WO1994026878A1 - Expansins, proteins that catalyze plant cell wall expansion and weakening of paper fiber connections - Google Patents
Expansins, proteins that catalyze plant cell wall expansion and weakening of paper fiber connections Download PDFInfo
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- WO1994026878A1 WO1994026878A1 PCT/US1994/005286 US9405286W WO9426878A1 WO 1994026878 A1 WO1994026878 A1 WO 1994026878A1 US 9405286 W US9405286 W US 9405286W WO 9426878 A1 WO9426878 A1 WO 9426878A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
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- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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- 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
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- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
- D21H17/00—Non-fibrous material added to the pulp, characterised by its constitution; Paper-impregnating material characterised by its constitution
- D21H17/20—Macromolecular organic compounds
- D21H17/21—Macromolecular organic compounds of natural origin; Derivatives thereof
- D21H17/22—Proteins
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Definitions
- the present invention relates to a new class of proteins, known as expansins, and isolation and utilization of same. These proteins have been identified in a wide variety of plant materials and have a variety of applications, including but not limited to, agricultural, food and industrial uses, such as use in paper industry as a catalyst for weakening the strength of paper products. For example, they can prove especially useful in the recycling of paper.
- T e paper products industry employs 3/4 million workers and is a $60-billion industry in the U.S. alone (plus $40 billion in retail sales). Recycling is a growing concern and will prove more important as the nation's landfill sites become more scarce and more expensive. According to 1992 testimony before congress on the problems and opportunities of paper recycling, the need for improved technology in paper recycling is urgent and of high priority. The use of expansins in this industry may be well received at this time.
- Paper derives its mechanical strength from hydrogen bonding between paper fibers, which are composed primarily of cellulose. During paper recycling, the hydrogen bonding between paper fibers is disrupted by chemical and mechanical means prior to re-forming new paper products. Expansins may be used to weaken the hydrogen bonding between the paper fibers of recycled paper. As demonstrated in this invention disclosure, expansins, at very low concentrations, in fact weaken commercial papers, including slick paper from magazines and catalogs. These latter types of papers are difficult to recycle because they are not easily disrupted in commercial recycling processes.
- the protein is nontoxic and environmentally innocuous; it could substitute for current harsh chemical treatments which are environmentally noxious.
- the protein is effective on paper products which are now recalcitrant to current recycling processes. Its use could expand the range of recyclable papers. Because the protein acts at moderate temperature and in mild chemical environments, degradation of paper fibers during recycling should be reduced. This should allow for recycled paper fibers with stronger mechanical properties and with the ability to be recycled more often than is currently practical. Moreover, savings in energy costs associated with heating and beating the paper may be realized.
- expansins may be useful in the production of paper pulp from plant tissues. Use of expansins could substitute for harsher chemicals now in use and thereby reduce the financial and environmental costs associated with disposing of these harsh chemicals. The use of expansins could also result in higher quality plant fibers because they would be less degraded than fibers currently obtained by harsher treatments.
- the proteins which can be characterized as catalysts of the extension of plant cell walls and the weakening of the hydrogen bonds in the pure cellulose paper are referred to as expansins.
- Two proteins have been isolated by fractionation techniques from washed wall fragments of the cucumber hypocotyls, referred to as “cucumber expansin-29” and “cucumber expansin-30” (abbreviated cEx-29 and cEx-30, with respect to their relative masses).
- Another expansin protein has been isolated from oat coleoptiles (oat expansin oEx-29).
- Expansins appear to be broadly distributed throughout the plant kingdom and have been identified in stem and leaf vegetables (i.e., broccoli, cabbage), fruit and seed vegetables (i.e., tomato), fiber crops and cereals (i.e., corn), and forest and ornamental crops (i.e., cotton). Also, expansin-like protein has been found in proteins obtained from the digestive track of snail and its feces (sEx). These novel proteins can find use in a variety of applications including the paper industry in production of paper pulp in preparation of virgin paper and in paper recycling as a preferred way of disruption of paper fibers due to their nontoxicity and environmental innocuosity in contrast with the harsh chemical treatments applied today, which are environmentally noxious.
- An object of this invention is to provide novel extractable proteins which can induce extension of isolated walls and can weaken commercial paper products.
- Another object of this invention is to provide a method of isolation of the said proteins from various sources including growing plant tissue.
- Fig.l Extension curves of native and reconstituted isolated cell wall specimens obtained using a constant load extensometer. Ten-mm sections of tissue were frozen and thawed, and lightly abraded prior to suspension in the extensometer. Tissues were clamped under an applied force of 20g and extension recorded using a linear voltage displacement transducer. The length of tissue between the clamps was 5 mm.
- Inactivated walls were treated for 15 sec with boiling water prior to suspension; as shown in the second line, this treatment eliminated acid induced extension.
- inactivated walls were suspended in 50 mM sodium acetate, pH 4.5, for 30 min. at which point the bathing solution was replaced by 0.5 mL of fresh solution (arrows) containing 2-3 mg of proteins extracted from growing cell walls (apical wall protein), or with soluble proteins from growing cells (apical soluble proteins), or proteins extracted from cell walls from the nongrowing cotyledon (cotyl. wall protein) or from walls of the basal hypocotyl (basal wall protein).
- Fig. 2 Fractionation of extension-inducing cucumber wall extracts by HPLC.
- Ammonium sulfate precipitates of salt extracted cucumber wall extracts were resuspended and loaded onto a C3 hydrophobic interactions column in 50 mM sodium acetate, pH 4.5, 20% (saturation) ammonium sulfate. Proteins were eluted in a descending gradient (20-0%) of ammonium sulfate. Fractions were desalted and checked for extension inducing activity with inactivated cucumber tissues as in Figure 1. A28O nTM is shown by a solid line and extension inducing activity by a broken line.
- Fig. 3 SDS-PAGE of ammonium sulfate precipitate (AS), and active fractions from C3 and sulfopropyl (SI and S2) HPLC separations of cucumber proteins.
- AS ammonium sulfate precipitate
- SI and S2 active fractions from C3 and sulfopropyl
- Protein samples were concentrated, desalted, and run on SDS-polyacrylamide gels according to the method of Laemmli. Gels were stained with Coomassie Brilliant Blue R 250. Arrows indicate the major protein bands at 29 and 30 kD in the SI and S2 fractions which appear to possess the extension-inducing activity.
- Fig. 4 The effects of DTT, metal ions, methanol and water boiling, and protease treatments on reconstituted extension.
- Reconstituted walls were incubated with 1000 units of trypsin or chymotrypsin for 4 hr at 30°C, in 50 mM Hepes, pH 7.3, or with 2 milligrams per mL of pronase or papain for 4 hr at 30°C, in 50 mM sodium acetate, pH 5.0. Controls were reconstituted and incubated in the same manner without the addition of proteases. At the end of the incubations tissues were clamped under constant load (as described in figure 1), first in 50 mM Hepes, pH 6.8. After 30 min. the bathing solution was replaced by 50 mM sodium acetate, pH 4.5. The difference in the two rates of extension was calculated. Data presented are the means of four experiments in each case.
- Fig. 6 The effects of cucumber expansins on cellulose paper.
- the bathing solution was replaced with either: 5 mg of SI protein in 15 mM Mes, pH adjusted to 4.5 with 1 M acetic acid (SI); 5 mg of SI protein, in the same buffer, which had been heated to 100°C for two minutes (Boiled SI); 100 mg of cellulase (from Trichoderma viride Boehringer Mannheim) in 50 mM sodium acetate (Cellulase), pH 4.5; or 50 mM sodium acetate pH 4.5 (Control). Extensions were recorded for a further 100 min. The figure shows representative traces from at least six independent experiments, all of which showed similar results.
- Paper strips of 2-3 mm width were cut and hung in our extensometer, such that a 5-mm long strip was put under 20-g force.
- the buffer contained 50 mM sodium acetate, pH 4.5 with or without added C3 protein (about 20 ⁇ g per paper strip was used).
- the length of the paper strips was recorded for up to 5 h.
- Four samples of each paper were used with each treatment (+/- protein).
- the untreated (control) paper showed very little change in length, and maintained its mechanical strength throughout the test period. In contrast, the treated papers began to extend and quickly broke once extension began. The time for breakage after addition of protein varied from 45 min. to 2.5 h, but all samples broke.
- Fig. 8 Acid-induced extension in native and heat-treated oat coleoptile walls.
- Fig. 9 Effect of dithiothreitol (DTT) addition to acid-induced extension of oat coleoptile walls.
- Fig. 10 Acid-induced extension in native oat coleoptile walls in the continuous presence of DTT.
- Fig. 11 Extension induced in oat, cucumber and pea walls by proteins extracted from oat coleoptiles walls (A) and cucumber walls (B).
- Coleoptile wall proteins and cucumber wall proteins were extracted with 1 M NaCl, precipitated with ammonium sulfate, desalted and partially purified on a DEAE Sephadex column. Heat-inactivated cell walls from oat coleoptiles, cucumber hypocotyls or pea epicotyls were clamped at 20-g tension in 50 mM sodium acetate, pH 4.5. After about 0.5 h, the solution was replaced with 0.4 mL of the same buffer containing about 10 mg proteins partially purified from oat or cucumber cell walls by DEAE-anion exchange chromatography. The data represent a typical experiment from at least four replicates. Fig. 12. Comparative responsiveness of oat and cucumber walls to exogenously added oat or cucumber proteins.
- Heat- inactivated oat and cucumber walls were prepared as in (A) and extended in sodium acetate, pH 4.5, for about 30 min prior to exchange of buffer for the same one containing 73 ⁇ g of crude oat coleoptile protein (ammonium sulfate precipitate) per sample holder (about 400 ⁇ L).
- the extension curves shown illustrate two examples with responses close to the mean (2.8% h ⁇ l increase for the cucumber wall and 1.1% h"l increase for the oat wall).
- the baseline extension rates prior to addition of protein averaged 2.4 % per h (oats) and 1.1 % per h (cucumber).
- Fig. 13 Effect of 1 M NaCl extraction on acid-induced extension of oat coleoptile walls.
- Fig. 14 pH dependence of the extension activity reconstituted by oat wall proteins.
- Heat-inactivated walls from cucumber hypocotyls were clamped in an extensometer as in Fig. 8 and placed in 50 mM sodium acetate buffers at pH 3.5, 4.0, 4.5, 5.0 or 5.5. After 20 min, the solutions were replaced with 0.4 mL of the corresponding buffer containing 10 mg oat-coleoptile wall proteins partially purified by DEAE-chromatography.
- the extension activity was calculated by subtracting the baseline rate without proteins from the linear rate (5 - 30 min) after the addition of the proteins, and expressed as % increase in length per h above the baseline rate.
- the average baseline rate prior to addition of protein ranged between 2.52 and 2.96 ⁇ m min" (or 3.0 to 3.55 % per h) for all pH groups.
- Fig. 15 Purification of oat expansin by high pressure liquid chromatography (HPLC) on a carboxymethyl (CM) cation exchange column.
- HPLC high pressure liquid chromatography
- CM carboxymethyl
- A Elution of proteins (absorbance at 280 ran) from a CM-column. Proteins were solubilized from the cell walls of etiolated coleoptiles by 1 M NaCl and then sequentially fractionated by ammonium sulfate precipitation, DEAE-chromatography, and ConA chromatography, prior to CM-HPLC.
- Fig. 16 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins from different purification steps.
- Active protein fractions were separated on a SDS-PAGE gradient gel (4-20%) and stained with Coomassie Blue R-250. Lane 1, 1 M NaCl extraction (20 mg); lane 2, ammonium sulfate precipitate (20 mg); lane 3, active DEAE-fraction (10 mg); lane 4, proteins passing through Con A column (5 mg); lane 5, active fraction from CM- HPLC (0.3 mg). The protein with apparent molecular mass of about 29 kD was designated as oat expansin (oat Ex29). Similar results were obtained in five trials, except in some of these an additional protein band at about 35 kD appeared in lane 5.
- Fig. 17 Western analysis of oat coleoptile proteins probed with antiserum against cucumber Ex29. Comparison of lane 1 (crude wall protein) with lane 2 (crude protoplasmic protein) shows that the antiserum recognizes an Ex29-like protein (arrow) specifically bound to the coleoptile cell wall. Comparison of lane 3 (crude wall protein) with lane 4 (purified protein with extension activity) shows that the Ex29-like protein co- purifies with the extension activity.
- Methods Lane 1 was loaded with 15 ⁇ g of crude wall protein (ammonium sulfae precipitate of 1 M NaCl extract). Lane 2 had 15 ⁇ g of the soluble protoplasmic protein fraction.
- Fig. 18 Extension induced in isolated cucumber walls by proteins from snails.
- Cucumber walls were prepared as in the description for Fig.l 1. Proteins prepared by dissolving snail acetone powder ( 10 mg /mL) in 50 mM sodium acetate, pH 4.5, is capable of inducing extension of isolated cucumber walls. The immediate response and the linearity of the extension are unique characteristics that indicate an expansin- like protein may be involved.
- the feces of Helix aspersa induced extension in cucumber walls.
- the feces were dissolved in 50 mM sodium acetate buffer, pH 4.5. The procedure was as described in Fig. 11.
- Fig.20 Western blot analysis of active HPLC- separated proteins fraction from snail.
- Acetone powder solution was probed with antibody which was raised against cucumber expansin -29.
- the analysis was performed as described in Fig. 17.
- the active fractions show a striking band at about 26 kD, which is similar to cucumber expansin-29.
- Fig.21 Western blot of the protein from Helix aspersa feces, probed with the antibody PA-1. Feces from Helix aspersa were dissolved as described in Fig. 19 and western blot analysis was performed as in Fig. 20. The single band indicates the presence of an protein antigenically similar to cucumber expansin-29.
- the present invention describes the first evidence of a new class of proteins named expansins.
- expansins a new class of proteins which we named expansins.
- Further research found expansins in oat coleoptile as well as in the proteins obtained from the digestive track of snails.
- the support for these new findings and ways of using it commercially are the object of the present invention.
- ConA Concanavalin A
- CM carboxymethyl
- DEAE diethylaminoethyl
- Hepes 4-(2- hydroxyethyl)-l-piperazineethanesulfonic acid
- DTT dithiothreitol
- Ex29 29-kD expansin
- Mes 2-(N-morpholino) ethanesulphonic acid
- HPLC high-pressure liquid chromatography
- PBST phosphate buffered saline with Tween-20
- SDS- PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- the extension induced by this extract mimicked the native extension activity in magnitude and kinetics, i.e. initially high rates decayed over a period of 2 hr to more stable rates of about 2 % per hour. These rates are lower than elongation rates of the living stem, but the stress applied to the isolated walls was only one-fifth of the equivalent longitudinal stress imposed on the walls by cell turgor. Like the endogenous extension activity of isolated cell walls, the reconstituted activity required an acid pH and was irreversible (i.e. upon removal of load, the segments did not return to their original length). The addition of protein to native cell walls did not substantially enhance extension, suggesting that endogenous activity was saturating or that binding sites were not accessible to the added material.
- the wall extract from growing cucumber walls did not induce extension of walls from the basal (non elongating) stem (Fig. 1A).
- the wall is biochemically modified so that it is not susceptible to extension by this material.
- peroxidative cross-linking of lignin or structural proteins such as extensin is involved in this loss of sensitivity.
- FIG. 1B shows that the cucumber wall extract was active on various dicot seedlings (pea, radish, cucumber and tomato) and on monocots of the Amaryllidaceae (onion and zephyr lily). In contrast, the extract had a much smaller effect on the coleoptile wall of graminaceous monocots (maize and barley). As graminaceous monocot cell walls differ from those of dicots by having less pectin and hydroxyproline-rich glycoprotein, and also by having a different type of hemicellulose, it may be that the active fraction extracted from cucumber walls interacts with one or more of these components to induce extension.
- grass cell walls are cross- linked (not necessarily covalentiy) in a manner that renders them immune to the cucumber extract.
- monocots of the Amaryllidaceae have a cell wall composition more similar to dicots than to the graminae, and this probably explains their susceptibility to the cucumber derived activity.
- the cucumber wall extract was separated by ammonium sulfate precipitation followed by sequential HPLC, shown in Figure 2, first using a hydrophobic interactions column, where a single peak of activity desiphated as the C3 proteins was obtained, and then using a cation exchange column from which the activity was eluted as two distinct peaks These fractions we have designated SI and S2 with respect to their order of elution.
- Figure 3 shows the analysis of the active fractions by SDS- PAGE which revealed a major band of relative molecular mass 29 kD associated with SI, while S2 contained a major band at 30 kD ( Figure 3).
- Active extracts have also been separated by native PAGE and by liquid chromatography with hydroxyapatite, gel filtration media, and DEAE anion exchangers, where activity was consistently associated with these two bands (data not shown).
- the SI fraction required only 0.3 -1.0 micrograms of protein to reconstitute extension rates similar to that of native extension, while it required 1.0 -2.0 micrograms of S2 to do this.
- Figure 8 shows an example of acid-induced extension of oat coleoptile walls.
- the extension rate was low 30 min after application of the load and could be greatly increased by replacing the neutral buffer with a buffer of pH 4.5.
- the wall extension rate decreased continuously with time after the change to acid buffer (Fig. 8B).
- cucumber hypocotyl walls as the "substrate" for measuring the extension activity of oat proteins.
- Cucumber walls were used because they were easier to prepare, broke less often, had lower baseline extension rates, and proved to be a more sensitive substrate for extension assays than did the walls from oat coleoptiles.
- the active oat fractions from the DEAE-column had a pH optimum between 4.5 and 5.0 (Fig. 14). At pH 3.5 or 5.5, the activity was reduced by about 50%. In contrast, cucumber expansins displayed a broader pH optimum, with high activity maintained at pH 3.5.
- oat coleoptile walls possess a protein that can mediate acid-induced extension of grass coleoptile walls and dicot walls.
- This protein resembles the cucumber 29-kD expansin in that it induces extension at acid pH but not at neutral pH, its activity is stabilized by dithiothreitol, it has a similar size as judged by SDS-PAGE, and it is specifically recognized by an antiserum raised against the cucumber protein. Because of these similarities between the oat and cucumber expansins, we propose that expansins are evolutionarily conserved proteins that underlie at least part of the acid-extension response common to the walls of many plant species.
- the similarity between oat and cucumber expansins is also surprising because the matrix components of the wall are believed to be important for wall loosening and extension, yet these components are quite different for grass walls and dicots.
- the major matrix polysaccharides of the coleoptile wall are (l->3, l->4)- ⁇ -D-glucans and arabinoxylans whereas dicots contain principally xyloglucans and pectins.
- Dicot walls contain hydroxyproline-rich glycoproteins of the extensin family whereas grass walls contain much lower amounts of such proteins. It may be that expansins interact directly with wall components common to both types of walls (e.g. cellulose or a minor matrix component) or that they can act on different glycans with similar functions, e.g. xyloglucans and (l->3, l->4)- ⁇ -D-glucans.
- oat Ex29 can induce an acid-dependent extension in coleoptile walls
- the acid response of native walls includes a large, but transient, burst in extension, which is mostly decayed away by 60-90 min. This burst is largely lacking in oEx29-reconstituted extensions, which resemble more closely the steady extensions which outlast the transient (e.g. Fig. 8 and 10).
- the pH dependence of reconstituted extension (Fig. 14) does not exactly match the pH dependence reported for acid-extension responses of isolated coleoptile walls.
- the reconstituted extensions displayed a maximum at pH 4.5 and fell off at lower pH values, whereas acid extensions of native coleoptile walls did not fall off at lower pH values.
- the maximum extension rate inducible with exogenous Ex29 was substantially less than acid-extension responses of native coleoptile walls (i.e. about 2 % h ⁇ l for reconstituted extensions versus about 5 % h ⁇ * for the stable component of native wall extensions or 20-30 % h"* for the immediate response of native walls).
- oat Ex29 is responsible for at least part of the long-term (> 1 h) acid-induced extension responses of oat coleoptiles.
- This view is strengthened by the findings that reconstituted wall extension and endogenous wall extension exhibit similar sensitivities to biochemical activators and inhibitors.
- exogenous expansins can restore extension rates as high or higher than the long-term extension rates in native walls exposed to acid pH.
- the expansin-like proteins in the snail digestive track may be synthesized by the snails or by an endosymbiont living in the digestive system of the snail.
- Such symbionts might be bacteria, fungi, protists or some other microbe.
- expansins could be much easier to interpret.
- S 1 protein showed clear effects on the mechanical properties of Whatman number 3 filter paper in both extensometer and stress relaxation assays. As our experiments show expansins are also able to disrupt slick paper (Fig 7).
- Expansins probably do not catalyze cell wall loosening by disrupting hydrogen bonds directly between microfibrils, as these structures appear to be separated from one another by matrix polysaccharides which are thought to coat the outside of microfibrils in the wall. However, it is possible that they could induce wall extension by disrupting hydrogen bonding between the matrix polysaccharides and the microfibrils, thus reducing the resistance to movement of the microfibrils.
- Seeds of cucumber (Cucumis sativus cv Burpee Pickler) were sown on Seed Germination Kimpack Paper K-22 (Seedburro Equipment Corporation, Chicago, Illinois) soaked with distilled water, in flats, 50 x 25 x6 cm, with lids of the same dimensions. Seedlings were grown in the dark for 4 days at 27°C. The apical 3 cm of hypocotyl was excised and frozen at -20°C for no more than 5 days and prepared for creep measurements as previously described (Cosgrove 1989). For bulk wall extractions, the apical 3-cm hypocotyl regions were collected on ice water and homogenized with 25 mM sodium acetate, 2 mM EDTA, pH 4.5 in a Waring blender. The wall fragments were collected and washed twice by filtration through Miracloth and subsequently used for protein extraction. Basal walls were from the lower 6 cm of the (15 cm long) hypocotyls.
- Oat seedlings were grown in moist vermiculite in complete darkness at 27°C.
- coleoptiles were from 4-day old oat seedlings (Avena sativus L. cv. Olge, from Carolina Biological Supply, Burlington, NC, USA).
- Seedlings were quickly harvested under room lights. For wall extension assays, the apical 2 cm region of the growing stem or coleoptile was excised, sealed in aluminum foil and frozen at -20°C prior to use. Coleoptiles were separated from primary leaves. Cuticles were abraded by rubbing the coleoptiles or stems between two fingers coated with a slurry of carborundum (320 grit, well washed prior to use;
- coleoptiles For oat coleoptiles, the cuticle was generally abraded prior to freezing, whereas for cucumber and pea stems the frozen segment was quickly abraded.
- coleoptile cuticles were removed by stripping the epidermis from the tissue with fine forceps and the remaining coleoptile cylinder was bisected longitudinally prior to freezing. Tissues were thawed, pressed under weight for 5-10 min to remove tissue fluids and clamped in an extensometer (5 mm between the clamps, corresponding to the apical 3-8 mm of the stem or coleoptile).
- Washed cucumber cell wall fragments (from 150-200 g of tissue) were extracted overnight in 20 mM Hepes, pH 6.8, 1 M NaCl at 4°C. Cell wall fragments were filtered on Miracloth and the salt-solubilized fraction precipitated with ammonium sulfate (the activity precipitated between 20 and 60% saturation with [NH4J2SO4). The precipitate was desalted on a 7-mL column of Bio-Gel P2 (Bio- Rad Laboratories) into 50 mM sodium acetate, pH 4.5. Protein concentration was 2 to 4 mg/mL, estimated by Coomassie Protein Assay Reagent (Pierce, Rockford, Illinois).
- soluble and wall associated proteins For the comparison of soluble and wall associated proteins, 100 g of tissue was harvested and homogenized with 100 mL of 25 mM sodium acetate, pH 4.5, 1 mM EDTA. Wall fragments were filtered out and the remaining solution designated as the soluble fraction. Wall fragments were cleaned as described above, and then extracted in 200 mL of 20 mM Hepes, pH 6.8, 1 M NaCl for one hour at 4°C. Aliquots of both solutions were dialyzed against 50 mM sodium acetate pH 4.5 and tested for reconstitution activity, as described below.
- the 20 to 60% (NH4)2SO4 precipitate pellet was resuspended in 2 mL of distilled water and insoluble material was removed by centrifugation and filtration through a Centricon 30 microconcentrator (Amicon, Beverly, Massachusetts) prior to being loaded onto a C3 hydrophobic interactions column (ISCO C-3/6.5 ⁇ m 10x250 mm) equilibrated with 50 mM sodium acetate, pH 4.5, 20% (saturation) (NH4)2SO4. Proteins were eluted from the column with a linear gradient from the equilibration buffer into 50 mM sodium acetate, pH 4.5, in 35 min. at a flow rate of 1 mL min.
- the active fractions from the C3 column were pooled and subsequently desalted and concentrated on a Centricon 30 microconcentrator (Amicon, Beverly, Massachusetts), the buffer being exchanged for 15 mM Mes NaOH, pH 6.5.
- the concentrated sample was then loaded (in a volume of 1.7 mL) onto a sulfopropyl cation exchange column (Bio-Rad HRLC MA7S 50x7.8 mm) equilibrated with 15 mM Mes NaOH, pH 6.5, and proteins eluted with an ascending gradient of NaCl (from 0 to 1.0 M over 45 min) in the same buffer at a flow rate of 1 mL min.
- A28O nm of eluting proteins was measured using a Dionex Variable Wavelength Detector (VDM-2).
- oat seedlings were rapidly cut under room lights and placed in ice water. The apical 2.5 cm (+/- 0.5 cm) of each coleoptile was then cut, separated from the primary leaf, and placed on ice while the other coleoptiles were harvested. About 500 coleoptiles were homogenized in 200 mL of 10 mM sodium phosphate, pH 6.0. In some instances the coleoptiles were collected in lots of 100-200 and frozen (-20°C) for 1 to 3 d prior to homogenization. The homogenate was filtered through a nylon screen (70 mm mesh), and the cell walls were collected and washed 4 times by resuspending in the homogenization buffer (300 mL) followed by filtration.
- Ionically-bound proteins were extracted for at least 1 hour at 4 °C with 50 mL of 1 M NaCl containing 20 mM Hepes (pH 6.8), 2 mM EDTA and 3 mM sodium metabisulfite. Wall fragments were removed by filtration or centrifugation and the wall proteins in the supernatant were precipitated with ammonium sulfate (0.4 g added to each mL). Precipitated proteins were dissolved in 1.5 mL of water and desalted on an Econo-Pac 10DG desalting column (BIO-RAD Laboratories, Richmond, CA, USA) which was equilibrated with 20 mM Tris-HCl, pH 8.0, containing 100 mM NaCl. Without NaCl the active proteins tended to bind to the desalting column, resulting in a lower recovery.
- Protein solution from the desalting column was centrifuged in a microcentrifuge for 3 min to remove precipitates. Proteins were then loaded onto a
- DEAE-column (Sephadex A-25, Sigma) equilibrated with 20 mM Tris-HCl, pH 8.0, containing 100 mM NaCl at 25 °C.
- the proteins bound to the DEAE-column were eluted by 1 M NaCl in 20 mM Tris-HCl buffer, pH 8.0.
- a 1-mL Concanavalin A column (Sigma) was equilibrated with 200 mM NaCl containing 1 mM each of Mg + 2, Ca + 2, and Mn + 2. Proteins from the DEAE-column were passed through this column. After the column was washed extensively with the same solution, the bound proteins were eluted with the same solution containing 0.5 M a-methyl manoside. Extension activity was associated with the fractions that did not bind to the column.
- CM carboxymethyl
- Proteins were eluted from the CM column at a flow rate of 1 mL/min with a gradient of 0 to 0%, 0 to 4%, 4 to 6% and 6 tol00% of 1.0 M NaCl in 10 mM MES, pH 5.5, from 0 to 5 min, 5 to 10 min, 10 to 30 min and 30 to 50 min, respectively, and detected by absorbance at 280 nm.
- Proteins were quantified colorimetrically using the Coomasie Protein Assay Reagent (Pierce, Rockford, IL, USA). For SDS-PAGE (Laemmli 1970), proteins were separated on a 14% polyacrylamide gel or a 4-20% gradient polyaciylamide gel (Bio-Rad Ready Gel, Richmond, CA, USA). For Western analysis, proteins were electrophoretically transferred to a nitrocellulose membrane in a solution of 192 mM glycine, 25 mM Tris, 20% methanol (v/v) at 10 volt cm for 3 h or in some cases 16 h.
- Table I Purification of oat Ex29 from etiolated oat coleoptiles. Activity was assayed as described in Fig. 4 and expressed as the initial increase in the extension rate of isolated cucumber cell walls upon addition of the protein fraction (e.g. 5 to 30 min after protein addition). Total activity was calculated by dividing the activity (measured in 1-3 extension assays) by the fraction of protein used for each assay. Specific activity was calculated by dividing the total activity by the total protein.
- Soluble protoplasmic proteins were obtained by homogenizing oat coleoptiles in 10 mM sodium phosphate buffer (in some cases, 10 mM Hepes, pH 6.8) and centrifuging at 4°C at 26,000 g for 10 min to remove paniculate matter.
- Acetone powders of the visceral humps of Helix pometia were purchased from Sigma Chemical Company. Live snails (Helix aspersa) were purchased from NASCO. Protein solution was made by dissolving snail acetone powder (10 mg ml) in 50 mM sodium acetate buffer, pH 4.5. Feces of Helix aspersa were dissolved in the same buffer.
- Extension measurements were performed using a constant load extensometer as described by Cosgrove (1989). Briefly, frozen/thawed tissues were abraded for a short time with carborundum to disrupt the cuticle, boiled in water for 15 sec (for reconstitution assays), and secured between two clamps (with about 5 mm between the clamps) under a constant tension of 20 g force (for assays with radish, tomato and onion tissues the tension was reduced to 10 g). Plastic cuvettes were fitted around the walls and filled with bathing solution. For reconstitution assays the bathing solution was first 50 mM sodium-acetate pH 4.5 for 30 min., followed by the protein fraction to be assayed in the same, or equivalent pH buffer.
- the protein solution obtained from snail acetone powder by dissolving snail acetone powder (10 mg /mL) into 50 mM sodium acetate, pH 4.5, is capable of inducing extension of isolated cucumber walls.
- Protein concentrations were estimated using Coomassie Protein Assay Reagent (Pierce, Rockford Illinois) using a standard curve constructed using BSA (Pierce). Wall specimens inactivated by boiling were preincubated with SI and S2 proteins at estimated concentrations of 5 micrograms per mL and then extended in the extensiometer, as described above, in 50 mM sodium acetate pH 4.5. After 1 hr enough dithiothreitol (DTT), from a 100 mM stock, was added to bring the bathing solution to 10 mM DTT.
- DTT dithiothreitol
- Inactivated stem sections were incubated with active C3 proteins, at an estimated concentration of 50 micrograms per mL, for 30 min at 25°C. Reconstituted stems were then incubated with 1000 units of trypsin or 1000 units of chymotrypsin in 50 mM Hepes-NaOH, pH 7.3 for 4 hr at 30°C, and with pronase (2 mg/mL), or papain (2 mg/mL) in 50 mM sodium acetate, pH 5.0, 1 mM DTT for 4 hr at 30°C. For a control C3 proteins were incubated at 30°C for 4 hr without the addition of proteases.
- Buffers in the range pH 3.0 to pH 6.5 were prepared by titrating 50 mM citric acid with 1 M K2HPO4 Inactivated stem sections were suspended in the extensiometer in buffer of appropriate pH. After 30 min of extension the bathing solution was replaced by a 1:1 dilution of C3 proteins (estimated final concentration 50 micrograms per mL) with the appropriate buffer, where necessary the pH was adjusted using either 1 M citric acid or 1 M K2HPO4. Change in rate of extension was calculated as the difference in extension rates before and after addition of proteins. All experiments were repeated eight times.
- SI protein (5 mg/mL) caused a marked increase in the relaxation rate of paper strips, while cellulase (100 mg mL) had almost no affect In both extensometer assays and stress relaxation tests, SI protein which had been boiled for two minutes did not induce the effects apparent with native SI proteins, indicating that the effects were associated with the activity of the protein. Similar results were obtained in assays with S2 proteins (data not shown).
- Slick paper was obtained from three sources: (a) the pages of "Nature” magazine (unprinted margins were used), (b) a commercial catalog for chromatography equipment (c) a colored advertising insert (Hills) in the Sunday paper. Paper strips of 2-3 mm width were cut and hung in our extensometer, such that a 5-mm long strip was put under 20-g force.
- the buffer contained 50 mM sodium acetate, pH 4.5 with or without added C3 protein (about 20 ug per paper strip was used). The length of the paper strips was recorded for up to 5 h.
- a stress relaxation assay was performed as described in Example 10.
- Fig. 20 presents a Western blot of active HPLC- separated protein fractions from snail acetone powder, probed with antibody PAl which was raised against cucumber expansin- 29.
- the active fractions schow a striking band at about 26 kD, which is similar to ( though slightly smaller than ) cucumber wxpansin-29. This results provide strong evidence that the wall extension activity found in the snail acetone powder is due to a protein with similar antigenic determinants as cucumber expansin-29.
- Fig. 21 presents a Western blot of the proteins from Helix aspersa feces. probed with the antibody PA-1.
- the single band indicates the presence of an protein antigenically similar to cucumber expansin-29.
- Expansins were identified and confirmed by use of the antibody assay described herein (having common antigenic epitomes with the antibody against ex29), and the extension activity analysis described herein.
- cucumber expansins appear to associate with the cellulose fraction of the cell wall. They do not exhibit polysaccharide hydrolysis under a number of assay conditions and they do not cause a progressive weakening of the wall. Expansins also appear to disrupt hydrogen bonds as particularly noted with cellulose paper.
- expansins could be used for processing of polysaccharides for control of physical properties.
- Hydrogen bonding is an important determinant of many physical properties of commercial products containing polysaccharides.
- Expansins may be incorporated into the polysaccharide products to modify hydrogen bonding and thereby modify the physical characteristics of the products. Examples include control of the viscosity and texture of polysaccharide thickeners used in foods and chemical products, control of stiffness and texture of paper products; and control of mechanical strength (e.g. tear strength) of paper products.
- the present invention is believed broadly applicable to alteration of various physical properties of polysaccarides. While plant polysaccarides represent a preferred embodiment of the invention, expansins of the invention are believed useful catalytic proteins for the treatment polysaccarides from a variety of sources (i.e., synthetic, bacterial or other microbial system, etc.). While it is reasonable to make a strong claim that expansins primarily operate by disruption of hydrogen bonds, the invention is not necessarily limited to this mode of action. Results concerning altered physical properties of polysaccardies are nonetheless produced by use of the novel expansins of the invention.
- Expansins may be used for de-inking paper, which is a significant limitation in current paper recycling operations.
- expansins may help loosen the bonding between surface polymers, which are stuck to the ink, and the remainder of the cellulose fibers.
- expansins may prove useful for large scale paper dissolution, or perhaps for alteration of the mechanical properties of dry paper. Treatment of dry paper could produce paper with novel properties.
- Expansins may be used in combination with other chemicals or enzymes to improve various processes.
- a major limitation in ethanol production from biomass is the degradation of cellulose.
- Expansins in concert with cellulose may act synergistically in the breakdown of cellulose. If expansins help celluloses gain access to glucan chains that make up microfibrils, then they could speed cellulose action.
- delignification is a major problem in industrial uses of many plant fibers.
- expansins may be used with lignases (peroxidases) for synergistically de-lignifying plant fibers.
- Expansins may also be useful in various bioremediation applications, either above or in combination with other biological or chemical materials.
- expansins have been found in cotton fibers and are probably found in native cell walls, but are likely not to be present in processed cotton fiber walls. Accordingly, expansins may be useful to the textile industry by virtue of alteration of either the wet or dry mechanical properties of cotton flax, or other natural fibers.
- expansins may be useful for insect control or as anti-fungal agents, either alone or in combination with other insecticidal or fungicidal agents.
- expansins could be for altering the mechanical strength of gels or otherwise affecting the gelling or other properties of gels (i.e., gelatin, gellum- gum/phytagel, agar, aarose, etc.). Such material are useful agents in foods, cosmetics, and other similar materials. Since hydrogen bonding is important for such gels and in view of the belief that expansins alter hydrogen bonding of wall glycons, expansins may alter the gelling properties of various gels.
- expansins may involve alteration of aggregation of hemicelluloseA and solubilized cellulose. In the event that expansins appropriately affect such aggregation, they may prove useful for industrial processes involving these materials, including cellulose processing and film making.
- Isolation of a novel protein allows one to attempt the cloning of the gene coding for this protein using the standard approach of establishing amono acids sequence of a fragment of the protein and designing oligonucleotides to screen the cDNA library.
- the gene for one or more expansins will need to be expressed in a bacterial or other system to obtain sufficient quantities for the commercial usefulness of the ideas listed above.
- Cloning will also be a necessary first step for the commercial uses requiring genetic manipulation of the protein in transgenic plants.
- expansins include increase of the growth of specific organs and tissues by selective enhanced expression of expansins during plant development.
- Control of selective expression would be by any means such as application of specific chemicals that promote transcription of expansins or by insertion into the genome of plants specific artificial genetic constructs (i.e. appropriate promoters, enhancers, structural genes and associated elements) to effect organ-specific, tissue-specific, and/or chemical-specific enhancement of expression of the introduced or endogenous genes for the expansin proteins.
- Examples of use might include enhancement of fruit production (grapes, citrus fruits, etc.), enhancement of leaf growth (lettuce, spinach, cabbage), enhancement of stem or petiole growth (sugarcane, celery, flower stalks), and enhancement of root growth.
- Tissues engineered to grow by expression of expansins might have enhanced desirable traits (size, succulence, texture, durability).
- Another direction of controlled growth would include decrease of the growth of specific organs and tissue by reducing the expression or effectiveness of endogenous expansins.
- the control of expression would be by any means such as application of specific chemicals that reduce transcription of expansins or by insertion of antisense genetic constructs that reduce mRNA levels of endogenous expansin genes, or by manipulation of the genetic control elements that regulate expression of endogenous expansin genes. Examples of such use might include dwarfing of stems for enhanced mechanical stability and genetic pruning, stunting, or elimination of undesirable plant organs.
- Control could be effected by addition of exogenous expansins or inhibitors of expansin action, by genetic regulation of endogenous expansin genes and their products, or by regulation of artificially inserted genes for expansin proteins, for antisense constructs against endogenous expansin mRNAs, or for proteins that regulate or modify expansin action.
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Abstract
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
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EP94916750A EP0753054A1 (en) | 1993-05-12 | 1994-05-12 | Expansins, proteins that catalyze plant cell wall expansion and weakening of paper fiber connections |
AU68320/94A AU6832094A (en) | 1993-05-12 | 1994-05-12 | Expansins, proteins that catalyze plant cell wall expansion and weakening of paper fiber connections |
JP6525696A JPH08510728A (en) | 1993-05-12 | 1994-05-12 | Weaker binding of expansins, proteins that catalyze plant cell wall elongation and paper fiber |
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US6094493A | 1993-05-12 | 1993-05-12 | |
US08/060,944 | 1993-05-12 |
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WO1994026878A1 true WO1994026878A1 (en) | 1994-11-24 |
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PCT/US1994/005286 WO1994026878A1 (en) | 1993-05-12 | 1994-05-12 | Expansins, proteins that catalyze plant cell wall expansion and weakening of paper fiber connections |
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Country | Link |
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EP (1) | EP0753054A1 (en) |
JP (1) | JPH08510728A (en) |
AU (1) | AU6832094A (en) |
CA (1) | CA2162721A1 (en) |
WO (1) | WO1994026878A1 (en) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999002693A2 (en) * | 1997-07-11 | 1999-01-21 | Genencor International, Inc. | Microbial swollenin protein; dna sequences encoding such swollenins and method of producing such swollenins |
WO2000012715A1 (en) * | 1998-08-29 | 2000-03-09 | Advanced Technologies (Cambridge) Limited | Modification of plant fibres |
US6140470A (en) * | 1995-06-30 | 2000-10-31 | Yale University | Human monoclonal anti-tumor antibodies |
US6218476B1 (en) | 1999-03-03 | 2001-04-17 | Eastman Chemical Company | Functionalized polypropylenes and process for production |
US7098380B2 (en) | 2001-09-27 | 2006-08-29 | Pioneer Hi-Bred International, Inc. | Manipulation of plant polysaccharide synthases |
CN107217047A (en) * | 2017-05-24 | 2017-09-29 | 华南理工大学 | A kind of method for reclaiming solution cellulase |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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US3844890A (en) * | 1971-09-30 | 1974-10-29 | Rikagaku Kenkyusho | Alkaline cellulase and preparation of the same |
US3966543A (en) * | 1972-10-30 | 1976-06-29 | Baxter Laboratories, Inc. | Enzyme-treated paper |
US4004976A (en) * | 1974-04-08 | 1977-01-25 | Deutsche Gold- Und Silber-Scheideanstalt Vormals Roessler | Process for recovering the main sapogenins from the roots of rhizomes of helleborus |
US5175275A (en) * | 1987-05-28 | 1992-12-29 | Tosco Co., Ltd. | Method for preparing powdery crystalline cellulose |
-
1994
- 1994-05-12 JP JP6525696A patent/JPH08510728A/en active Pending
- 1994-05-12 WO PCT/US1994/005286 patent/WO1994026878A1/en not_active Application Discontinuation
- 1994-05-12 AU AU68320/94A patent/AU6832094A/en not_active Abandoned
- 1994-05-12 CA CA002162721A patent/CA2162721A1/en not_active Abandoned
- 1994-05-12 EP EP94916750A patent/EP0753054A1/en not_active Withdrawn
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3844890A (en) * | 1971-09-30 | 1974-10-29 | Rikagaku Kenkyusho | Alkaline cellulase and preparation of the same |
US3966543A (en) * | 1972-10-30 | 1976-06-29 | Baxter Laboratories, Inc. | Enzyme-treated paper |
US4004976A (en) * | 1974-04-08 | 1977-01-25 | Deutsche Gold- Und Silber-Scheideanstalt Vormals Roessler | Process for recovering the main sapogenins from the roots of rhizomes of helleborus |
US5175275A (en) * | 1987-05-28 | 1992-12-29 | Tosco Co., Ltd. | Method for preparing powdery crystalline cellulose |
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6140470A (en) * | 1995-06-30 | 2000-10-31 | Yale University | Human monoclonal anti-tumor antibodies |
WO1999002693A2 (en) * | 1997-07-11 | 1999-01-21 | Genencor International, Inc. | Microbial swollenin protein; dna sequences encoding such swollenins and method of producing such swollenins |
WO1999002693A3 (en) * | 1997-07-11 | 1999-04-22 | Genencor Int | Microbial swollenin protein; dna sequences encoding such swollenins and method of producing such swollenins |
US6458928B1 (en) | 1997-07-11 | 2002-10-01 | Genecor International, Inc. | Microbial swollenin protein, DNA sequences encoding such swollenins and method of producing such swollenins |
US6967246B2 (en) | 1997-07-11 | 2005-11-22 | Genencor International, Inc. | Microbial swollenin protein, DNA sequences encoding such swollenins and method of producing such swollenins |
WO2000012715A1 (en) * | 1998-08-29 | 2000-03-09 | Advanced Technologies (Cambridge) Limited | Modification of plant fibres |
AU777447B2 (en) * | 1998-08-29 | 2004-10-14 | British American Tobacco (Investments) Limited | Modification of plant fibres |
US6218476B1 (en) | 1999-03-03 | 2001-04-17 | Eastman Chemical Company | Functionalized polypropylenes and process for production |
US7098380B2 (en) | 2001-09-27 | 2006-08-29 | Pioneer Hi-Bred International, Inc. | Manipulation of plant polysaccharide synthases |
US7612254B2 (en) | 2001-09-27 | 2009-11-03 | Pioneer Hi-Bred International, Inc. | Manipulation of plant polysaccharide synthases |
CN107217047A (en) * | 2017-05-24 | 2017-09-29 | 华南理工大学 | A kind of method for reclaiming solution cellulase |
CN107217047B (en) * | 2017-05-24 | 2020-05-22 | 华南理工大学 | Method for recovering cellulase in solution |
Also Published As
Publication number | Publication date |
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JPH08510728A (en) | 1996-11-12 |
EP0753054A1 (en) | 1997-01-15 |
AU6832094A (en) | 1994-12-12 |
CA2162721A1 (en) | 1994-11-24 |
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