WO2002048350A1 - Levure modifiee possedant sur la couche de surface cellulaire un domaine de liaison a la cellulose - Google Patents

Levure modifiee possedant sur la couche de surface cellulaire un domaine de liaison a la cellulose Download PDF

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Publication number
WO2002048350A1
WO2002048350A1 PCT/JP2001/007207 JP0107207W WO0248350A1 WO 2002048350 A1 WO2002048350 A1 WO 2002048350A1 JP 0107207 W JP0107207 W JP 0107207W WO 0248350 A1 WO0248350 A1 WO 0248350A1
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cellulose
yeast
binding domain
amino acid
acid sequence
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PCT/JP2001/007207
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English (en)
Japanese (ja)
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Atsuo Tanaka
Mitsuyoshi Ueda
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Mitsubishi Chemical Corporation
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Publication of WO2002048350A1 publication Critical patent/WO2002048350A1/fr

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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
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • C12N11/12—Cellulose or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention relates to a transformed yeast displaying a cellulose-binding domain on a cell surface, and to the transformed yeast immobilized on a cellulosic carrier. More specifically, the present invention relates to a transformed yeast, which is obtained by introducing a yeast expression vector containing a cellulose-binding domain of cellulase into yeast, expressing the cellulose-binding domain on the cell surface, and a cell mouth. The present invention relates to the transformed yeast immobilized on a raw carrier.
  • Immobilization of various chemical substances including proteins having biological activity is important for industry, and various immobilization methods have been developed in recent years.
  • most of these immobilization methods require chemical modification of the solid phase matrix. That is, these chemical modifications require the covalent attachment of the ligand to the matrix, often resulting in problems such as loss of ligand activity and the use of toxic organic compounds before the solid phase matrix can be used as a drug. Had to be removed.
  • Protein A-Sepharose which is used in the purification of IgG by affinity chromatography, and in many diagnostic applications.
  • a method using a chimeric protein that is, a protein having a functional domain (for example, a catalytic protein or the like) together with a binding domain is also known, which is very useful in a protein purification method.
  • the glutathione S-transferase gene fusion system is designed to express a gene of interest fused to the C-terminus of glutathione S-transferase.
  • the recombinant protein obtained by expressing this gene is glucanthione-sepharose It can be purified by affinity chromatography using a column.
  • the problem to be solved by the present invention is a novel method for immobilizing yeast using a cellulose-binding domain, that is, a cell-binding domain is presented on the cell surface by genetic engineering, and a cell is expressed by affinity between cellulose and the cellulose-binding domain. It is intended to provide a method for immobilizing to a cell-like carrier.
  • Another object of the present invention is to provide a yeast capable of expressing and displaying a cellulose binding domain on a cell surface.
  • Still another object of the present invention is to provide an immobilized yeast in which a yeast capable of expressing and displaying a cellulose binding domain on a cell surface is immobilized on a cellulosic carrier.
  • the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, by transforming a gene encoding a cellulose binding domain into yeast using a certain yeast expression vector, the cellulose binding domain was transformed into yeast. They have found that they can be expressed and presented on the cell surface of yeast, and have completed the present invention.
  • a transformed yeast displaying the cellulose-binding domain of cellulase on the cell surface.
  • the transformed yeast according to any one of (1) to (3) which is obtained by transforming a yeast expression vector having a DNA encoding a cellulose binding domain of cellulase into yeast. .
  • the yeast expression vector contains the promoter, secretory signal sequence, DNA encoding the cellulose binding domain, yeast-derived aglutinin gene or a fragment thereof in this order in the 5 'to 3' direction.
  • the transformed yeast according to (4) which is characterized in that:
  • cellulose binding domain is a cellulose binding domain of cellulase derived from Tricoderma reesei.
  • (C) an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 1 or 2, which has a cellulose binding activity.
  • FIG. 1 is a diagram showing the construction of a chromosome-integrated plasmid pICBD1 expressing the cellulose-binding domain (CBD1) of CBHI.
  • FIG. 2 is a diagram showing the construction of a multicopy plasmid pMCBD1 expressing the cellulose binding domain (CBD1) of CBHI.
  • FIG. 3 is a diagram showing the structure of a chromosome-integrated plasmid pICBD1 expressing the cellulose-binding domain (CBD1) of CBHI.
  • FIG. 4 is a diagram showing the structure of a chromosome-integrated plasmid PICBD2 expressing the cellulose-binding domain (CBD2) of CBHII.
  • FIG. 5 is a diagram showing the structure of the multicopy plasmid pMCBD1 expressing the cellulose binding domain (CBD1) of CBHI.
  • FIG. 6 is a diagram showing the structure of the multicopy plasmid pMCBD2 expressing the cellulose-binding domain (CBD2) of CBHII.
  • FIG. 3 is a diagram showing the structure of pICBD3.
  • the cellulose-binding domain of cellulase has an affinity for cellulose.
  • the present inventors succeeded in displaying a cellulose binding domain on the yeast cell surface for the first time.
  • By presenting the cellulose-bound domain on the surface of yeast cells it becomes possible to immobilize the yeast on a cell-like carrier, and to provide yeast cells that specifically adsorb to cellulose.
  • the yeast cell of the present invention can be applied to immobilization of a library, a self-propagating immobilized bioreactor, a biosensor, and the like.
  • the yeast of the present invention is a transformant obtained by transforming a yeast expression vector having DNA encoding a cellulose binding domain into yeast.
  • the yeast of the present invention can display the cellulose binding domain on the cell surface.
  • the expression vector used in the present invention contains a yeast-derived aglutinin gene or a fragment thereof.
  • yeast for obtaining the aglutinin gene include a group of microorganisms described as yeast in "Classification and Identification of Microorganisms", edited by Takeharu Hasegawa (Society Press, 1975).
  • preferred yeasts are those belonging to the genus Saccharomyces, and include, for example, Saccharomyces cerevisiae MT8-1.
  • the detailed properties of yeast, T are described in Taima, M. et al., Yeast 1, 67-77 (1985).
  • the agglutinin may be any of a-agglutinin and hiagglutinin.
  • the molecular structure of ⁇ -agglutinin has, in order from the ⁇ terminus, a secretory signal sequence, an agglutinin active region, a cell surface internal region, and a GPI anchor (glycosyl phosphatidylinositol) attachment signal region.
  • the fragment of the aglutinin gene region can express a heterologous protein on the yeast cell surface. (Including the cell surface internal region), but a particularly preferred example is a gene sequence encoding 320 amino acids from the C-terminus of Saccharomyces hiaglutinin and a 3 'untranslated sequence comprising 446 bases And a DNA fragment containing the region.
  • This DNA fragment contains the GPI anchor attachment signal region.
  • This DNA fragment can also be obtained by cutting out a plasmid pGA11 containing the fragment described in Murai, T. et al. Applied and Environmental Microbiology, 63, 1362-1366 (1997) with a restriction enzyme. .
  • the expression vector of the present invention containing the agglutinin gene or a fragment thereof and DNA encoding the cellulose binding domain can be obtained by introducing the gene into an expression vector usually used in yeast.
  • the expression vector include, for example, pI CAS 1 (Murai et al., Appl. Environ. Microbiol., 64, 4857-4861 (1998)), p CAS 1 (Murai et al., Appl. Environ. Microbiol., 64, 4857-4861 (1998)).
  • the expression vector used in the present invention usually contains a promoter and a secretory signal sequence (for example, a secretory signal sequence derived from Chinese cabbage).
  • the promoter is not particularly limited as long as it is a promoter generally used for expression in yeast, but a preferred example is glyceroaldehyde triphosphate dehydrogenase of Saccharomyces cerevisiae. Drogenase (GAPDH) Promote Overnight (Sawani-Hatanaka, H., T. et al., Biosci. Biotechnol.
  • a signal region usually used for expression and secretion in yeast for example, a secretory signal region of a glucoamylase gene derived from Rhizopus oryzae can be used.
  • the above promoter and secretion signal sequence can be prepared by known methods, for example, by using It can be obtained, for example, by subjecting the genomic DNA of the above microorganism to type I using an image and performing a PCR method.
  • DNA encoding a cellulose binding domain is used as DNA encoding a protein to be expressed and displayed on the cell surface.
  • the cellulose binding domain generally refers to the cellulose binding domain of cellulase.
  • CBD Cellulose binding domain
  • cellulose binding domains are classified into nine families (Family I-IX) based on their amino acid sequence homology (Tome, P. etal., Adv. Microbiol. Physiol., 37, 1-81 (1995)).
  • an appropriate one can be selected from these cellulose-binding domains according to the purpose of use in consideration of the affinity and selectivity of binding to cellulose, and the like.
  • CBH cellobiohydrolase
  • CBH II cellobiohydrolase
  • H. Okada et al. Appl. Microbiol. Biotechnol. (1998) 49,301.
  • CBH Cellobiohydrolase
  • SEQ ID NO: 1 and SEQ ID NO: 2 The amino acid sequence of the cellulose binding domain of biohydrolase (CBH) II is set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • a cellulose binding domain having the amino acid sequence of SEQ ID NO: 1 or 2 can be used, but its variant or homolog, that is, 1% in the amino acid sequence of SEQ ID NO: 1 or 2, An amino acid sequence in which several amino acids have been deleted, substituted, added and / or inserted from the amino acid sequence having a cellulose-binding activity; or the amino acid sequence described in SEQ ID NO: 1 or 2, An amino acid sequence having 0% or more homology and having a cellulose binding activity can also be used.
  • 1 to several amino acids are deleted, substituted, added and Z or inserted
  • amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 1 or 2 refers to 60% or more, preferably 7% or more, of the amino acid sequence of SEQ ID NO: 1 or 2. It means an amino acid sequence having 0% or more, more preferably 80% or more, further preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more homology.
  • DNA encoding the above mutant or homolog may be used.
  • DNAs encoding such mutants and homologues can be prepared by any method known to those skilled in the art, such as chemical synthesis, genetic engineering techniques, and mutagenesis. Specifically, a desired DNA can be obtained by using a DNA having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and introducing a mutation into these DNAs.
  • a DNA having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is brought into contact with a mutagenic drug.
  • the method can be performed using a method, a method of irradiating ultraviolet rays, a genetic engineering technique, or the like.
  • Site-directed mutagenesis which is one of the genetic engineering techniques, is useful because it is a technique that can introduce a specific mutation at a specific position, and is useful in Molecular Cloning 2nd Edition, Current, Protocols ⁇ Molecular ⁇ Biology, Nucleic Acids Research, 10, 6487, 1982, Nucleic Acids Research, 12, 9441, 1984, Nucleic Acids Research, 13, 4431, 1985, Nucleic Acids Research, 13, 8749, 1985, Proc. Natl. Acad. Sci. USA, 79, 6409, 1982, Proc. Natl. Acad. Sci. USA, 82, 488, 1985, Gene, 34, 315, 1985, Gene, 102, 67, 1991, etc. Can be done.
  • promoter In a preferred embodiment of the expression vector used in the present invention, promoter, secretory signal sequence, DNA encoding a cellulose binding domain, yeast-derived aglutinin gene or a fragment thereof (for example, DNA encoding a C-terminal fragment of hyaglutinin) Fragments) in this order in the 5 to 3 'direction.
  • Such an expression vector is introduced, for example, by introducing the promoter, secretory signal sequence, multicloning site and agglutinin gene or its fragment into the above-described plasmid vector in this order. After that, it can be constructed by introducing DNA encoding a cellulose binding domain into the multicloning site.
  • Conventional recombinant DNA technology can be used to construct such a recombinant expression vector.
  • the expression vector used in the present invention may contain a selection marker gene such as an antibiotic resistance gene.
  • the yeast to be transformed by the above-described recombinant vector is described as a yeast in, for example, "Classification and Identification of Microorganisms", edited by Takeharu Hasegawa (Society Press Center, 1975). Microbial groups.
  • the type of yeast is not particularly limited, and includes, for example, genus Sapalomyces, genus Schizosaccharomyces, genus Candida, genus Pichia, genus Hansenula, and the like.
  • Transformation of yeast with the expression vector can be performed by a conventional method, and a transformant can be obtained by, for example, a lithium acetate method, an electric pulse method, a protoplast method, or the like. Methods for culturing transformants are also known and described, for example, in "M. D. Rose et al. ,, Methods in Yeast Genetics", Cold Spring Harbor Laboratory Press (1990).
  • a method for immobilizing a transformed yeast on a cellulosic carrier which comprises contacting the transformed yeast produced by the above method with a cellulosic carrier.
  • the type and form of the cellulosic carrier used in the present invention are not particularly limited, and any one can be used as long as it can adsorb and immobilize the transformed yeast of the present invention.
  • the cellulosic carrier is preferably a porous material.
  • Preferred examples of the porous material include a cellulose porous material, a cellulose-chitin composite porous material, and a material obtained by adding various functional groups thereto (for example, a porcine acetylamino esterified cellulose material, a carboxymethylated material).
  • Such a porous material can be easily produced by a known method. For example, when obtaining a porous cellulose material, viscose and a foaming agent such as calcium carbonate are mixed, extruded from a nozzle, dropped into an acid bath in the form of droplets, and cellulose regeneration and foaming are performed simultaneously. Manufactured by At this time, by changing the aging conditions of the viscose and the conditions of the acid bath, several types of porous materials having different pore and pore structures can be obtained. Normally, the pores are radial, but depending on the state of foaming, they may be open cells.
  • the shape of the porous material having the skin layer produced by being ejected from the nozzle is generally spherical or flat spherical. Like this The close shape is very convenient for increasing the packing ratio of the porous material in the culture tank during culturing.
  • the diameter of the particles of the porous material can be any size, but usually:! 110 mm.
  • cellulosic carriers such as CellSnow TM (1 mm cubic manufactured by Kirin) and Avicel (manufactured by Huriki).
  • CellSnow TM has three types (cells with polyethyleneimine bound to cellulose; cells with collagen bound to cellulose; cell adhesion-active peptides) to improve cell adhesion. (A certain RGDS bonded to cellulose)).
  • the method of culturing yeast and the type of culture tank are appropriately selected in consideration of the type of yeast used, the type of porous material, and the like. Any of a batch type, a semi-batch type and a continuous culture type may be used.
  • the culture tank type any of a tower type fermentation tank (bubble tower type, spout tank type, draft tube type, etc.), a stirring tank and the like may be used, and any type can be used.
  • the culture conditions are not particularly limited, but generally the temperature is 25-45.
  • the culture is performed at pH 4 to 9 and culture time of about 1 to 14 days.
  • the culture may be performed under aerobic conditions or anaerobic conditions.
  • Useful substances industrially produced by yeast include yeast aerobic metabolic systems such as (1) organic acids, (2) vitamins, (3) proteins such as enzymes, and (4) hydroxy fatty acids. There are many things.
  • the useful substances as described above can be produced using the transformed yeast of the present invention and the immobilization method.
  • Example 1 Cellulosic biohydrolase (CBH) I or cellobiohydrolase (CBH) II from eukaryotic fungus Trkoderma reesei, or cellulose binding of both Construction of plasmid expressing domain
  • Plasmid pT7Blue containing the gene in C-dish I is transformed into type I, and the CBHI-CBD portion is amplified by PCR using primers 1 and 2 below.
  • CBD Sac 11 aataattccccgcgggaacccagtctcactacggccagtgcgg (sequence number 3)
  • CBD Xhol attattccgctcgagcccaggcactgagagtagtaagggttca (SEQ ID NO: 4)
  • This PCR product is digested with SacI I and Xhol.
  • the pICAS 1 digested with SacI I and Xhol and the CBHI-CBD fragment digested with SacIK Xhol are ligated.
  • the resulting plasmid is digested with Xhol.
  • DNA having the following linker sequences 9 and 10 is annealed, and the resulting linker fragment and the plasmid fragment obtained by digesting with Xhol are ligated.
  • Linker ⁇ ' tcgagcctccaccagaacctccgccagaaccgccacctga
  • Plasmid pKF containing the CBHII gene is transformed into type III, and the CBHII-CBD portion is amplified by PCR using the following primers 4 and 5. The following procedure was followed in the same manner as described above to finally construct a chromosome-integrated plasmid PICBD2 expressing the cellulose-binding domain (CBD2) of CBHII (Fig. 4).
  • CBD Sac 11 aataattccccgcgggacaagcttgctcaagcgtctggccaa (SEQ ID NO: 6)
  • the pICBD1 obtained above was digested with SacII and Xhol, separated into 230 bp and 6300 bp fragments by electrophoresis, the 23 O bp fragment was eluted, and pCASl digested with SacII and Xhol Then, a multicopy plasmid pMCBD1 expressing the cellulose binding domain (CBD1) of CBHI was constructed (FIG. 5).
  • CBD Xhol attattccgctcgagcccaggcactgagagtagtaagggttca (SEQ ID NO: 4)
  • CBD Bglll ggcggaagatctcctggcaccaccaccacccgccgcccagcc (system U number 5)
  • the BglII, Xhol digested fragment of pCASl and this fragment are ligated.
  • the plasmid obtained by this procedure is digested with SacII and Bglll.
  • the plasmid pKF containing the CBHI I gene is transformed into type III, and the DNA fragment amplified by PCR is digested with SacI I and Bglll using the following primers 4 and 6.
  • CBD Sac 11 aataattccccgcgggacaagcttgctcaagcgtctggccaa (SEQ ID NO: 6)
  • CBD Bgll l gcttgaagatctagcgccgggaagacactgggagtaatag (SEQ ID NO: 8)
  • This fragment is ligated with the plasmid fragment obtained by digestion with Sac11 and BglII.
  • This plasmid is digested with Xhol.
  • the DNAs having the linker sequences 9 and 10 (SEQ ID NOS: 11 and 12) are annealed, and the resulting linker fragment and the plasmid fragment obtained by digesting with Xhol are ligated.
  • the ligated brassmid is digested with Sacl I.
  • RGS His 5 ' gggaagaggatcccatcaccatcaccatcacggttctggctcaggaggtgc (SEQ ID NO: 9)
  • pICBD1 and pICBD2 were each linearized at bal and transformed into the yeast Saccharomyces cerevisiae by the lithium metal method. Integration into the genome was confirmed by PCR after isolating each genomic DNA.
  • CBD1 CBHI cellulose-binding domain
  • CBD2 CBHI I cellulose-binding domain
  • pMCBD1, pMCBD2 and pMCBD3 were directly transformed into yeast Saccharomyces cerevisiae by the metal lithium method.
  • Example 3 Confirmation of cell surface presentation by antibody staining
  • pICBD K A strain having pICBD 2, pMCBD 1, pMCBD 2, pMCBD 3, pICAS 1 (control), and pCAS 1 respectively is named as wisteria 1 ⁇ recite 2 ⁇ MCBD1 ⁇ MCBD2, MCBD3, ICAS1, and CAS1 strains.
  • RGS (ms) 4 antibody As the primary antibody, RGS (ms) 4 antibody, as the secondary antibody, was used the Alexa Fluor TM 488 goat anti-mouse IgG (H + L) conjugate antibody, in ICBD1, ICBD2, M hide ⁇ MCBD2, MCBD3 strain, Fluorescence was observed on the cell surface, but not in ICAS1 and CAS1 strains. From this, CBD was successfully displayed on the cell surface as intended.
  • Example 4 Evaluation of binding ability of yeast displaying CBD cell surface to cellulose substrate
  • the cells were collected the same amount of yeast.
  • the yeast was washed twice with 0.05 M KPB (pH 7.4) + 0.05 M NaCl Buffer, and 0.2 g of Avicel and the washed yeast were put into a spinner tube and made up to 10 ml. .
  • the yeast and Avicel were reacted while mixing at a constant speed using a Rotator. After the reaction, a spinner tube was set up and allowed to stand for 5 minutes. After 5 minutes 3 ml of the supernatant was sampled just below the meniscus, and 3 ml of the sampled (yeast not bound to Avicel) was collected. . It was measured.
  • Reaction conditions yeast culture time: 30 hours, Reaction temperature: 4 ° C, Reaction time: 12 hours Table 2
  • the yeast of the present invention can display the cellulose-binding domain of cellulase on the cell surface, and by using this, it has become possible to provide a novel method for immobilizing yeast.
  • the yeast of the present invention is used for an immobilized bioreactor for industrially producing useful substances such as (1) organic acids, (2) vitamins, (3) proteins such as enzymes, and (4) hydroxy fatty acids. It can be used for fixing a library, or applied to a biosensor.

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Abstract

L'invention concerne un nouveau procédé d'immobilisation d'une levure au moyen d'un domaine de liaison à la cellulose, soit, un procédé qui consiste à présenter un domaine de liaison à la cellulose sur la couche de surface cellulaire par une technique d'ingénierie génétique et à immobiliser des cellules sur un support cellulosique en profitant de l'affinité de la cellulose pour le domaine de liaison à la cellulose. L'utilisation de ce procédé permet de fournir une levure modifiée qui possède sur sa couche de surface cellulaire le domaine de liaison à la cellulose de la cellulase.
PCT/JP2001/007207 2000-12-12 2001-08-23 Levure modifiee possedant sur la couche de surface cellulaire un domaine de liaison a la cellulose WO2002048350A1 (fr)

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
WO2003106490A1 (fr) * 2002-06-14 2003-12-24 Norsk Hydro Asa Proteine hybride comprenant un domaine de liaison a la cellulose
US10081802B2 (en) 2013-07-29 2018-09-25 Danisco Us Inc. Variant Enzymes
US10167460B2 (en) 2013-07-29 2019-01-01 Danisco Us Inc Variant enzymes
US10479983B2 (en) 2013-07-29 2019-11-19 Danisco Us Inc Variant enzymes

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