WO1995026398A1 - Cellulase modifiee et preparation enzymatique la contenant - Google Patents

Cellulase modifiee et preparation enzymatique la contenant Download PDF

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Publication number
WO1995026398A1
WO1995026398A1 PCT/DK1995/000132 DK9500132W WO9526398A1 WO 1995026398 A1 WO1995026398 A1 WO 1995026398A1 DK 9500132 W DK9500132 W DK 9500132W WO 9526398 A1 WO9526398 A1 WO 9526398A1
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Prior art keywords
modified
cellulase
enzyme
modified cellulase
enzyme preparation
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PCT/DK1995/000132
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English (en)
Inventor
Arne Agerlin Olsen
Niels Munk
Henrik Lund
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Novo Nordisk A/S
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Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to AU19468/95A priority Critical patent/AU1946895A/en
Publication of WO1995026398A1 publication Critical patent/WO1995026398A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38645—Preparations containing enzymes, e.g. protease or amylase containing cellulase
    • 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
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
    • D06M16/003—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic with enzymes or microorganisms
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
    • D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes

Definitions

  • the present invention relates to a chemically modified cellulase, an enzyme preparation comprising the modified cellulase and methods for using the modified cellulase and/or enzyme preparation e.g. in the pulp and paper industry, the textile industry, for animal feed and human food and for baking purposes.
  • Enzymes have been used for a long time for a variety of industrial applications. For instance important uses of enzymes are in papermaking pulp processing, in the baking industry for improving the properties of flour, in the wine and juice industries for the degradation of /3-glucans, in the textile industry for bio-polishing of cellulosic fabrics, i.e. for obtaining a soft and smooth fabric by subjecting the cellulosic fabrics to treatment by cellulolytic enzymes during their manufacture, and in animal feed for improving the digestibility of vegetable protein sources.
  • the desired process conditions are quite often in the weakly acidic to alkaline range, e.g. in order to obtain a higher solubility of reaction products, or in order to avoid use of acid and thus minimize the corrosiveness of the reaction mixture.
  • enzymes having relatively acidic pi e.g. below 5
  • the overall performance of the enzymes may be limited under the process conditions.
  • Some enzymes at least might be expected to show an improved performance if the pi of the enzymes is shifted to a value approximating that of the pH during application.
  • the lignocellulosic fibers may be subjected to enzymatic hydrolysis by cellulolytic enzymes.
  • Cellulolytic enzyme preparations for fibre modification may besides cellulases also contain hemi- cellulases, for efficient degradation of carbohydrate material in the fibre walls or in fibre debris.
  • the electrostatic repulsion may be reduced through modification of the enzyme molecules in stead of modifying the substrate.
  • the amount of substrate is typically at least 100 times more than the mass of the enzyme product used in enzymatic processes, e.g. for treatment of the lignocellulosic fibers. Accordingly, it is much more economical to modify the enzyme instead of the lignocellulosic fibers.
  • the present invention relates to a modified cellulase having a pi which is as least one pi unit higher than that of the parent or native cellulase, the modification being obtained by chemical modification.
  • the modified cellulase of the invention is believed to have an improved performance due to an increased pi value and/or an increased surface activity.
  • the present invention further relates to an enzyme preparation comprising one or more modified cellulases.
  • the enzyme preparation of the invention may contain one or more modified cellulase components (e.g. endo-/3-l,4-glucanases and cellobiohydrolases) , either alone or in combination with other enzymes which have or have not been subjected to a chemical modification or an amino acid substitution with the purpose of obtaining an increased pi value and/or an increased surface activity.
  • the term "improved performance" is intended to indicate that the modified enzyme, when subjected to the same standard test conditions as the parent enzyme, exhibits an improved effect compared to the parent enzyme.
  • the performance of a modified cellulase preparation is evaluated from the amount of soluble hydrolysis products, e.g. short chained carbohydrates such as cello- oligomers which are dissolved from the pulp during a treatment with said cellulase preparation minus the amount that is dissolved in a control treatment without addition of cellulase.
  • the dissolved carbohydrates may be determined by analysis with anion exchange chromatography with pulsed amperometric detection (Dionex Corporation, Sunnyvale, CA, USA : Technical Note 20, 1989) or with the orcinol total reducing sugar method (see Vasseur, E. , Acta Chem . Scand. , "A spectrophotometric study on the orcinol reaction with carbohydrates, " 2 : 693 (1948) ) .
  • the dry matter weight loss compared to a control is often also used as an additional measure of cellulase performance.
  • the isoelectric point, pi is defined as the pH value at which the enzyme molecule is neutral, i. e. the sum of electrostatic charges (net electrostatic charge) is equal to zero. In this sum of course consideration of the positive or negative nature of the individual electrostatic charges must be taken into account.
  • the pi may conveniently be determined experimentally by isoelectric focusing or by titrating a solution containing the enzyme.
  • the pi of the modified enzyme should preferably be at least one pi unit, more preferably at least two pi units, most preferably at least three pi units, higher than that of the parent enzyme.
  • a (parent) cellulase having a pi of 5.5 may be chemically modified as described herein resulting in a modified cellulase having a pi of 9.
  • cellulase and “cellulase component” are intended to mean an enzyme that hydrolyses cellulose.
  • the cellulase or cellulase component may be a component occurring in a cellulase system produced by a given microorganism, such a cellulase system mostly comprising several different cellulase enzyme components including those usually identified as e.g. cellobiohydrolases, endo- ⁇ -1,4- glucanases, 3-glucosidases.
  • the cellulase component may be a single component, i.e.
  • a component essentially free of other cellulase components usually occurring in a cellulase system produced by a given microorganism the single component being a recombinant component, i.e. produced by cloning of a DNA sequence encoding the single component and subsequent cell transformed with the DNA sequence and expressed in a host.
  • the host is preferably a heterologous host, but the host may under certain conditions also be the homologous host.
  • the native or unmodified cellulase or cellulase component may be derived from microorganisms which are known to be capable of producing cellulolytic enzymes, e.g. species of Humicola.
  • the derived component may be either homologous or heterologous component.
  • the component is homologous.
  • a heterologous component which is immunologically reactive with an antibody raised against a highly purified cellulase component and which is derived from a specific microorganism is also preferred.
  • a preferred example of a native or parent or unmodified cellulase component is an endoglucanase component which is im- munologically reactive with an antibody raised against a highly purified ⁇ 43kD endoglucanase derived from Humicola insolens , DSM 1800, or which is a derivative of the ⁇ 43kD endoglucanase exhibiting cellulase activity.
  • a preferred endoglucanase component has the amino acid sequence disclosed in PCT Patent Application No. W091/17243, SEQ ID#2, which is hereby in ⁇ corporated by reference.
  • Another preferred endoglucanase component is the core enzyme corresponding to the amino acid sequence disclosed in PCT Patent Application No. W091/17243, SEQ ID#2, but having the amino acid sequence corresponding to position 1-213, i.e. truncated at position 213.
  • Yet another preferred endoglucanase component is an endoglucanase component which is immunologically reactive with an antibody raised against a highly purified ⁇ 26kD endogluca ⁇ nase derived from Aspergillus aculeatus , CBS101.43, or which is a derivative of the ⁇ 26kD endoglucanase exhibiting cellulase activity.
  • 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.
  • a preferred endoglucanase component is the Endoglucanase III disclosed in International Patent Application PCT/DK93/00444, which is hereby incorporated by reference, the Endoglucanase III being believed to exhibit a substantial homology with the sequence disclosed by Ooi et al., 1990, in "Complete nucleotide sequence of a gene coding for Aspergillus aculeatus cellulase (FI- CMCase)", Nucleic Acids Research, Vol. 18, No. 19: 5884.
  • cellulosic fabric is intended to include fabric originating from cellulose fibers, e.g. from wood pulp, and cotton.
  • Examples of cellulosic fabrics are cotton, viscose (rayon) ; lyocell; all blends of viscose with other fabrics such as viscose/polyester blends, viscose/cotton blends, viscose/wool blends; flax (linen) and ramie and other fabrics based on cellulose fibers, including all blends of cellulosic fabrics with other fabrics such as wool and polyester, e.g. viscose/polyester blends, viscose/cotton blends, viscose/wool blends, viscose/cotton/polyester blends, flax/cotton blends etc.
  • the enzyme is chemically modified by coupling an amine ligand to the carboxyl group of glutamic acid or aspartic acid residues in the enzyme.
  • the amine ligand is preferably an aminated sugar, aminated alcohol or aminated polyalcohol.
  • suitable aminated sugars are glucosamine, isomeric forms thereof with the general formula r oligomers and polymers of the general formula for example polymers of glucosamines such as chitosans. Oligomers and polymers may be either branched or linear.
  • an aminated alcohol is used for coupling to the carboxyl group, it should generally contain at least 3 carbon atoms.
  • aminated alcohols are a inopropanol or aminobutanol. More preferably the amine ligand is an aminated polyalcohol. Polyalcohols should generally contain at least 3 carbon atoms, and may for instance contain 6 carbon atoms. Examples of suitable aminated polyalcohols are glucamine, isomeric forms thereof with the general formula CgHjsOsN, or oligomers and polymers thereof with the general formula [CgH ⁇ O ⁇ ],,, wherein n>l.
  • amine substituted alkanes and derivatives thereof are amine substituted alkanes and derivatives thereof.
  • Preferred examples of amine substituted alkanes and their derivatives are amino acids such as lysine, polylysine; esters of amino acids; spermine; spermidine; putrescine; and the like.
  • the amine ligand such as an aminated sugar, alkane, alcohol or polyalcohol and polymer thereof, should have at least one amino group per monomeric unit, but should not be considered to be limited to having only one amino group per monomeric unit.
  • the coupling of the amine to the carboxyl group of glutamic acid or aspartic acid residues is mediated by a crosslinking agent capable of binding a carboxyl group and an amino group.
  • the coupling reaction may suitably be carried out by standard methods as described by S.S. Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press, Boca Raton, Florida, USA, 1991, in particular Chapter 2, IV, C, Chapter 4, IV and Chapter 5, II; or Wong and Wong, Enzyme Microb.Technol. 14, November 1992, pp. 866-873.
  • a particularly preferred crosslinking agent for the coupling reaction is a carbodiimide, e.g. l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC).
  • Methods of conjugating proteins with ligands using EDC can be implemented according to manufacturer's description (e.g. Pierce Instructions 0475 C, 22980 X; 22981 X; EDC) using either the protocol for "Use of EDC for coupling of Haptens/small ligands to carrier Proteins" or "Protocol for Efficient Two- Step coupling of Proteins in Solution Using EDC and N- hydroxysuccinimide or sulfo-N-hydroxysucciminide".
  • the enzyme may be dissolved, or transferred by dialysis or desalting by size exclusion chromatography in a coupling buffer, such as, for instance 50 mM MES pH 5.0 containing 200 mM sodium chloride.
  • the ligand e.g. glucosamine, may be dissolved in coupling buffer as well.
  • the conjugation reaction may proceed by mixing enzyme and ligand to a final concentration of 3 mg/ml for both enzyme and ligand followed by mixing with 5 mg of EDC per mg of enzyme.
  • the conjugation reaction then runs for 2 hours at room temperature with continuous stirring.
  • the reaction is terminated by removal of surplus reagent either by desalting by size exclusion chromatography or by extensive dialysis, e.g. against 0.2 M ammonium acetate pH 6.9 at 5°C.
  • the resulting derivative may then be stored at 5°C.
  • the degree of modification or incorporation of ligands may, of course, be controlled by adjustments in the initial enzyme, ligand and/or carbodiimide concentration. Variations in pH or temperature of the coupling buffer may also be included to optimise the conjugation reaction for a specific enzyme.
  • substrate analogues and reversible inhibitors may be used to control of the modification reaction.
  • the preparation comprises one or more cellulases modified by substitution of one or more amino acids as disclosed in PCT/DK93/00327 which is hereby incorporated by reference. Preparation of modified cellulases by amino acid substitution
  • the DNA sequence encoding a parent enzyme may be isolated from any cell or microorganism producing the enzyme in question by various methods, well known in the art. First a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the enzyme to be studied. Then, if the amino acid sequence of the enzyme is known, homologous, labelled oligonucleotide probes may be synthesized and used to identify enzyme-encoding clones from a genomic library prepared from the organism in question. Alter ⁇ natively, a labelled oligonucleotide probe containing sequences homologous to a known enzyme could be used as a probe to identify enzyme-encoding clones, using hybridization and washing conditions of lower stringency.
  • Yet another method for identifying enzyme-encoding clones would involve inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming enzyme-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing a substrate for enzyme thereby allowing clones expressing the enzyme to be identified.
  • an expression vector such as a plasmid, transforming enzyme-negative bacteria with the resulting genomic DNA library
  • the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by S.L. Beaucage and M.H. Caruthers, Tetrahedron Letters 22, 1981, pp. 1859-1869, or the method described by Matthes et al., The EMBO J. 3., 1984, pp. 801-805.
  • oligonucleo ⁇ tides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and clon
  • the DNA sequence may be of mixed genomic and synthe- tic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) , the fragments corresponding to various parts of the entire DNA sequence, in accordance with standard techniques.
  • the DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or R.K. Saiki et al.. Science 239. 1988, pp. 487-491.
  • mutations may be intro ⁇ quizzed using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligonucleotide synthesis.
  • a single-stranded gap of DNA, bridging the enzyme-encoding sequence is created in a vector carrying the enzyme gene. Then the synthetic nucleotide, bearing the desired mutation, is annealed to a homologous portion of the single-stranded DNA.
  • a mutated enzyme-encoding DNA sequence produced by methods described above, or any alterna ⁇ tive methods known in the art can be expressed, in enzyme form, using an expression vector which typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.
  • the recombinant expression vector carrying the DNA sequence encoding a modified enzyme of the invention encoding 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, a bacteriophage or an extrachromosomal element, minichromosome or an artificial chromosome.
  • 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 should be operably connected to a suitable promoter 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.
  • suitable promoters for directing the transcription of the DNA sequence encoding a cellulase variant as described herein, especially in a bacterial host are the promoter of the lac operon of E.
  • the Streptomyces coelicolor agarase gene dagrA promoters the promoters of the Bacillus licheniformis ⁇ - amylase gene (amyL) , the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM) , the promo ⁇ ters of the Bacillus Amyloliquefaciens ⁇ -amylase (amyQ) , the promoters of the Bacillus subtilis xylA and xylB genes etc.
  • useful promoters are those derived from the gene encoding A.
  • oryzae TAKA amylase Rhizomucor miehei aspartic proteinase, A. niger neu ⁇ tral ⁇ -amylase, A. niger acid stable ⁇ -amylase, A. niger glu- coamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.
  • the expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, poly- adenylation sequences operably connected to the DNA sequence encoding the enzyme variant of the invention. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
  • the vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBHO, pE194, pAMBl and pIJ702.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the dal genes from B .subtilis or B .licheniformi ⁇ , or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance.
  • a selectable marker e.g. a gene the product of which complements a defect in the host cell, such as the dal genes from B .subtilis or B .licheniformi ⁇ , or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance.
  • the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, e.g. as described in WO 91/17243.
  • the cell of the invention either comprising a DNA construct or an expression vector as defined above is advantageously used as a host cell in the recombinant production of an enzyme variant of the invention.
  • the cell may be transformed with the DNA con ⁇ struct encoding the modified enzyme, conveniently by inte ⁇ grating the DNA construct in the host chromosome. This integra- tion is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g. by homologous or heterologous recombination.
  • the cell may be transformed with an expression vector as described below in connection with the different types of host cells.
  • the host cell may be a cell of a higher organism such as a mammal or an insect, but is preferably a microbial cell, e.g. a bacterial or a fungal (including yeast) cell.
  • suitable bacteria are gram-positive bacteria such as Bacillus subtilis , Bacillus licheniformi ⁇ , Bacillus lentus , Bacillus brevis , Bacillus stearothermophilus, Bacillus alkalo- philus, Bacillus amyloliquefacien ⁇ , Bacillus coagulans , Bacillus circulans , Bacillus lautus , Bacillus megaterium, Ba ⁇ cillus thuringiensi ⁇ , or Streptomyces lividan ⁇ or Streptomyce ⁇ murinu ⁇ , or gram-negative bacteria such as E. coli .
  • the trans ⁇ formation of the bacteria may for instance be effected by protoplast transformation or by using competent cells in a manner known per se.
  • the yeast organism may favourably be selected from a species of Saccharomyce ⁇ or Schizosaccharomyces , e.g. Saccharomyce ⁇ cerevi ⁇ iae .
  • the filamentous fungus may advantageously belong to a species of Aspergillus, e.g. A ⁇ pergillu ⁇ oryzae, A ⁇ pergillu ⁇ awamori, A ⁇ pergillu ⁇ aculeatu ⁇ 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 . A suitable procedure for transformation of A ⁇ pergillu ⁇ host cells is described in EP 238 023.
  • the modified enzyme may be produced by cultivating a host cell as described above under conditions conducive to the production of the modified enzyme and recovering the modified enzyme from the cells and/or culture medium.
  • the medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the modified enzyme of the invention.
  • Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection) .
  • the modified cellulase secreted from the host cells may con ⁇ veniently be recovered from the culture medium by well-known procedures including separating the cells from the medium by centrifugation or filtration, and 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.
  • a salt such as ammonium sulphate
  • chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
  • the enzyme preparation of the invention may comprise a combination of one or more modified cellulases with enzymes selected from the group consisting of unmodified or modified amylases, lipases, proteases, oxidoreductases and hemicellulases.
  • the cellulase and/or enzyme preparation according to the invention may be applied advantageously e.g. as follows:
  • pretreatment with the cellulase and/or enzyme preparation according to the invention may degrade the cambium layer prior to debarking in mechanical drums resulting in advantageous energy savings.
  • treatment of a material containing cellulosic fibers with the cellulase and/or enzyme preparation of the invention prior to refining or beating may result in reduction of the energy consumption due to the hydrolysing effect of the cellulase on the interfibre surfaces.
  • Use of the cellulase and/or enzyme preparation of the invention may result in improved energy savings as compared to the use of unmodified enzymes, since it is believed that the modified cellulase may possess a higher ability to penetrate fibre walls.
  • fibre modification i.e. improvement of fibre properties where partial hydrolysis across the fibre wall is needed which requires deeper penetrating enzymes (e.g. in order to make coarse fibers more flexible) .
  • Deep treatment of fibers has so far not been possible for high yield pulps e.g. mechanical pulps or mixtures of recycled pulps. This has been ascribed to the nature of the fibre wall structure that prevents the passage of enzyme molecules due to physical restriction of the pore matrix of the fibre wall. It is contemplated that the modified (i.e. derivatised) cellulases of the invention are capable of penetrating into the fibre wall.
  • the drainability of papermaking pulps may be improved by treatment of the pulp with hydrolysing enzymes, e.g. cellulases.
  • hydrolysing enzymes e.g. cellulases.
  • Use of the modified cellulase and/or enzyme preparation according to the invention may be more effective, e.g. result in a higher degree of loosening bundles of strongly hydrated micro-fibrils in the fines fraction (consisting of fibre debris) that limits the rate of drainage by blocking hollow spaces between fibers and in the wire mesh of the paper machine.
  • CSF Canadian standard freeness
  • increases and the Schopper-Riegler drainage index decreases when pulp in subjected to cellulase treatment see e.g. US patent 4,923,565; TAPPI T227, SCAN C19:65 which are hereby incorporated by reference.
  • Partial hydrolysis of recycled paper during or upon pulping by use of hydrolysing enzymes such as cellulases are known to facilitate the removal and agglomeration of ink particles.
  • Use of a modified cellulase and/or enzyme preparation according to the invention may give a more effective loosening of ink from the surface structure due to a better penetration of the enzyme molecules into the fibrillar matrix of the fibre wall, thus softening the surface whereby ink particles are effectively loosened.
  • the agglomeration of loosened ink particles are also improved, due to a more efficient hydrolysis of cellulosic fragments found attached to ink particles originating from the fibres.
  • lignocellulosic pulp may, e.g., be performed as described in WO 91/14819, WO 91/14822, WO 92/17573 and WO 92/18688. Textile applications
  • the present invention relates to use of the modified cellulase and/or enzyme preparation according to the invention in the bio-polishing process.
  • Bio-Polishing is a specific treatment of the yarn surface which improves fabric quality with respect to handle and appearance without loss of fabric wettability. The most important effects of Bio- Polishing can be characterized by less fuzz and pilling, increased gloss/luster, improved fabric handle, increased durable softness and altered water absorbency.
  • Bio-Polishing usually takes place in the wet processing of the manufacture of knitted and woven fabrics. Wet processing comprises such steps as e.g. desizing, scouring, bleaching, washing, dying/printing and finishing. During each of these steps, the fabric is more or less subjected to mechanical action.
  • Desizing is the act of removing size from textiles.
  • warp yarns Prior to weaving on mechanical looms, warp yarns are often coated with size starch or starch derivatives in order to increase their tensile strength. After weaving, the size coating must be removed before further processing the fabric in order to ensure a homogeneous and wash-proof result.
  • size starch or starch derivatives Prior to weaving on mechanical looms, warp yarns are often coated with size starch or starch derivatives in order to increase their tensile strength. After weaving, the size coating must be removed before further processing the fabric in order to ensure a homogeneous and wash-proof result. It is known that in order to achieve the effects of Bio-Polishing, a combination of cellulolytic and mechanical action is required. It is also known that "super-softness" is achievable when the treatment with cellulase is combined with a conventional treatment with softening agents.
  • Bio-polishing may be obtained by applying the method described e.g. in WO 93/20278. Stone-washing of cellulosic fabric
  • the present invention relates to use of the modified cellulase and/or enzyme preparation according to the invention in "stone-washing" of dyed fabric.
  • the modified cellulase or the enzyme preparation of the invention is most beneficially applied to cellulose-containing fabrics, such as cotton, viscose, rayon, ramie, linen, lyocell (Tencel) or mixtures thereof, or mixtures of any of these fibres.
  • the fabric is denim.
  • the fabric may be dyed with vat dyes such as indigo, direct dyes such as Direct Red 185, sulphur dyes such as Sulfur Green 6, or reactive dyes fixed to a binder on the fabric surface.
  • the fabric is indigo-dyed denim, including clothing items manu ⁇ factured therefrom.
  • the amount of modified cellulase or cellulase preparation used to treat the dyed fabric typically depends on the ratio of cellulolytic enzyme and optionally buffer and pumice or perlite in the composition and the dry weight of the dyed fabric to be washed.
  • the composition used in the process of the invention contain a minimum of 20 ECU of endoglucanase and, in case of using perlite, a minimum of 20 w/w% of perlite to obtain the stone-washed look.
  • the dyed fabric may be contacted with about 40-150 ECU of endoglucanase per litre of washing liquor for 75 minutes at about 55°C.
  • the preferred pH is dependent on the pH optimum of the cellulolytic enzyme, i.e. whether an acid, neutral, or alkaline cellulase is applied.
  • the present invention relates to use of the modified cellulase and/or enzyme preparation in baking flour so as to improve the development, elasticity and/or stability of dough and/or the volume, crumb structure and/or anti-staling properties of the baked product.
  • the enzyme preparation may be used for the preparation of dough or baked products prepared from any type of flour or meal (e.g. based on rye, barley, oat, or maize)
  • the modified cellulase and/or enzyme preparation of the invention have been found to be particularly useful in the preparation of dough or baked products made from wheat or comprising substantial amounts of wheat.
  • the baked products produced with the modified cellulase and/or enzyme preparation of the invention includes bread, rolls, baguettes and the like.
  • the enzyme preparation of the invention may be used as having cellulase as the major enzymatic activity, or may be used in combination with other enzymes such as a lipase, an amylase, an oxidase (e.g. glucose oxidase, peroxidase) , a laccase and/or a protease; the lipase, amylase, oxidase and laccase optionally being modified as described in Danish patent application no. DK 0259 / 94 .
  • the present invention relates to use of a modified cellulase and/or enzyme preparation according to the invention in the beer brewing industry in particular to improve the filterability of wort e.g. containing barley and/or sorghum malt.
  • the cellulase and/or enzyme preparation may be used in the same manner as pentosanases conventionally used for brewing, e.g. as described by Vietor et al., 1993, J. Inst. Brew. , May-June, 99, pp. 243-248, and EP 227 159.
  • the modified cellulase and/or enzyme preparation of the invention may be used for treatment of brewers spent grain, i.e. residuals from beer wort production containing barley or malted barley or other cereals, so as to improve the utiliz- ation of the residuals for, e.g., animal feed.
  • the present invention relates to use of a modified cellulase and/or enzyme preparation according to the invention for degradation of plant material e.g. cell walls.
  • modified cellulase and/or enzyme preparation of the invention is useful in the preparation of wine, fruit or vegetable juice in order to increase yield.
  • Cellulases according to the invention may also be applied for enzymatic hydrolysis of various plant cell-wall derived materials or waste materials, e.g. agricultural residues such as wheat-straw, corn cobs, whole corn plants, nut shells, grass, vegetable hulls, bean hulls, spent grains, sugar beet pulp, and the like.
  • the plant material may be degraded in order to improve different kinds of processing, facilitate purification or extraction of other components like purification of beta-glucan or beta-glucan oligomers from cereals, improve the feed value, decrease the water binding capacity, improve the degradability in waste water plants, improve the conversion of e.g. grass and corn to ensilage, etc.
  • the present invention relates to use of a cellulase and/or enzyme preparation according to the invention in animal feed (or for the treatment of animal feed prior to ingestion by the animal) .
  • Modified cellulase and/or enzyme preparation is preferably added to the feed in an amount which is efficient for improving the digestibility of vegetable protein sources, e.g. cereals and legumes.
  • the cellulase and/or enzyme preparation of the present invention may be used for modification of animal feed and may exert its effect either in vitro (by modifying components of the feed) or in vivo.
  • Endoglucanase III the recombinant endoglucanase denoted Endoglucanase III as disclosed in International Patent Application PCT/DK93/00444, with glucosamine through carbodiimide mediated coupling was performed according to standard procedures.
  • An enzyme stock solution was prepared by dissolving approximately 100 mg/ml of endoglucanase III in water, followed by size exclusion chromatography on Superdex 75 (from Pharmacia AB, Sweden) in coupling buffer (50 mM MES pH 5.0 containing 250 mM sodium chloride) . The glucosamine was dissolved in coupling buffer as well.
  • the conjugation reaction proceeded by mixing enzyme and glucosamine to a final concentration of about 1 mg/ml for both enzyme and glucosamine followed by addition of 5 mg of EDC per mg of enzyme to mediate the reaction.
  • the reaction mixture also contained 200 mM of cellobiose for active site protection.
  • the conjugation reaction continued for 1 hour at room temperature with continuous magnetic stirring.
  • the reaction was terminated by desalting on a Sephadex G-15 column equilibrated with 0.2 M ammonium acetate, pH 6.9, at room temperature.
  • the derivative was stored at 5°C.
  • the endoglucanase-glucosamine derivative prepared according to the above described procedure was shown to be monomeric by size-exclusion chromatography on a TSK-G2000SW column, has a pi value of about 9 as determined by isoelectric focusing and 26% residual cellulase activity when compared to the parental (native) enzyme which has a pi of 5.5.
  • the activity was measured according to the standard Novo Nordisk cellulase methods AF-275-GB/AF-295-GB (available from Novo Nordisk A/S on request) which are hereby incorporated by reference.
  • Figure 1 illustrates the effect of glucosamination on the A/W- adsorption of cellulase by showing the surface pressure (mN/m) as a function of the time after addition of enzyme (at the time 2 min; 50 mM glycin/500 mM NaCl; temperature 298K) .
  • the bold line illustrates the effect of glucosaminated cellulase and the skip line illustrates the zero effect of the native cellulase.
  • the surface pressure is defined as - ⁇ surface tension when the surface tension of the pure buffer solution and the enzyme containing buffer solution, respectively, is compared.
  • Treatment of kraft pulp with endoglucanase is applied e.g. in enzymatic deinking of office waste paper or for improving interfibre bonding in kraft liner.
  • the example demonstrates how the activity of a cellulase, i.e. an endoglucanase, may be increased by modification according to the invention.
  • the ECU assay quantifies the amount of catalytic activity present in the sample by measuring the ability of the sample to reduce the viscosity of a solution of carboxy-methylcellulose (CMC) .
  • the assay is carried out at 40°C, pH 7.5 using a relative enzyme standard for reducing the viscosity of the CMC substrate and the resulting reduction in viscosity may be determined by a vibration viscosimeter (e.g. MIVI 3000 from Sofraser, France) .
  • Determination of the cellulolytic activity, measured in terms of ECU may be determined according to the analysis method AF 301.1 which is available from the Applicant upon request.
  • Table 1 Results from treatment of oxygen bleached hardwood kraft pulp at pH 5.2 and 6. The concentration of dissolved glucose is a measure of the performance of the cellulase. The reference has been treated with the unmodified parent endo ⁇ glucanase in a dosage equal to what is applied for the derivatised endoglucanase. Table 1
  • Table 2 As Table 1 but the substrate is oxygen bleached softwood kraft pulp.
  • Cotton materials may be treated with cellulolytic enzymes e.g. to create a smoother fibre surface or to remove impurities from the surface.
  • cotton fluff and woven fabric was treated similarly to the kraft pulp samples as described in example 3 except for the repulping step, that was omitted.
  • the woven fabric was treated in a Launder-Ometer.
  • the concentration of dissolved glucose as measured by the 5 orcinol method was used as a measure of the effect of the treatment.
  • Table 3 Results from treatment of cotton fluff at pH 5.0 and 5.9.
  • the concentration of dissolved glucose is a measure of the performance of the cellulase.
  • the reference has been treated 15 with the unmodified parent endo-glucanase in a dosage equal to what is applied for the derivatized endo-glucanase.
  • a commercial oxygen bleached kraft pulp made from Scandinavian softwood was treated 3 hours at 10% consistency with cellulase preparations.
  • the treatments were carried out at pH 7 and 8.8, at 55°C in a 0.025 M phosphate buffer. After the treatment time a sample of the water phase was collected and the reaction was stopped by washing the pulp with boiling water.
  • the enzymes were dosed on an equal activity basis, the dosage was 4000 ECU/kg pulp.
  • the final content of total glucose as determined by the orcinol method, is listed in the table. As can be seen derivation enhances the performance at high pH.
  • the cellulase preparations A and B, respectively, used in this example were highly purified 43kD endoglucanase derived from Humicola in ⁇ olen ⁇ , DSM 1800, "B" being a modified endoglucanase of the invention prepared as described in Example 1.
  • the cellulolytic activity of the preparations were:

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Abstract

Des enzymes cellulolytiques (cellulases) modifiées par voie chimique, donnent des résultats améliorés et un pI supérieur d'au moins une unité à celui de la cellulase parente ou native. Elles sont modifiées par exemple par couplage d'une amine au groupe carboxyle de leurs restes d'acide glutamique ou aspartique. Des préparations enzymatiques contenant de telles cellulases modifiées s'utilisent par exemple dans les industries papetière et textile, pour les denrées alimentaires destinées à l'homme et à l'animal, et pour la cuisson au four.
PCT/DK1995/000132 1994-03-28 1995-03-27 Cellulase modifiee et preparation enzymatique la contenant WO1995026398A1 (fr)

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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0897667A2 (fr) † 1997-07-31 1999-02-24 Gist-Brocades B.V. Composition d'amélioration du pain
US6001639A (en) * 1995-03-17 1999-12-14 Novo Nordisk A/S Endoglucanases
EP0982396A2 (fr) * 1998-06-23 2000-03-01 Korea Research Institute Of Chemical Technology Enzyme modifié et son procédé de modification
US6146494A (en) * 1997-06-12 2000-11-14 The Procter & Gamble Company Modified cellulosic fibers and fibrous webs containing these fibers
US6261828B1 (en) * 1995-11-15 2001-07-17 Novo Nordisk A/S Process for combined desizing and “stone-washing” of dyed denim
WO2002022943A1 (fr) * 2000-09-14 2002-03-21 Meiji Seika Kaisha, Ltd. Procede pour desencrer des vieux papiers au moyen d'une cellulase, sans diminuer la resistance du papier, et procede d'evaluation
WO2002099091A2 (fr) 2001-06-06 2002-12-12 Novozymes A/S Endo-beta-1,4-glucanase
JP2003501021A (ja) * 1999-05-28 2003-01-14 ノボザイムス アクティーゼルスカブ 新規エンド−β−１，４−グルカナーゼ
WO2003055974A2 (fr) 2001-12-22 2003-07-10 Henkel Kommanditgesellschaft Auf Aktien Nouvelle protease alcaline obtenue a partir de bacillus sp. (dsm 14392) et agents de lavage et de nettoyage contenant cette protease alcaline
WO2003056000A1 (fr) * 2001-12-21 2003-07-10 Unisearch Enzyme modifiee et procede de modification
US7256030B1 (en) 1999-05-28 2007-08-14 Novozymes A/S Family 9 endo-β-1,4-glucanases
DE102007032111A1 (de) 2007-07-09 2009-01-15 Henkel Ag & Co. Kgaa Neue Proteasen und Wasch- und Reinigungsmittel enthaltend diese Proteasen
DE102007036756A1 (de) 2007-08-03 2009-02-05 Henkel Ag & Co. Kgaa Neue Proteasen und Wasch- und Reinigungsmittel, enthaltend diese neuen Proteasen
EP2261359A1 (fr) 1998-06-10 2010-12-15 Novozymes A/S Mannanases
US8309338B2 (en) 2005-11-16 2012-11-13 Novozymes A/S Polypeptides having endoglucanase activity and polynucleotides encoding same
CN110184316A (zh) * 2019-05-24 2019-08-30 华南理工大学 一种利用改性β-葡萄糖苷酶强化木质纤维素酶解的方法

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0149520A2 (fr) * 1984-01-17 1985-07-24 Bellex Corporation Lipase modifiée
US4923565A (en) * 1986-09-22 1990-05-08 La Cellulose Du Pin Method for treating a paper pulp with an enzyme solution
EP0405901A1 (fr) * 1989-06-26 1991-01-02 Unilever Plc Compositions détergentes enzymatiques
WO1991000910A1 (fr) * 1989-07-07 1991-01-24 Unilever Plc Enzymes et compositions detergentes enzymatiques
WO1991014819A1 (fr) * 1990-03-29 1991-10-03 Novo Nordisk A/S Procede de desencrage enzymatique
WO1991016424A1 (fr) * 1990-04-25 1991-10-31 Hans Ulrich Geyer Procede de modification appropriee de proprietes d'enzymes par transformation chimique et enzymes chimiquement transformes
WO1991017243A1 (fr) * 1990-05-09 1991-11-14 Novo Nordisk A/S Preparation de cellulase comprenant un enzyme d'endoglucanase
US5290474A (en) * 1990-10-05 1994-03-01 Genencor International, Inc. Detergent composition for treating cotton-containing fabrics containing a surfactant and a cellulase composition containing endolucanase III from trichoderma ssp
WO1994007998A1 (fr) * 1992-10-06 1994-04-14 Novo Nordisk A/S Variantes de cellulase

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0149520A2 (fr) * 1984-01-17 1985-07-24 Bellex Corporation Lipase modifiée
US4923565A (en) * 1986-09-22 1990-05-08 La Cellulose Du Pin Method for treating a paper pulp with an enzyme solution
EP0405901A1 (fr) * 1989-06-26 1991-01-02 Unilever Plc Compositions détergentes enzymatiques
WO1991000910A1 (fr) * 1989-07-07 1991-01-24 Unilever Plc Enzymes et compositions detergentes enzymatiques
WO1991014819A1 (fr) * 1990-03-29 1991-10-03 Novo Nordisk A/S Procede de desencrage enzymatique
WO1991016424A1 (fr) * 1990-04-25 1991-10-31 Hans Ulrich Geyer Procede de modification appropriee de proprietes d'enzymes par transformation chimique et enzymes chimiquement transformes
WO1991017243A1 (fr) * 1990-05-09 1991-11-14 Novo Nordisk A/S Preparation de cellulase comprenant un enzyme d'endoglucanase
US5290474A (en) * 1990-10-05 1994-03-01 Genencor International, Inc. Detergent composition for treating cotton-containing fabrics containing a surfactant and a cellulase composition containing endolucanase III from trichoderma ssp
WO1994007998A1 (fr) * 1992-10-06 1994-04-14 Novo Nordisk A/S Variantes de cellulase

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EP1683860A2 (fr) 1995-03-17 2006-07-26 Novozymes A/S Nouvelles endoglucanases
US6855531B2 (en) 1995-03-17 2005-02-15 Novozymes A/S Endoglucanases
US7226773B2 (en) 1995-03-17 2007-06-05 Novozymes A/S Endoglucanases
US6001639A (en) * 1995-03-17 1999-12-14 Novo Nordisk A/S Endoglucanases
US9023620B2 (en) 1995-03-17 2015-05-05 Novozymes A/S Method of providing color clarification of laundry
US6387690B1 (en) 1995-03-17 2002-05-14 Novozymes A/S Endoglucanases
US8642730B2 (en) 1995-03-17 2014-02-04 Novozymes A/S Endoglucanases
EP2431462A2 (fr) 1995-03-17 2012-03-21 Novozymes A/S Nouvelles endoglucanases
US6261828B1 (en) * 1995-11-15 2001-07-17 Novo Nordisk A/S Process for combined desizing and “stone-washing” of dyed denim
US6146494A (en) * 1997-06-12 2000-11-14 The Procter & Gamble Company Modified cellulosic fibers and fibrous webs containing these fibers
EP0897667A2 (fr) † 1997-07-31 1999-02-24 Gist-Brocades B.V. Composition d'amélioration du pain
EP0897667B2 (fr) † 1997-07-31 2008-11-12 DSM IP Assets B.V. Composition pour améliorer le pain
EP2284272A1 (fr) 1998-06-10 2011-02-16 Novozymes A/S Mannanases
EP2261359A1 (fr) 1998-06-10 2010-12-15 Novozymes A/S Mannanases
EP2287318A1 (fr) 1998-06-10 2011-02-23 Novozymes A/S Mannanases
EP0982396A2 (fr) * 1998-06-23 2000-03-01 Korea Research Institute Of Chemical Technology Enzyme modifié et son procédé de modification
EP0982396A3 (fr) * 1998-06-23 2001-02-28 Korea Research Institute Of Chemical Technology Enzyme modifié et son procédé de modification
US7256030B1 (en) 1999-05-28 2007-08-14 Novozymes A/S Family 9 endo-β-1,4-glucanases
JP2003501021A (ja) * 1999-05-28 2003-01-14 ノボザイムス アクティーゼルスカブ 新規エンド−β−１，４−グルカナーゼ
JP4730933B2 (ja) * 2000-09-14 2011-07-20 Ｍｅｉｊｉ Ｓｅｉｋａファルマ株式会社 紙力低下を伴わない古紙のセルラーゼ脱墨法及びその評価方法
US7297224B2 (en) 2000-09-14 2007-11-20 Meiji Seika Kaisha, Ltd. Method of deinking waste paper using cellulase without lowering paper strength and method of evaluating the same
WO2002022943A1 (fr) * 2000-09-14 2002-03-21 Meiji Seika Kaisha, Ltd. Procede pour desencrer des vieux papiers au moyen d'une cellulase, sans diminuer la resistance du papier, et procede d'evaluation
JPWO2002022943A1 (ja) * 2000-09-14 2004-01-22 明治製菓株式会社 紙力低下を伴わない古紙のセルラーゼ脱墨法及びその評価方法
WO2002099091A2 (fr) 2001-06-06 2002-12-12 Novozymes A/S Endo-beta-1,4-glucanase
WO2003056000A1 (fr) * 2001-12-21 2003-07-10 Unisearch Enzyme modifiee et procede de modification
WO2003055974A2 (fr) 2001-12-22 2003-07-10 Henkel Kommanditgesellschaft Auf Aktien Nouvelle protease alcaline obtenue a partir de bacillus sp. (dsm 14392) et agents de lavage et de nettoyage contenant cette protease alcaline
US8309338B2 (en) 2005-11-16 2012-11-13 Novozymes A/S Polypeptides having endoglucanase activity and polynucleotides encoding same
DE102007032111A1 (de) 2007-07-09 2009-01-15 Henkel Ag & Co. Kgaa Neue Proteasen und Wasch- und Reinigungsmittel enthaltend diese Proteasen
DE102007036756A1 (de) 2007-08-03 2009-02-05 Henkel Ag & Co. Kgaa Neue Proteasen und Wasch- und Reinigungsmittel, enthaltend diese neuen Proteasen
CN110184316A (zh) * 2019-05-24 2019-08-30 华南理工大学 一种利用改性β-葡萄糖苷酶强化木质纤维素酶解的方法

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