• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
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  • 优先权
    • 专利摘要:
    The present invention is Bacillus sp. It relates to cellulase compositions obtainable from CBS 670.93. Preferred cellulase has an output molecular weight of about 50 kD, an isoelectric point of about 4 calculated and an optimal pH for CMC of about 7 at about 6-10 and 60 ° C at 40 ° C.
    公开号:KR19990008130A
    申请号:KR1019970707655
    申请日:1996-04-26
    公开日:1999-01-25
    发明作者:솔링엔 피터 반
    申请人:마가렛 에이. 혼；제넨코 인터내셔널, 아이엔씨.；
    IPC主号:
    专利说明:

    Alkaline Cellulase and Method of Making the Same
    Cellulase is an enzyme capable of hydrolyzing 1,4β-D-glucoside bonds in cellulose. Celluloid enzymes have traditionally been classified into subgroups of endoglucanases, exoglocanases, and β-glucosidases (Knowles, J et al. (1987), TIBTETH 5, 255-261) and have been found in many bacteria, yeasts. And it is known to be produced by fungi.
    Initial applications made for the use of celluloid enzymes are therefore known to be useful in cellulase compositions to remove dirt, ie in cleaning detergent compositions. For example, UK applications 2,075, 028, 2,095,275 and 2,094,826 illustrate improved cleaning performance when the detergent comprises cellulase. Additionally, British Patent No. 1,358,599 illustrates the use of cellulase in detergents to reduce the roughness of cotton-containing fabrics.
    Another property of cellulase useful in fabric processing is the ability to recover the fabric used by making the color sharper. For example, repeated washing of cotton-containing fabrics can cause a whitish tint to the fibers, which may be due to broken irregular fine fibers, sometimes called pill, caused by mechanical action. It is fed. This grayish hue can be discerned especially on the colored fibers. Thus, the ability of cellulase to remove irregular top layers of fibers to improve the overall appearance of the fibers was valuable.
    Despite the knowledge known in the art for many cellulase compositions having some or all of the above properties, for example, textile treatments, as components of detergent compositions, various properties useful for the treatment of pulp and paper and for the conversion of biomass. New cellulase with is constantly being desired. Applicants have discovered some cellulase useful in these known cellulase products and supplementing these properties.
    [Summary of invention]
    It is an object of the present invention to provide novel cellulase with advantageous properties for use in detergents, textile processing and pulp and paper production.
    Cellulase or derivatives thereof of the present invention are Bacillus sp. It may be derived from or obtained from CBS 670.93. CBS 670.93 has been deposited with accession number CBS 670.93 at Centraalbureau voor Schimmelcultures (CBS), at night, December 23, 1993. (CBS 670.93). The novel cellulase preferably comprises the amino acid sequence of FIG. 2 (SEQ ID NO: 2) or derivatives thereof having at least 89%, preferably at least 95% sequence identity thereto. The invention also relates to a novel cellulase comprising the amino acid sequence of FIG. 2 (SEQ ID NO: 2) or a derivative thereof having at least 92.5%, preferably at least 97% sequence similarity thereto.
    In a second embodiment there is provided a composition comprising DNA encoding the amino acid sequence of FIG. 2 (SEQ ID NO: 2) or a derivative thereof having at least 89%, preferably at least 95% sequence identity thereto. Alternatively, a composition is provided comprising DNA encoding the amino acid sequence of FIG. 2 (SEQ ID NO: 2) or a derivative thereof having at least 92.5%, preferably at least 97% sequence similarity thereto.
    In a third embodiment of the invention, there is provided a method for transforming a suitable microorganism having a DNA encoding the amino acid sequence of the invention. Alternatively, microorganisms transformed with the present invention DNA are provided.
    In a particularly preferred embodiment of the invention, the cellulase is Bacillus sp. With a calculated molecular weight of about 50 kD. Cellulase derived from CBS 670.93. The cellulase of about 50 kD has an optimum isoelectric point of about 4 and an optimal pH for CMC of about 6-10 at 60 ° C. and about 7 at 60 ° C.
    The present invention relates to novel cellulase compositions. The present invention also relates to novel cellulase compositions, preferably derived from Bacillus sp. The present invention is also known in the art as an additive in detergent compositions, advantageously added to the cellulase as an additive in the treatment of fibers containing cellulose, in the treatment of pulp and paper and in the treatment of starch for preparing high fructose corn-syrup or ethanol. To the use of the new cellulase in the prepared compositions.
    1 shows the pH profile activity of about 50 kD cellulase derived from CBS 670.93 at 40 ° C. and 60 ° C. FIG.
    FIG. 2 shows the DNA sequence (SEQ ID NO: 1) and the deduced amino acid sequence (SEQ ID NO: 2) for 50 kD cellulase derived from CBS 670.93 (where the leader peptide sequence is underlined and cleaved upon secretion to mature enzyme ).
    Derivatives are methods of adding one or more amino acids to one or both of the C- and M-terminus of the original protein, substituting one or more amino acids of one or more different sites in the original amino acid sequence, of one or both ends of the original protein. Derived from the original protein by a method of removing one or more amino acid sequences or by inserting one or more amino acids in one or more sites of the original amino acid sequence. The preparation of enzyme derivatives is preferably obtained by modifying the DNA sequence encoding the original protein and expressing the modified DNA sequence by transforming said DNA sequence into a suitable host. Derivatives of the invention comprise altered amino acid sequences compared to precursor enzyme amino acid sequences (e.g., wild type or other native enzymes of the present invention) and possess altered properties in some particular aspects while retaining the characteristic enzymatic properties of the precursor enzyme. Peptides. For example, a changed cellulase may have increased optimal pH and increased temperature resistance but retain its characteristic cellulosic activity. Derivatives also include amino acid residues that are chemically modified in the enzyme molecule.
    Cellulase can be obtained from Bacillus 670.93 if the cellulase has an amino acid sequence corresponding to the amino acid sequence of cellulase that can be obtained from organism Bacillus 670.93. Thus, cellases having the same amino acid sequence for 50kD cellulase of the invention derived from different Bacillus may be obtained from Bacillus 670.93.
    By host cell is meant a cell capable of acting as a host and expression carrier for the recombinant DNA vector of the invention. In a preferred embodiment of the invention, host cell means Bacillus cell.
    A DNA construct or DNA vector refers to a nucleotide sequence comprising one or more DNA fragments encoding a novel kind of cellulase or a cellulase derivative described above.
    In a preferred embodiment the cellulase can be obtained from Centraalbureau voor Schimmelcultures (CBS), Night, Netherlands, microbial deposit No. CBS 670.93 (described in PCT / EP94 / 04312) deposited under the Budapest Treaty on 23 December 1993. . As used herein, the deposited species will be referred to as CBS 670.93. In a more preferred embodiment, the cellulase of the invention is about 50 kD of cellulase (calculated based on the amino acid sequence of mature protein) derived from CBS 670.93 (herein referred to as 50 kD cellulase). A cellulase of about 50 kD has an optimal pH for the calculated p1 for a mature protein of about 4 and a CMC of about 7 at 60 ° C. and about 6-10 at 60 ° C.
    Genes encoding about 50 kD cellulase amino acid sequences were analyzed by comparing sequence data from various collections (GenBank, Swiss-Prot, EMBL, and PIR) using the CAOS / CAMM center of the University of Nizmegan, Holland. A database comparing cellulase encoded by a published or known cellulase gene sequence with cellulase encoded by the DNA sequence of the present invention was examined. Bacillus sp. A significant amount of amino acid identity was found in cellulase CelA of N-4 (Fukumori et al., J. Bacter., Vol 168, pp. 479-485 (1986)).
    About 50 kD cellulase is selected from Proc, Nat, Acad. Sci., Vol. 85, pp.2444-2448 showed 89% sequence identity and 92.5% sequence similarity to the closest cellulase sequence published when using the TFastA program as described by Pearson Lipman. The TFastA search program is available as sequencing software package version 6.0 (Wisconsin 53705, Madison, University of Wisconsin Biotechnology Center, Genetic Computer Group), therefore, the present invention provides the amino acid sequence of FIG. 2 (SEQ ID NO: 2) or 89 Cellulase having an amino acid sequence having at least%, preferably at least 97% sequence similarity. The invention also includes cellulase having an amino acid sequence having at least 92.5%, preferably at least 97% sequence similarity to the amino acid sequence of FIG. 2 (SEQ ID NO: 2).
    The invention also describes a method of making cellulase. In one embodiment, the cellulase is for example Bacillus sp. It may be prepared by culturing a suitable organism such as CBS 670.93. Such conditions preferably include those generally suggested for culturing Bacillus to maximize the production of cellulase and include the use of cellulose derived substrates in combination with the salts, ions and other well known ingredients required as an energy source. do. In general, the medium used to culture the cells may be any conventional medium suitable for growing bacteria. The cells may be cultured under aerobic conditions in a nutrient medium containing assimilable carbon and nitrogen and other essential nutrients. Suitable carbon sources are carbohydrates such as sucrose, glucose and starch, or carbohydrates containing substances such as cereal grains, malt, rice and bovine gums. The concentration of carbohydrates incorporated in the medium may vary, for example up to 25%, 1-5%, but usually 8-10% is appropriate (percent is calculated as glucose equivalent). The nitrogen source in the nutrient medium may be organic and / or inorganic. Suitable inorganic nitrogen sources are nitrate and ammonium salts. Among the organic nitrogen sources commonly used in the fermentation process for culturing bacteria are soy flour, cotton seed flour, peanut flour, casein, corn, corn liquor, yeast extract, urea and albumin. In addition, the nutritional medium should contain standard trace substances.
    Cellulase may be recovered from the medium by conventional procedures including separating cells from the medium by centrifugation or filtration. If necessary, the cells are disrupted, followed by precipitation of the components containing the protein of the filtrate or supernatant, for example using salts such as ammonium sulfate, followed by various chromatographic procedures such as exchange chromatography, affinity chromatography or similar techniques. Purification by means of procedures is recognized. It is preferable that the alkaline cellulase of the present invention is cultured under alkaline conditions using a medium containing an cellulose-based energy source.
    Cellulase of the invention is preferably prepared using genetic engineering techniques by transforming a suitable host cell with a gene encoding the cellulase and expressing it under conditions suitable for host cell growth and cellulase expression. As a first step, chromosomal DNA may be obtained from a donor bacterium strain by methods of Saito and Minura (Saito Minura, Biochem. Biophys. Acta., Vol. 72, pp. 619 (1963)) or similar methods. . Thus, the resulting chromosomal DNA is cleaved by a restriction enzyme to provide a DNA fragment containing an alkaline cellulase gene. For this purpose, any of the restriction enzymes may be used, provided that the gene region is not cleaved. However, alternatively, restriction enzymes that cleave genes using reduced enzyme concentrations or incubation times may only allow partial catabolism. Preferred restriction enzyme is Sau3A. Isolating the appropriate fragment (2-6 kb) from the resulting catabolism mixture, a DNA construct comprising a DNA fragment of about 1.9 kb encoding the appropriate host cell DNA construct (e.g., the inventive 50 kD cellulase ligated to the appropriate vector sequence). ) Can be used for transformation. The ligation mixture is then transformed into appropriate host cells.
    Genes that cancer the cellulase of the present invention is E. Coli host cells and λ-phage (expression) vectors can be used to replicate (otherwise, PCR replication using sympathetic primers designed on conserved regions may be used). Applicants have transformed the gene encoding the cellulase of the present invention. It has been found that active proteins are obtained by expression in collie. this. After the first replication step in Collie, the cellulase genes of the invention can be transferred to more preferred industrial expression hosts such as fibrous fungi such as Bacillus or Streptomyces, Aspergillus or Trichoderma or yeast such as Saccharomyces. The high degree of expression and secretion that can be obtained from these host organisms allows the cellulase to accumulate in the culture medium where the cellulase can be recovered later.
    The expression host cell preferably comprises Bacillus sp., More preferably Bacillus rickeniformis or Bacillus subtilis. In a particularly preferred embodiment, the transgenic host for the protease gene is removed so that the resulting cellulase is not proteolyzed in fermentation broth or concentrates thereof. Preferred general transformation and expression protocols for proteases removed from Bacillus strains are described in US Pat. No. 5,264,366 to Ferrari et al., Incorporated herein by reference. Also preferably, the transformed Bacillus host is fermented at a pH of about 6.9.
    Transformation and expression in Aspergillus are described, for example, in US Pat. No. 5,364,770 to Berka et al., Which is incorporated herein by reference. Preferred promoters are aprE promoters when the transforming host cell is Bacillus.
    About 50 kD of this cellulase derived from CBS 670.93 has been shown to be useful in buffer systems including glycine, ammonium acetate, borex and / or tris. Such cellulase has also been found to be activated on CMC by the presence of magnesium and inhibited by the presence of calcium. It has also been found that the ratio of calcium to magnesium of about 750: 250 ppm gives an active advantage.
    The cellulase compositions of the invention described above may be used in detergent compositions according to methods known in the art of using cellulase in detergents. The good activity of the present cellulase at alkaline pH makes the cellulase particularly useful in high pH detergents.
    The invention will be described in more detail in the following examples provided for illustrative purposes, which should not be construed as limiting the invention.
    Cellulase Separation and Screening from Alkaline Soil and Water Samples
    Two methods were applied to isolate cellulase-producing microorganisms from alkaline soil and water samples. In a first method, soil and water samples were suspended in 0.85% saline containing solution and used directly for carboxymethyl cellulose (CMC) -agar diffraction analysis for detection of cellulase producing colonies.
    In a second method, incubation in a liquid minimal medium or GAM-medium containing cellulase for 1-3 days at 40 ° C. enriched the strain containing cellulase in soil and water samples. For detection of cellulase producing colonies, cell cultures showing bacterial growth were assayed for cellulase activity using the CMC-agar diffraction assay. The CMC-agar diffraction assay and the procedure for enriching the strain consisted of 1% KNO 3 , 0.1% yeast extract (Difco), 0.1% KH 2 PO 4 , 0.02% MgSO 4 · 7H 2 O, 1% Na A preparation was used with a minimal medium of about pH 9.7 comprising 2 C0 3 , 4% NaC and 0.25% CMC (Sigma C-4888). Agar of 1.5% was added to solidify.
    One of two procedures was used for the CMC-agar diffraction analysis, depending on whether the colony or liquid fraction was tested. To test colonies, cell suspensions in 0.85% saline solution were plated in CMC-containing minimal medium.
    After incubation at 40 ° C. for 1-3 days, the plates were plated repeatedly and the mother plate was flooded with 0.1% Congo Red for 15 minutes. The plate was destained with 1M NaCl for 30 minutes. Strains showing wash zones around colonies were isolated as potential cellulase producing microorganisms. Liquid fractions were analyzed by pipetting 40 μl of enzyme solution or fermentation broth fraction into wells punched with a 5 mm minimum medium layer in a Petri dish. Cellulase activity was detected by Congo Red / NaCl treatment after incubation at 40 ° C. for 16 hours. The diameter of the wash zone is a measure of CMCase activity.
    Strains showing wash zones were selected for growth and isolation of cellulase using one of two screening methods. Colonies were fermented in 100 ml of 250 rpm incubator shaker (New Brunswick Scientific, NJ, USA) for 72 hours at 40 ° C. in 25 ml of GAM-medium in shake flasks. CMCase activity was measured at pH 9 and 40 ° C. to vary the presence of cellulase in fermentation broth. The complex medium (GAM) used for enzyme production was 0.5% peptone (Difco), 0.5% yeast extract (Difco), 1% glucose H 2 O, 0.1% KH 2 PO 4 , 0.02% MgSO 4 .7H 2 O , Na 2 CO 3 1%, NaCl 4%. The pH was adjusted to 9.5 with 4M HCl and then 1% CMC was added.
    Using the method described above, cellulase producing microorganisms, which are characterized by small straight rods, which sometimes occur in pairs and are mobilized, were isolated. The spore sac obviously swells and the terminal spores become elliptical. Colonies on GAM-agar appeared creamy white and had a dull (i.e. not shiny) appearance with irregular surfaces with fibrous edges. Based on 16S rRNA sequencing, this microorganism was classified into Bacillus. The organism is referred to herein as CBS 670.93 and has been deposited with the accession number to Centraalbureau voor Schimmelcultures, Chestnut, The Netherlands.
    Example 2
    DNA Isolation, Cellulase Transformation and Expression
    this. The pro-alkaline bacilli strain CBS 670.93 was selected as the donor strain for intracollisal expression replication. Biochem. Biophys. Chromosomal DNA was isolated by Acta., Vol. 72, pp. 619-629 (1963) described by Saito and Minura.
    Partially chromosomal DNA by restriction enzyme Sau3A using an enzyme solution that was continuously diluted at 37 ° C. for 1 hour using React Buffer (Gettersburg, Maryland, Gettysburg, USA) under recommended conditions Catabolized. The catalyzed DNA was fractionated by agarose gel electrophoresis and a suitable fraction (2-6 kb) was extracted from the gel using the QIAquick Gel Extraction Kit according to the protocol described by Chatsworth QIAGEN Inc., California, USA. Separated.
    Sau3A fragments of chromosomal DNA are used to construct a genomic gene library in a catalyzed CIAP treated ZAP expression vector, BamH1, according to a protocol described by the supplier (Stratagene Cloning Systems, Lazola, Calif., USA).
    PBK-CMV phagmids containing cloned DNA inserts were cut from the ZAP Express ™ vector and e. Coli strain XLOLR was transformed.
    Meth recombinant clones. Screened by agar diffraction described by Wood et al. In Enzym., Vol. 160, pp. 59-74 (1988). Strains showing wash zones around colonies are isolated. After 48 hours of fermentation in 4 * YEP-medium consisting of 4% yeast extract (Difco), 8% peptone (Difco), 0.2% lactose and 100 μg / ml of ampicillin, the CMCase activity of the isolated recombinant was measured. The recombinant protein is purified (Example 3) and the amino acid sequence (SEQ ID NO: 2) is measured.
    The plasmid DNA of the cellulase that produces the recombinant is isolated using the QIAprep Plasmid Kit according to the protocol described by the supplier (QIAGEN Inc.). The plasmid contained about 1.9 kb of chromosomal DNA insert. The oligonucleotide sequence of the 1933 bp fragment is measured using a series of degenerated oligonucleotides derived from the N-terminal amino acid sequence as primers to position the gene on the 1.9 kb insert. The 1933 bp fragment contains an open reading frame of 1422 bp from which 467 amino acid proteins can be inferred to include 26 amino acid leader sequences. The nucleotide sequence of the gene encoding the cellulase (SEQ ID NO: 1) and the deduced amino acid sequence of the isolated single cellulase (SEQ ID NO: 2) may then be measured and this is illustrated in FIG.
    Example 3
    Purification of Cellulase
    Cellulase producing clones obtained in Example 2 were grown on complex media (4 * YEP) consisting of 4% yeast extract (Difco), 8% peptone (Difco), 0.2% lactose, and 100 μl of ampicillin. The fermentation broth was separated from the culture by centrifugation (8000 rpm). Cellulase in the supernatant was precipitated with ammonium sulfate (65% saturated). The precipitate was dissolved in 25 mM phosphate buffer pH 7 + 5 mM EDTA until a conductivity of 7 mS / cm was obtained. This solution was applied to a Q-Sepharose FF (5 cm in diameter, 10 cm long) anion exchange column and the column was washed with 25 mM phosphate buffer pH 7 + 5 mM EDTA to an absorption of 0.2 AU. The column was subjected to a 0-0.5 M NaCl gradient in 25 mM phosphate pH 7 after about 80 followed by a 0.5-1 M NaCl gradient after 10 minutes. Elution took place on the first slope. After elution the column was washed with 1 M NaOH (upstream) and again equilibrated with 25 mM phosphate pH 7 + 5 mM EDTA. The cellulase obtained according to the elution profile had a purity of about 80% or less.
    Example 4
    Characteristics of the Cellulase of the Invention
    Cellulase activity was measured for CMC at various pH and temperature values to determine the pH / temperature profile of the inventive 50 kD cellulase.
    Solutions containing about 50 kD cellulase were combined and diluted in 10 mM phosphate buffer (pH 7) in buffer (pH was adjusted to 4, 5, 6, 7, 8, 9, or 10 using 4 M NaOH). And adjust the pH using a buffer containing a mixture of 100 ml 1 M phosphonic acid, 100 ml citric acid and 600 ml dilution water to 1 L with dilution water). The enzyme solution was diluted at pH 7 and 40 ° C. until the measurement was 0.05 U / ml. 0.5 ml buffer, 0.5 ml substrate (1% CMC) and 0.1 ml 10 mM phosphate buffer were mixed and each buffer system tested at the actual pH of the seedlings. The actual pHs for pH 4, 5, 6, 7, 8, 9 and 10 are 4.2, 5.2, 6.2, 7, 8, 8.7 and 9.9, respectively.
    This result is exemplified in FIG. 1 showing the good alkaline activity of the present cellulase. The slope of the scale curve depends on the pH of the enzyme substrate mixture because at each pH two glucose standards are taken (500 mg glucose H2) / 100 ml 10 and diluted 25-fold.
    Cellulase activity may be analyzed using the modified PAHBAH method (Lever M. Anal. Biochem. 1972, 47, 273-279 and Levwr M. Anal. Biochem. 1977, 81, 21-27) as follows. . pH / temperature profiles may be determined using fixed enzyme concentrations that fit the linear range of dose response profiles measured at pH 7 and 40 ° C. Such enzyme concentrations may be used for activity measurements under all other determined conditions. The test tube is filled with 250 μl of 2.5% CMC (CMC-low viscosity purchased from Sigma) and 250 μl of 50 kD cellulase fraction in 50 mM glycine buffer pH 9 and diluted with appropriate buffer. Incubate the test tubes in a water bath at 40 ° C. for 30 minutes and then in 1.5 ml of PAHBAH solution (1% PAHBAH in 100 ml 0.5 M NaOH) and bismuth solution (48.5 g bismuth nitrate, 28.2 g potassium sodium tartrate and 100 ml 12.0 g of NaOH). The mixture is heated at 70 ° C. for 10 minutes and then cooled on ice for 2 minutes. Absorption is measured at 410 nm. To eliminate background absorption of the enzyme sample, a control experiment is performed as follows: The tube containing the substrate is incubated as a test tube under the same conditions. Incubate 1.5 ml of PAHBAH before adding the enzyme preparation (in this order). One unit (U) is defined as the amount of enzyme that produces 1 μmol of glucose from a CMC equivalent measured as decreasing sugar per gram of product per minute.
    Sequence listing
    (1) General Information:
    (i) Applicant:
    (A) Name: Gist-brocades
    (B) Distance: Wateringseweg 1
    (C) city: Delft
    (E) Country: Netherlands
    (F) ZIP code: 2611 XT
    (ii) Title of the Invention: Novel Cellulase and Its Application
    (iii) SEQ ID NO: 2
    (iv) form of computer reading:
    (A) Medium form: floppy disk
    (B) Computer: IBM PC Compatibility
    (C) operating system: PC-DOS / MS-DOS
    (D) Software: PatentIn Release # 1.0. Version # 1.25 (EPO)
    (2) SEQ ID NO: 1
    (i) Sequence Properties:
    (A) Length: 1404 base pairs
    (B) Form: Hexane
    (C) chain: double chain
    (D) topology: linear
    (ii) Molecular form: DNA (genome)
    (iii) Hypothesis: None
    (iii) anti-sense: none
    (vi) original source
    (A) organism: Bacillus sp.
    (C) individual isolate: CBS 670.93
    (ix) Features:
    (A) name / key: sig-peptide
    (B) Location: 1..78
    (ix) Features:
    (A) name / key: mat-peptide
    (B) Location: 79..1404
    (D) Other information: /action=Endoglucanase/EC-Number=3.2.1.4/Product=BCE103 Cellulase
    (ix) Features:
    (A) Name / Key: CDS
    (B) Location: 1..1404
    (xi) Description of sequence # 1:
    (2) Information about sequence number 2:
    (i) Sequence Properties:
    (A) Length: 467 amino acids
    (B) form: amino acid
    (D) topology: linear
    (ii) Molecular form: protein
    (xi) description for sequence 2
    权利要求:
    Claims (16)
    [1" claim-type="Currently amended] Bacillus sp. Cellulase or derivative thereof derived from or obtainable from CBS 670.93.
    [2" claim-type="Currently amended] A composition comprising a cellulase comprising the amino acid sequence of SEQ ID NO: 1 or a derivative thereof having at least 89% sequence identity.
    [3" claim-type="Currently amended] The composition of claim 2, wherein said cellulase has at least 95% sequence identity to SEQ ID NO: 1.
    [4" claim-type="Currently amended] A composition comprising a cellulase comprising the amino acid sequence of SEQ ID NO: 1 or a derivative thereof having at least 92.5% sequence similarity.
    [5" claim-type="Currently amended] 6. The composition of claim 5, wherein said cellulase has at least 97% sequence similarity.
    [6" claim-type="Currently amended] The method of claim 1, wherein the cellulase is Bacillus sp. Composition obtained from CBS 670.93.
    [7" claim-type="Currently amended] A composition comprising DNA encoding the amino acid sequence of claim 2.
    [8" claim-type="Currently amended] A composition comprising DNA encoding the amino acid sequence of claim 3.
    [9" claim-type="Currently amended] An expression vector comprising the DNA composition of claim 7.
    [10" claim-type="Currently amended] An expression vector comprising the DNA composition of claim 8.
    [11" claim-type="Currently amended] A host cell transformed with the DNA composition of claim 7.
    [12" claim-type="Currently amended] A host cell transformed with the DNA composition of claim 8.
    [13" claim-type="Currently amended] (a) transforming a suitable microorganism with DNA encoding the amino acid sequence of claim 2 or 4;
    (b) preparing a fermentation broth containing the appropriate microorganisms under conditions suitable for expression of the DNA;
    (c) maintaining the fermentation broth for a while under the above conditions to express the desired amount of cellulase;
    (d) collecting the fermentation broth containing the cellulase
    Method for expressing a cellulase comprising.
    [14" claim-type="Currently amended] A detergent composition comprising the cellulase of claim 1, 2 or 4.
    [15" claim-type="Currently amended] A method of treating a fabric comprising contacting the fabric with the cellulase of claim 1.
    [16" claim-type="Currently amended] A method of treating pulp mainly of cellulose comprising contacting the pulp mainly of cellulose with the cellulase of claim 1, 2 or 4.
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    GR3035573T3|2001-06-29|
    EP0828840A2|1998-03-18|
    JPH11503902A|1999-04-06|
    DE69611524D1|2001-02-15|
    NZ306973A|1999-10-28|
    WO1996034108A2|1996-10-31|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1995-04-28|Priority to EP95201115.3
    1995-04-28|Priority to EP95201115A
    1996-04-26|Application filed by 마가렛 에이. 혼, 제넨코 인터내셔널, 아이엔씨.
    1999-01-25|Publication of KR19990008130A
    2004-07-19|Application granted
    2004-07-19|Publication of KR100423670B1
    2011-01-31|First worldwide family litigation filed
    优先权:
    申请号 | 申请日 | 专利标题
    EP95201115.3|1995-04-28|
    EP95201115A|EP0739982A1|1995-04-28|1995-04-28|Bacillus cellulase and its applications|
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