular application. It is thereby possible to design enzyme combinations, which only degrade specific parts of the plant cell. This specific degradation has not previously been possible to obtain with commercially available cellulase, hemicellulase and/or pectinase preparations.
• the present invention provides endoglucanases of a wide specificity range.
• an endoglucanase of type II of the invention has been found to be highly specific to xyloglucan, whereas an endoglucanase of type IV has been found to degrade cellulose and cellulose derivatives like carboxymethylcellulose and hydroxyethylcellulose, and mixed ⁇ -1,3-1,4 glucans like cereal ⁇ -glucans.
• an endoglucanase type IV of the invention has been found to have a very high ⁇ -glucan degrading capability.
• endoglucanases useful (either alone or in combination) in applications where modification or full or partial degradation of cellulose, xylo-glucans and mixed ⁇ -1,3-1,4 glucans or derivatives of these substrates are desired.
• the activity towards mixed ⁇ -1,3-1,4 glucans makes EG IV and homologous thereof useful for brewing purposes as the enzymes degrades the barley ⁇ -glucan and thereby reduces the viscosity and improves the filterability of the wort.
• ⁇ -glucans In brewing the high specificity for ⁇ -glucans is an advantage as compared to other endoglucanases as the viscosity caused by ⁇ -glucan can be reduced without simultaneous degradation of the cellulose struc- tures which are essential for the filterability of the wort where brewers spent grains act as filter-aid. Finally, the activity towards mixed ⁇ -1,3-1,4 glucans makes the enzyme useful for addition to animal feed to improve the feed-uptake.
• endoglucanase type II enzymes of the invention towards xyloglucans and the activity of endoglucanase type IV enzymes towards cellulose are useful for treatment of fruits and vegetables.
• the endoglucanases may for example be used in treatment of e.g. apple and pear mash for high yielding juice processes. The juices can be made clear or cloud-stable depending on the used combination of enzymes.
• the endoglucanses may be used in mash treatment of grapes for higher yields and better aroma and colour of wine.
• the endoglucanases may be used to facilitate pectin extraction from e.g.
• the hemicellulose like xyloglucan has to be removed from plant fibres like cotton, flax, hemp and jute before these can be used for textiles.
• endoglucanase of type II has the advantage that it specifically removes the xyloglucan without damaging the cellulose.
• This endoglucanase may be used alone or together with other enzymes (e.g. pectinases) active on the pectic substances on the fibres.
• endoglucanases of the invention and analogous thereof may be used to treat cellulose fibres or cellulose- fibre rich material.
• the endoglucanases may e.g. be used in the paper industry to improve the drainage of pulp, and to treat fabrics such as cotton fabrics, to give a more smooth fabric.
• the endoglucanases of the invention may also be used to produce oligosaccharides from e.g. plant material with mixed ⁇ -1,3-1,4 glucan and xyloglucan.
• the resulting oligosaccharides may be used as bulking agents in e.g. food.
• a preferred type of enzyme preparation of the invention is an enzyme preparation which is enriched in an endoglucanase according to the invention, e.g. an endoglucanase of type II and/or an endoglucanase of type IV, preferably with an enrichment factor of at least 1.1. In this manner a boosting of the cell wall degrading ability of the enzyme preparation can be obtained.
• the enzyme preparation having been enriched with an enzyme of the invention may e.g. be an enzyme preparation comprising multiple enzymatic activities, in particular an enzyme preparation comprising multiple plant cell wall degrading enzymes such as Pectinex ® , Pectinex Ultra SP ® , Celluclast or Celluzyme (all available from Novo Nordisk A/S).
• the term "enriched" is intended to indicate that the endoglucanase activity of the enzyme preparation has been increased, e.g. with an enrichment factor of 1.1, conveniently due to addition of an enzyme of the invention prepared by the method described above.
• the enzyme preparation enriched in an enzyme exhibiting endoglucanase activity may be one which comprises an enzyme of the invention as the major enzymatic component, e.g. a mono-component enzyme preparation.
• the enzyme preparation may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry preparation.
• the enzyme preparation may be in the form of a granulate or a microgranulate.
• the enzyme to be included in the preparation may be stabilized in accordance with methods known in the art.
• the enzyme preparation of the invention may, in addition to an endoglucanase of the invention, contain one or more other plant cell wall degrading enzymes, for instance those with cellulytic, xylanolytic or pectinolytic activities such as xylanase, arabinanase, rhamnogalacturonase, pectin acetylesterase, galactanase, polygalacturonase, pectin lyase, pectate lyase, endo glucanase or pectin methylesterase.
• other plant cell wall degrading enzymes for instance those with cellulytic, xylanolytic or pectinolytic activities such as xylanase, arabinanase, rhamnogalacturonase, pectin acetylesterase, galactanase, polygalacturonase, pectin lyase, pe
• the additional enzyme(s) may be producible by means of a microorganism belonging to the genus Aspergillus , preferably Aspergillus niger, Aspergillus aculeatus , Aspergillus awamori or Aspergillus oryzae .
• a microorganism belonging to the genus Aspergillus preferably Aspergillus niger, Aspergillus aculeatus , Aspergillus awamori or Aspergillus oryzae .
• Such a preparation is able to provide an extraordinary good total liquefaction power and thus a marked viscosity decrease of apple mash and similar biological materials.
• an enzyme preparation may be used for the above mentioned purposes.
• the dosage of the enzyme preparation of the invention and other conditions under which the preparation is used may be determined on the basis of methods known in the art.
• Fig. 1 is a restriction map of plasmid pYHD17
• Fig. 2 a restriction map of plasmid pHD 414
• Fig. 3 illustrates the pH optimums for EG II, EG III and EG IV measured in citrate/phosphate buffers.
• the optimal activity for each enzyme is defined as 100%
• Fig. 4 the pH stability for for EG II and EG III measured as the remaining activity after 1 h at different pH values compared to the activity of fresh enzyme (100%),
• Fig. 5 the temperature optimum for EG II and EG III.
• the optimal temperature for each enzyme is defined as 100%, and
• Fig. 6 The temperature stability of EG II and EG III measured as the remaining activity after preincubation in water for 1 h at different temperatures compared to the activity of fresh enzyme (100%).
• Donor organism mRNA was isolated from Aspergillus aculeatus , CBS 101.43, grown in a soy-containing fermentation medium with agitation to ensure sufficient aeration. Mycelia were harvested after 3-5 days' growth, immediately frozen in liquid nitrogen and stored at -80°C.
• Yeast strains The Saccharomyces cerevisiae strain used was yNG231 (MAT alpha, leu2, ura3-52, his4-539, pep4-delta 1, cir+) or JG169 (MAT ⁇ ; ura 3-52; leu 2-3, 112; his 3-D200; pep 4-113; prcl::HIS3; prbl:: LEU2; cir+).
• Plasmid Construction of an expression plasmid The commercially available plasmid pYES II (Invitrogen) was cut with Spel, filled in with Klenow DNA polymerase + dNTP and cut with Clal. The DNA was size fractionated on an agarose gel, and a fragment of about 2000 bp was purified by electroelution. The same plasmid was cut with Clal/PvuII, and a fragment of about 3400 bp was purified by electroelution. The two fragments were ligated to a blunt-ended Sphl/EcoRI fragment containing the yeast TPI promoter. This fragment was isolated from a plasmid in which the TPI promoter from S . cerevisiae (cf. T.
• RNA isolations were baked at 220°C for at least 12 h. Eppendorf tubes, pipet tips and plastic columns were treated in 0.1% diethylpyrocarbonate (DEPC) in EtOH for 12 h, and autoclaved. All buffers and water (except Tris-contain- ing buffers) were treated with 0.1% DEPC for 12 h at 37°C, and autoclaved. Extraction of total RNA: The total RNA was prepared by extraction with guanidinium thiocyanate followed by ultracen- trifugation through a 5.7 M CsCl cushion (Chirgwin et al . , 1979) using the following modifications.
• DEPC diethylpyrocarbonate
• RNA extraction buffer (4 M GuSCN, 0.5% Na-laurylsarcosine, 25 mM Na-citrate, pH 7.0, 0.1 M ⁇ -mercaptoethanol). The mixture was stirred for 30 min. at RT° and centrifuged (30 min., 5000 rpm, RT°, Heraeus Megafuge 1.0 R) to pellet the cell debris.
• the supernatant was collected, carefully layered onto a 5.7 M CsCl cushion (5.7 M CsCl, 0.1 M EDTA, pH 7.5, 0.1% DEPC; autoclaved prior to use) using 26.5 ml supernatant per 12.0 ml CsCl cushion, and centrifuged to obtain the total RNA (Beckman, SW 28 rotor, 25 000 rpm, RT°, 24h). After centrifugation the supernatant was carefully removed and the bottom of the tube containing the RNA pellet was cut off and rinsed with 70% EtOH.
• RNA pellet was transferred into an Eppendorf tube, suspended in 500 ⁇ l TE, pH 7.6 (if difficult, heat occasionally for 5 min at 65°C), phenol extracted and precipitated with ethanol for 12 h at -20°C (2.5 vols EtOH, 0.1 vol 3M NaAc, pH 5.2). The RNA was collected by centrifugation, washed in 70% EtOH, and resuspend ed in a minimum volume of DEPC-DIW. The RNA concentration was determined by measuring OD 260/280 .
• poly(A) + RNAs were isolated by oligo(dT)-cellulose affinity chromatography (Aviv & Leder, 1972). Typically, 0.2 g of oligo(dT) cellulose (Boehringer Mannheim) was preswollen in 10 ml of 1 x column loading buffer (20 mM Tris-Cl, pH 7.6, 0.5 M NaCl, 1 mM EDTA, 0.1% SDS), loaded onto a DEPC-treated, plugged plastic column (Poly Prep Chromatography Column, Bio Rad), and equilibrated with 20 ml 1 x loading buffer.
• 1 x column loading buffer (20 mM Tris-Cl, pH 7.6, 0.5 M NaCl, 1 mM EDTA, 0.1% SDS
• RNA sample was heated at 65oC for 8 min., quenched on ice for 5 min, and after addition of 1 vol 2 x column loading buffer to the RNA sample loaded onto the column.
• the eluate was collected and reloaded 2-3 times by heating the sample as above and quenching on ice prior to each loading.
• the oligo(dT) column was washed with 10 vols of 1 x loading buffer, then with 3 vols of medium salt buffer (20 mM Tris-Cl, pH 7.6, 0.1 M NaCl, 1 mM EDTA, 0.1% SDS), followed by elution of the poly (A) + RNA with 3 vols of elution buffer (10 mM Tris-Cl, pH 7.6, 1 mM EDTA, 0.05% SDS) preheated to 65°C, by collecting 500 ⁇ l fractions. The OD 260 was read for each collected fraction, and the mRNA containing fractions were pooled and ethanol precipitated at -20°C for 12 h.
• the poly (A) + RNA was collected by centrifugation, resuspended in DEPC-DIW and stored in 5-10 ⁇ g aliquots at -80°C.
• Double-stranded cDNA was synthesized from 5 ⁇ g of A. aculeatus poly(A) + RNA by the RNase H method (Gubler & Hoffman 1983, Sambrook et al., 1989) using the hairpin modification.
• the poly(A) + RNA (5 ⁇ g in 5 ⁇ l of DEPC-treated water) was heated at 70°C for 8 min., quenched on ice, and combined in a final volume of 50 ⁇ l with reverse transcriptase buffer (50 mM Tris-Cl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, Bethesda Research Laboratories) containing 1 mM each dNTP (Pharmacia), 40 units of human placental ribonuclease inhibitor (RNasin, Promega), 10 ⁇ g of oligo(dT) 12-18 primer (Pharmacia) and 1000 units of Superscript II RNase H- reverse transcriptase (Bethesda Research Laboratories).
• First-strand cDNA was synthesized by incubating the reaction mixture at 45°C for 1 h.
• Second strand synthesis After synthesis 30 ⁇ l of 10 mM Tris- Cl, pH 7.5, 1 mM EDTA was added, and the mRNA:cDNA hybrids were ethanol precipitated for 12 h at -20°C by addition of 40 ⁇ g glycogen carrier (Boehringer Mannheim) 0.2 vols 10 M NH 4 Ac and 2.5 vols 96% EtOH.
• 40 ⁇ g glycogen carrier Boehringer Mannheim
• hybrids were recovered by centrifugation, washed in 70% EtOH, air dried and resuspended in 250 ⁇ l of second strand buffer (20 mM Tris-Cl, pH 7.4, 90 mM KCl, 4.6 mM MgCl2, 10 mM (NH 4 ) 2 SO 4 , 16 ⁇ M ⁇ NAD + ) containing 100 ⁇ M each dNTP, 44 units of E. coli DNA polymerase I (Amersham), 6.25 units of RNase H (Bethesda Research Laboratories) and 10.5 units of E. coli DNA ligase (New England Biolabs).
• second strand buffer 20 mM Tris-Cl, pH 7.4, 90 mM KCl, 4.6 mM MgCl2, 10 mM (NH 4 ) 2 SO 4 , 16 ⁇ M ⁇ NAD + ) containing 100 ⁇ M each dNTP, 44 units of E. coli DNA polymerase I (Amersham), 6.25
• Second strand cDNA synthesis was performed by incubating the reaction tube at 16°C for 3 h, and the reaction was stopped by addition of EDTA to 20 mM final concentration followed by phenol extraction.
• Mung bean nuclease treatment The double-stranded (ds) cDNA was ethanol precipitated at -20°C for 12 h by addition of 2 vols of 96% EtOH, 0.1 vol 3 M NaAc, pH 5.2, recovered by centrifugation, washed in 70% EtOH, dried (SpeedVac), and resuspended in 30 ⁇ l of Mung bean nuclease buffer (30 mM NaAc, pH 4.6, 300 mM NaCl, 1 mM ZnSO4 , 0.35 mM DTT, 2% glycerol) containing 36 units of Mung bean nuclease (Bethesda Research Laboratories).
• the single-stranded hair-pin DNA was clipped by incubating the reaction at 30°C for 30 min, followed by addition of 70 ⁇ l 10 mM Tris-Cl, pH 7.5, 1 mM EDTA, phenol extraction, and ethanol precipitation with 2 vols of 96% EtOH and 0.1 vol 3M NaAc, pH 5.2 at -20°C for 12 h.
• T4 DNA polymerase The ds cDNA was blunt- ended with T4 DNA polymerase in 50 ⁇ l of T4 DNA polymerase buffer (20 mM Tris-acetate, pH 7.9, 10 mM MgAc, 50 mM KAc, 1 mM DTT) containing 0.5 mM each dNTP and 7.5 units of T4 DNA polymerase (Invitrogen) by incubating the reaction mixture at 37°C for 15 min. The reaction was stopped by addition of EDTA to 20 mM final concentration, followed by phenol extraction and ethanol precipitation.
• T4 DNA polymerase buffer 20 mM Tris-acetate, pH 7.9, 10 mM MgAc, 50 mM KAc, 1 mM DTT
• Adaptor ligation and size selection After the fill-in reaction the cDNA was ligated to non-palindromic BstX I adaptors (1 ⁇ g/ ⁇ l, Invitrogen) in 30 ⁇ l of ligation buffer (50 mM Tris-Cl, pH 7.8, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, 25 ⁇ g/ml bovine serum albumin) containing 600 pmol BstX I adaptors and 5 units of T4 ligase (Invitrogen) by incubating the reaction mix at 16°C for 12h.
• ligation buffer 50 mM Tris-Cl, pH 7.8, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, 25 ⁇ g/ml bovine serum albumin
• the reaction was stopped by heating at 70°C for 5 min, and the adapted cDNA was size-fractionated by agarose gel electrophoresis (0.8% HSB-agarose, FMC) to separate unligated adaptors and small cDNAs.
• the cDNA was size-selected with a cutooff at 0.7 kb, and the cDNA was electroeluted from the agarose gel in 10 mM Tris-Cl, pH 7.5, 1 mM EDTA for 1 h at 100 volts, phenol extracted and ethanol precipitated at -20°C for 12 h as above.
• a large-scale ligation was set up in 40 ⁇ l of ligation buffer containing 9 units of T4 ligase, and the reaction was incubated at 16°C for 12h. The ligation reaction was stopped by heating at 70°C for 5 min, ethanol precipitated at -20°C for 12 h, recovered by centrifugation and resuspended in 10 ⁇ l DIW.
• One ⁇ l aliquots were transformed into electrocompetent E. coli 1061 cells using the same electroporation conditions as above, and the transformed cells were titered and the library plated on LB + ampicillin plates with 5000-7000 c.f.u. /plate. To each plate was added 3 ml of medium.
• yeast transformants To ensure that all the bacterial clones were tested in yeast, a number of yeast transformants 5 times larger than the number of bacterial clones in the original pools was set as the limit.
• the vector pHD414 is a derivative of the plasmid p775 (described in EP 238 023). In contrast to this plasmid, pHD 414 has a string of unique restriction sites between the promoter and the terminator. The plasmid was constructed by removal of an approximately 200 bp long fragment (containing undesirable RE sites) at the 3 'end of the terminator, and subsequent removal of an approximately 250 bp long fragment at the 5'end of the promoter, also containing undesirable sites.
• the 200 bp region was removed by cleavage with Narl (positioned in the pUC vector) and Xbal (just 3' to the terminator), subsequent filling in the generated ends with Klenow DNA polymerase +dNTP, purification of the vector fragment on gel and religation of the vector fragment.
• This plasmid was called pHD413.
• pHD413 was cut with StuI (positioned in the 5'end of the promoter) and PvuII (in the pUC vector), fractionated on gel and religated.
• the plasmid pHD 414 is shown in Fig. 2 which is a map of plasmid pHD414, wherein "AMG Terminator” indicates the A. niger glucoamylase terminator, and "TAKA Promoter" indicates the A. oryzae TAKA amylase promoter. Transformation of Aspergillus oryzae or Aspergillus niger (general procedure)
• YPD Yeast et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory, 1981
• the mycelium is harvested by filtration through miracloth and washed with 200 ml of 0.6 M MgSO 4 .
• the suspension is cooled on ice and 1 ml of buffer containing 120 mg of Novozym ® 234, batch 1687 is added.
• Protoplasts are spread on the appropriate plates.
• YPD 10 g yeast extract, 20 g peptone, H 2 O to 810 ml. Autoclaved, 90 ml 20% glucose (sterile filtered) added.
• YPG-agar 25 g/l Bactoagar, 15 g/l glucose, 5 g/l K 2 PO 4 , 0.5 g/l MgSO 4 -7H 2 O, pH adjusted to 5.0.
• 10 x Basal salt 66.8 g yeast nitrogen base, 100 g succinic acid, 60 g NaOH, H 2 O ad 1000 ml, sterile filtered.
• SC-URA 90 ml 10 x Basal salt, 22.5 ml 20% casamino acids, 9 ml 1% tryptophane, H 2 O ad 806 ml, autoclaved, 3.6 ml 5% threonine and 90 ml 20% glucose added.
• SC-H agar 7.5 g/l yeast nitrogen base without amino acids, 11.3 g/l succinic acid, 6.8 g/l NaOH, 5.6 g/l casamino acids without vitamins, 0.1 g/l tryptophan and 20 g/l agar (Bacto).
• YNB-1 agar 3.3 g/l KH 2 PO 4 , 16.7 g/l agar, pH adjusted to 7. Autoclaved for 20 min. at 121°C. After autoclaving, 25 ml of a 13.6% yeast nitrogen base without amino acids, 25 ml of a 40% glucose solution, 1.5 ml of a 1% L-leucine solution and 1.5 ml of a 1% histidine solution were added per 450 ml agar.
• YNB-1 broth Composition as YNB-1 agar, but without the agar.
• FG-4-Agar 35 g/l agar, 30 g/l Soy bean meal, 15 g/l maltodextrin (Glucidex 6), 5 g/l Bacto pepton, pH 7.
• FG-4 medium 30 g/l Soy bean meal, 15 g/l maltodextrin (Glucidex 6), 5 g/l Bacto pepton.
• AZCL xyloglucan available from Megazyme, Australia.
• AZCL-HE cellulose available from Megazyme, Australia.
• SDS-PAGE electrophoresis was performed in a Mini-Leak 4 electrophoresis unit (Kem-En-Tec, Copenhagen) as a modified version of the Laemli procedure (Laemmli, 1970; Christgau, 1991). Briefly, the separation gel was cast with 12% acrylamide; 0.2% BIS acrylamide; 0.1% SDS; 0.375 M Tris pH 8.8; 0.04% APS (ammonium-persulphate) & 0.04% TEMED.
• the stacking gel was cast with 4.5% w/w Acrylamide; 0.075% BIS-acrylamide; 0.1% SDS; 66.5 mM Tris pH 6.8; 0.4% w/w APS (ammonium persulphate) & 0.4% TEMED.
• the electrode chambers are filled with running buffer : 25 mM Tris-base; 0.192 M glycine & 0.05% SDS, whereafter the samples containing sample buffer are loaded, and the gel is run at 2-4 mA/gel for over-night running and 10-30 mA/gel for fast running. The gel is subsequently removed and stained by either commassie or silver staining.
• Isoelectric focusing is carried out on Ampholine PAG plates pH 3.5-9.5 (Pharmacia, Upsala) on a Multiphor electrophoresis unit according to the manufactures instructions. After electrophoresis the gel is either commassie stained or silver stained.
• Enzyme incubations are carried out in triplicate. A blank is produced in which enzyme is added but inactivated immediately. After centrifugation the absorbance of the supernatant is measured in microtiter plates at 620nm and the blank is subtracted. The activities of the enzymes are measured on different pure polysaccharides: xyloglucan and ⁇ -glucan from MegaZyme, CMC (Blanose from Aqualon) and Avicell (microcrystaline cellulose from Merck). Before use Avicell is swelled in 85% ortho- phosphoric acid for 1 hour at room temperature and washed with acetone and water.
• 0.5% solutions/suspensions of the different substrates are made in 0.1M acetate buffer ( if not otherwise specified) of the optimal pH, 10 ⁇ l enzyme solutions are added to 1ml of substrate, incubations are carried at 30°C for 15 minutes before heat-inactivation as above.
• Reducing sugars are determined by reaction, in microtiter plates, with a PHBAH reagent comprising 0.15 g of para hydroxy benzoic acid hydra- zide (Sigma H-9882), 0.50 g of potassium-sodium tartrate (Merck 8087) and 2% NaOH solution up to 10.0 ml. Results of blanks are subtracted. Glucose is used as a standard.
• pH optimum is measured on substrates from MegaZyme (EG II on AZCL-xyloglucan, EG III on pure 0-glucan, and EG IV on AZCL- ⁇ - glucan).
• 0.5ml of 0.4% substrate is mixed with 0.5ml 0.1M citrate/phosphate buffer of varying pH and 10 ⁇ l of a suitably diluted enzyme solution is added. Incubations are carried out as described above.
• Temperature optimum is measured by incubating the enzyme with AZCL- ⁇ -glucan substrate at varying temperatures for 15 minutes at the optimal pH.
• Temperature stability is measured by leaving the enzyme, diluted in water, at various temperatures for 1 hour before incubation at 30°C with the relevant substrate.
• DNA was isolated from 20 individual clones from the library and subjected to analysis for cDNA insertion.
• the insertion fre quency was found to be >90% and the average insert size was approximately 1400bp.
• DNA from some of the pools was transformed into yeast, and 50- 100 plates containing 200-500 yeast colonies were obtained from each pool. The colonies were scraped off and stored in glycerol at -80°C.
• Yeast cells from the library were spread onto YNB agar to a total of about 250,000 colonies. The number of colonies per plate varied from 250 to 500.
• the agar plates were replica plated onto several sets of SC-H agar plates and three different endoglucanases were identified:
• One set of the replica plates contained 0.1% AZCL xyloglucan (Megazyme). These plates were incubated for 2-4 days at 30°C. Endoglucanase II and Endoglucanase III positive colonies were identified as colonies surrounded by a blue halo.
• Another set of replica plates contained 0.1% AZCL-HE cellulose. These plates were incubated for 2-4 days at 30°C.
• Endoglucanase III and Endoglucanase IV positive colonies were indentified as colonies surrounded by a blue halo.
• Cells from enzyme-positive colonies were spread for single colony isolation on agar, and an enzyme-producing single colony was identified and selected by the above described methods.
• the positive clones were obtained as single colonies, the cDNA inserts were amplified directly from the yeast colony using biotinylated polylinker primers, purified by magnetic beads (Dynabead M-280, Dynal) system and characterized individually by sequencing the 5' -end of each cDNA clone using the chain-termination method (Sanger et al., 1977) and the Sequenase system (United States Biochemical).
• the partial DNA sequence of the enzyme gene obtained from screening with AZCL xyloglucan is shown in claim 2 and the partial DNA sequence of the enzyme gene obtained from screening with AZCL-HE cellulose is shown in claim 6.
• a partial DNA sequence was obtained for Endoglucanase III.
• Endoglucanase III is included in the following examples in order to illustrate the difference existing between the endoglucanases of the present invention and a prior art endoglucanase.
• the isolate was inoculated into 20 ml YNB-1 broth in a 50 ml glass test tube. The tube was shaken for 2 days at 30°C. The cells were harvested by centrifugation for 10 min. at 3000 rpm. The cells were resuspended in 1 ml 0.9 M sorbitol, 0.1 M EDTA, pH 7.5. The pellet was transferred to an Eppendorf tube, and spun for 30 seconds at full speed. The cells were resuspended in 0.4 ml 0.9 M sorbitol, 0.1 M EDTA, 14 mM 0-mercaptoethanol.
• the supernatant was transferred to a fresh tube which was filled with EtOH (room temp.) followed by thorough but gentle mixing and spinning for 30 seconds.
• the pellet was washed with cold 70% EtOH, spun for 30 seconds and dried at room temperature.
• the pellet was resuspended in 50 ⁇ l TE (Tris-EDTA) and spun for 15 minutes.
• the supernatant was transferred to a fresh tube.
• 2.5 ⁇ l 10 mg/ml RNase was added, followed by incubation at 37°C for 30 minutes and addition of 500 ⁇ l isopropanol with gentle mixing. The mixture was spun for 30 seconds, and the supernatant was removed.
• the pellet was rinsed with cold 96% EtOH and dried at room temperature.
• the DNA was dissolved in 50 ⁇ l water to a final concentration of approximately 100 ⁇ l/ml.
• the DNA was transformed into E . coli by standard procedures. Two
• E. coli colonies were isolated and analysed with the restriction enzymes Hindlll and Xbal which excised the DNA insert.
• DNA sequences of several of the positive clones were determined.
• a partial DNA sequence of the gene encoding endoglucanase II of the invention is shown in claim 2 and a partial DNA sequence of the gene encoding endoglucanase IV of the invention is shown in claim 6.
• the DNA encoding the endoglucanase is digested with Hindlll/Xbal, size fractionated on gel, and a fragment corresponding to the endoglucanase gene is purified.
• the gene is subsequently ligated to Hindlll/Xbal digested pHD414 resulting in the plasmids pAEG II and pAEG IV.
• the plasmids pAEG II and pAEG IV are transformed into Aspergillus oryzae as described above.
• Each of the transformants was inoculated on FG agar in the centre of a Petri dish. After 4 days of incubation at 30°C, 4 mm diameter plugs were removed by means of a corkscrew.
• One set of plugs was embedded in a xyloglucan overlayer gel containing 0.1% AZCL xyloglucan and 1% agarose in a buffer with an appropriate pH, and incubated overnight at 30°C.
• the endoglucanase II and endoglucanase III activity, respectively, was identified as described above.
• the best transformant had a halo with a diameter significantly larger than the A. oryzae background. This demonstrates efficient expression of endoglucanase II in A. oryzae .
• Another set of plugs was embedded in a HE-cellulose overlayer gel containing 0.1% AZCL-HE cellulose and 1% agarose in a buffer with an approprioate pH, and incubated overnight at 30°C.
• the endoglucanase III and Endoglucanase IV activity, respectively, was identified as described above.
• the best transformant had a halo with a diameter significantly larger than the A. oryzae background. This demonstrates efficient expression of endoglucanase IV in A . oryzae .
• EG II, EG III and EG IV were produced by fed batch fermentation of A . oryzae expressing the enzymes.
• the medium used for the fermentation comprised maltodextrin as a carbon source, urea as a nitrogen source and yeast extract.
• the fed batch fermentation was performed by innoculating a shake flask culture of the A. oryzae host cells in question into a medium comprising 3.5% of the carbon source and 0.5% of the nitrogen source. After 24 hours of cultivation at pH 5.0 and 34°C the continuous supply of additional carbon and nitrogen sources were initiated. The carbon source was kept as the limiting factor and it was secured that oxygen was present in excess. The fed batch cultivation was continued for 4 days, after which the enzymes could be recovered.
• the culture supernatant from fermentation of Aspergillus oryzae expressing the recombinant enzyme was centrifuged at 5000 xg and filtered through a 0.2 ⁇ m filter to remove the mycelia. 35- 50 ml of the filtered supernatant were ultrafiltrated in a Filtron ultracette or Amicon ultrafiltration device with a 10 kDa membrane to achieve 10 fold concentration. This concentrate was diluted 100 times in 20 mM Tris pH 7.0 in two successive rounds of ultrafiltration in the same device. This ultrafiltra- ted sample was loaded at 2 ml/min on a Pharmacia HR16/10 Fast Flow Q Sepharose anion exchanger equilibrated in 20 mM Tris pH 7.0.
• the column was washed with two column volumes 20 mM Tris pH 7.0, and bound proteins were eluted with a linear increasing NaCl gradient from 0 to 0.4 M NaCl in 20 mM Tris pH 7.0.
• the protein elutes at approx. 50 mM NaCl.
• the molecular weight of the enzyme was determined to about 35 kDa by SDS-PAGE and the isoelectric point to 3.4 by IEF.
• the culture supernatant from fermentation of Aspergillus oryzae expressing the recombinant enzyme (the expression level should be at least 0.5 mg enzyme/ml supernatant) was centrifuged at 5,000 xg for 20 min and filtered through a 0.2 ⁇ m filter to remove the mycelia. 35-50 ml of the filtered supernatant was ultrafiltrated in a Amicon YM03 ultrafiltration device with a 3 kDa membrane to achieve 10 fold concentration. This concentrate is diluted 100 times in 20 mM Tris pH 8.0 in two successive rounds of ultrafiltration in the same device.
• This ultrafiltratred sample is loaded at 1.5 ml/min on a Pharmacia HR16/10 Fast Flow Q Sepharose anion exchanger equilibrated in 20 mM Tris pH 8.0. After the sample has been applied, the Endoglucanase III is washed through the column, whereas the contraminating impurities remains bound to the column. The Endoglucanase III in this fraction is more than 95% pure. The molecular weight of the enzyme was determined to about 26 kDa by SDS-PAGE and the isoelectric point to 5.5 by IEF.
• the purified enzyme is used for characterization.
• EG IV the fermentation supernatant is used for characterization. pH optimum
• the pH optimums of the different enzymes can be seen in Fig. 3.
• the EG II has a pH optimum of about 3.0, EG IV of about 3.5.
• EG II and EG IV are both acidic endoglucanases showing optimal activity from pH 2.5 to 4.0 whereas EG III is optimal from pH 4.0 to 6.0.
• EG II and EG III have similar temperature optimums (optimal activity between 30°C and 60°C) and temperature stability (stable for lh up to 60°C) but EG III is more stable at high pH than EG II.
• ORGANISM Aspergillus aculeatus

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