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WO2003056003A1 - Amelioration de la stabilite d'enzymes - Google Patents

Amelioration de la stabilite d'enzymes Download PDF

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Publication number
WO2003056003A1
WO2003056003A1 PCT/AU2002/001487 AU0201487W WO03056003A1 WO 2003056003 A1 WO2003056003 A1 WO 2003056003A1 AU 0201487 W AU0201487 W AU 0201487W WO 03056003 A1 WO03056003 A1 WO 03056003A1
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Prior art keywords
enzyme
amino acid
xylanase
positively
uncharged
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PCT/AU2002/001487
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English (en)
Inventor
Khawar Sohail Siddiqui
Ricardo Cavicchioli
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Unisearch Limited
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Priority to AU2002335938A priority Critical patent/AU2002335938A1/en
Publication of WO2003056003A1 publication Critical patent/WO2003056003A1/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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03001—Alkaline phosphatase (3.1.3.1)

Definitions

  • the invention relates to improvements in the stability of an enzyme by chemical modification.
  • the invention also provides a stabilised enzyme produced by said method.
  • xylan is a polymer of xylopyranose residues joined by ⁇ -1,4 linkages.
  • the ⁇ -1,4 linkages are hydrolysed by the enzyme xylanase to yield short-chain xylo-oligosaccharides of varying lengths.
  • Xylanases are used in many industrial processes, including paper manufacture, baking and in the treatment of feeds. In baking, xylanase may be added to the flour to impart flavour to the dough and to the bread itself. Addition of xylanase also results in increased loaf volume and better textural characteristics, such as break and shred quality and crumb quality.
  • xylanase In paper manufacture, xylanase is used in kraft pulp bleaching to improve the bleachability of the pulp by removing xylan which precipitates during the pulp process.
  • xylanase may be used to treat feeds containing cereals (e.g. barley, wheat, maize, rye or oats) or cereal by-products.
  • cereals e.g. barley, wheat, maize, rye or oats
  • xylanase improves the breakdown of plant cell walls which leads to better utilization of the plant nutrients by the animal. This leads to improved growth rate and feed conversion by the animal. Also, the viscosity of the feeds containing xylan can be reduced.
  • thermostability and enzyme activity at elevated temperature is the result of a combination of a number of features including hydrophobic interactions, compact packing, salt bridges, reduction of conformational strain, reduction of the entropy of unfolding, ⁇ -helix stabilization, hydrogen bonding, disulfide bridges, metal binding, surface loop stabilization and resistance to covalent degradation.
  • thermostable xylanases have focused on isolating naturally occurring xylanases from thermostable organisms (see, for example, US Pat. No. 6,083,733) on the theoretical basis that organisms which grow in high temperature environments will have evolved enzymes also adapted to function at high temperatures.
  • thermostable xylanases While such an approach has yielded thermostable xylanases, culturing of the organisms and isolation of the enzyme can be problematic.
  • thermostability to xylanases independent of the source of the xylanase would be of significant benefit, as the xylanase could be isolated from readily available sources.
  • the inventors have found that by linking an uncharged or positively-charged molecule to a side chain of an amino acid residue of a xylanase, the thermostability of the enzyme is improved.
  • the invention provides an enzyme capable of cleaving ⁇ -1, 4-xylosidic bonds of xylan which comprises a xylanase which has been modified by having an uncharged or positively-charged molecule linked to: (a) a side chain of an amino acid of the xylanase; and/or (b) to a carboxy-terminal amino acid of the xylanase.
  • the enzyme functions at an elevated temperature and/or has an extended half-life compared to the corresponding unmodified xylanase.
  • the enzyme optionally further comprises a further uncharged or positively-charged molecule linked to a side chain of an amino acid residue of the enzyme, and/or to a carboxy-terminal amino acid residue of the enzyme.
  • the uncharged or positively- charged molecule is linked to a carboxyl group of a side chain of an amino acid and/or to the carboxy terminal amino acid.
  • the linkage is via an amide bond.
  • the uncharged or positively-charged molecule is linked to an aspartate residue, a glutamate residue and/or a carboxy-terminal amino acid residue.
  • the uncharged or positively- charged molecule is linked to the side chain of a tyrosine residue.
  • the uncharged or positively-charged molecule is linked to a hydroxyl group of a side chain of a tyrosine residue.
  • the uncharged or positively-charged molecule may be any uncharged or positively-charged molecule that improves the capacity of the enzyme to cleave ⁇ -1, 4-xylosidic bonds of xylan at elevated temperature, and/or extends the half- life of the enzyme.
  • the uncharged or positively- charged molecule is an aromatic molecule.
  • the aromatic molecule is selected from the group consisting of aniline, 2 -guanidino-benzimidazole and a heterocyclic amine.
  • the heterocyclic amine is selected from the group consisting of cytosine, cytidine and pyridine.
  • the uncharged or positively- charged molecule is an amine-containing compound.
  • the amine- containing compound is an amine-containing carbohydrate such as glucosamine or chitosan.
  • the chitosan is oligo-chitosan.
  • the oligo-chitosan has a molecular weight of about 5000. More preferably the oligo-chitosan is 75%-85% deacetylated.
  • the amine-containing compound is an aliphatic amine-containing molecule, such as arginine methyl ester.
  • the amine-containing compound is cytidine or cytosine.
  • the enzyme may have the amino acid sequence of a naturally occurring xylanase.
  • the xylanase is produced by an organism selected from the group consisting of vertebrates, invertebrates, angiosperms, fungi, yeast, bacteria, archeae and algae.
  • the organism is a psychrophilic or a esophilic organism. More preferably, the organism is a fungus. Even more preferably the fungus is Aspergillus sp. or Tr ⁇ choderma sp. Most preferably the organism is Trichoderma 1 on gibrachi aturn.
  • the enzyme may have an amino acid sequence of a xylanase encoded by a recombinant nucleic acid molecule.
  • the recombinant nucleic acid molecule may be obtained from an organism selected from the group consisting of vertebrates, invertebrates, angiosperms, fungi, yeast, bacteria, archeae and algae.
  • the recombinant nucleic acid is obtained from a psychrophilic, a mesophilic or a thermophilic organism. More preferably, the organism is a f ngus. Even more preferably the fungus is Aspergillus sp.or Trichoderma sp.
  • the organism is Trichoderma longibrachi a turn .
  • the invention provides a process for producing an enzyme capable of cleaving ⁇ -1,4 - xylosidic bonds of xylan at elevated temperature and/or having an extended half-life, the process comprising the step of contacting an enzyme capable of cleaving ⁇ -1,4 - xylosidic bonds of xylan with a compound which comprises an uncharged or positively-charged molecule, in conditions sufficient for linking the uncharged or positively-charged molecule to a side chain of an amino acid residue of the enzyme, and/or to a carboxy-terminal amino acid residue of the enzyme .
  • the uncharged or positively-charged molecule is linked to a carboxyl group of a side chain of an amino acid, and/or to the carboxy terminal amino acid.
  • the uncharged or positively- charged molecule is linked to the side chain of an aspartate residue, a glutamate residue and/or to a carboxy terminal amino acid residue of the enzyme.
  • the compound which comprises an uncharged or positively-charged molecule is a nucleophile.
  • the nucleophile is selected from the group consisting of aromatic nucleophile, carbohydrate nucleophile, aliphatic amine nucleophile, heterocyclic amine nucleophile and cytidine nucleophile. In particularly preferred embodiments;
  • the aromatic nucleophile is aniline hydrochloride or 2 -guanidino-benzimidazole dihydrochloride
  • the heterocyclic nucleophile is selected from the group consisting of pyridine hydrochloride, cytidine and cytosine;
  • the carbohydrate nucleophile is glucosamine or chitosan.
  • the uncharged or positively-charged molecule is linked to the enzyme by activating carboxyl groups of the enzyme.
  • the carboxyl groups may be activated by any compound that provides sufficient conditions for an uncharged or positively-charged molecule to be linked to the side chain of an amino acid residue of the enzyme, and/or linked to the carboxy terminal amino residue acid of the enzyme.
  • carboxyl groups are activated by carbodiimide.
  • the carbodiimide is l-ethyl-3 (3-dimethylaminopropyl) carbodiimide or 1- (3-dimethylaminopropyl) -3 -ethyl carbodiimide ethiodide.
  • the process optionally also comprises the step of contacting the enzyme with an agent for controlling the linkage of the uncharged or positively-charged molecule to a side chain of an amino acid residue or a terminal amino acid residue located in a catalytic site of the enzyme.
  • the agent is an inhibitor of the enzyme .
  • the inhibitor is a short chain xylo- oligosaccharide .
  • the agent is a substrate of the enzyme.
  • the substrate may be any oligomer of ⁇ -1,4 linked xylopyranose residues.
  • the invention provides a product produced by the process of the second aspect of the invention.
  • the invention provides a composition comprising an enzyme according to the first or third aspects of the invention and a suitable carrier.
  • the invention provides a method of cleaving the ⁇ -1, 4-xylosidic bonds of xylan, comprising the step of exposing a compound or composition comprising xylan to an enzyme according to the invention.
  • a method of cleaving the ⁇ -1, 4-xylosidic bonds of xylan comprising the step of exposing a compound or composition comprising xylan to an enzyme according to the invention.
  • an enzyme includes a plurality of such enzymes
  • an amino acid is a reference to one or more amino acids.
  • the present invention relates to an enzyme which comprises a modified xylanase for cleaving the ⁇ -1,4- xylosidic bonds of xylan at elevated temperature and/or having an extended half-life.
  • a modified xylanase for cleaving the ⁇ -1,4- xylosidic bonds of xylan at elevated temperature and/or having an extended half-life.
  • uncharged or positively-charged molecules were linked to the side chains of amino acids of xylanase and/or to the carboxy-terminal amino acid residue and the resulting modified xylanase was then able to function at elevated temperature and/or have an extended half-life.
  • the expression "elevated temperature” refers to a temperature above that at which the corresponding unmodified xylanase not having an uncharged or positively- charged molecule linked to the side chain of an amino acid of the enzyme or to a carboxy-terminal amino acid exhibits maximum activity.
  • a xylanase not having an uncharged or positively-charged molecule linked to the side chain of an amino acid or to a carboxy terminal amino acid residue would lose activity at 55°C, while the same enzyme having an uncharged or positively-charged molecule linked to the side chain of an amino acid or to a carboxy terminal amino acid residue may retain activity, or will lose activity at 55°C at a slower rate.
  • corresponding unmodified xylanase refers to a xylanase having the same amino acid sequence as the enzyme but not having an uncharged or positively-charged molecule linked to the side chain of an amino acid or to a carboxy- terminal amino acid of the enzyme.
  • ⁇ -1,4-xylosidic bonds refers to the ⁇ -1,4 bonds formed between xylopyranose residues in a molecule of xylan.
  • the first step in preparing the enzyme of the invention involves selecting the xylanase to which the uncharged or positively-charged molecule is to be linked.
  • xylanases are classified according to their ability to cleave xylan. While xylanases may be isolated from different organisms and therefore have slightly different activities and/or properties, the overall classification is the same. In other words, a xylanase isolated from one organisms will have very similar properties to a xylanase isolated from a different organism.
  • the "unmodified” xylanase can be "wild- type", “naturally-occurring” or “recombinant” xylanase or variant thereof obtained from any suitable origin, such as vertebrate, invertebrate, angiosperm, fungus, yeast, prokaryotes including bacteria, archeaebacteria and eubacteria, or a mesophilic organism. Origin can further be psychrotolerant, psychrotrophic, mesophilic or extremophilic (psychrophilic, psychrotrophic, thermophilic, barophilic, alkalophilic, acidophilic, halophilic, etc.). Purified or non-purified forms of these xylanases may be used.
  • mutants of wild-type xylanase are mutants of wild-type xylanase. Mutants can be obtained eg. by protein and/or genetic engineering, chemical and/or physical modifications of wild-type xylanase. Common practice as well is the expression of the xylanase via host organisms in which the genetic material responsible for the production of the xylanase has been cloned. Examples of suitable sources of xylanase include Aspergillua sp., Trichoderma sp.. Bacillus sp., Microtetraspora sp., Scopulariopsia sp., Arachniotus sp. , Actinomodura sp., Cryptococcus albidis , Thermonospora fucsa, Butyrivibrio fibrisolvens ,
  • Lactobacillus plantarum Streptomyces sp . , Humicola, Coprrinuc, Thielavia , Myceliopthora, Fusarium,
  • Streptomyces, Scopuloropsis or Rhizopus sp. examples of particular species from which xylanases may be isolated include Humicola insolens, Coprinus cinereus, Fusarium oxysporum, Myceliophthora thermophila , Meripilus giganteus , Thielavia terrestris , Acremonium sp. ,
  • the organism from which the xylanase is isolated is a fungus.
  • the fungus is Aspergillus sp . , Scopulariopsis sp. or Trichoderma sp. Even more preferably the organism is Trichoderma longibrachia tum .
  • unmodified xylanase per se may be isolated de novo , or obtained through commercial means as described below, unmodified xylanase may also be obtained by recombinant means.
  • recombinant means have been used to obtain xylanases from Actinomadura sp. FC7 (Ethier, J. -F. et al., in: Industrial
  • Microorganisms Basic and Applied Molecular Genetics, R. Baltz et al . , eds, (Proc. 5th ASM Conf. Gen. Mol . Biol . Indust. Microorg., Oct. 11-15, 1992, Bloomington, nd., poster C25) ; bacteria (e.g. Ghangas, G. S. et al . , J. Bacteriol. 171:2963-2969 (1989); Lin, L. -L., Thomson, J. A., Mol. Gen. Genet. 228:55-61 (1991); Shareck, F. et al., Gene 107:75-82 (1991); Scheirlinck, T. et al . , Appl
  • amino acids sensitive to oxidation or amino acids that affect the surface charges are of interest.
  • the isoelectric point of such enzymes may also be modified by the substitution of some charged amino acids, eg. an increase in isoelectric point may help to improve compatibility with anionic surfactants.
  • the stability of the enzymes may be further enhanced by the creation of eg. additional salt bridges and enforcing metal binding sites to increase chelant stability.
  • amino acid refers to any of the naturally occurring amino acids, as well as optical isomers (enantiomers and diastereomers) , synthetic analogs and derivatives thereof.
  • ⁇ -Amino acids comprise a carbon atom to which is bonded an amino group, a carboxyl group, a hydrogen atom, and a distinctive group referred to as a "side chain.”
  • ⁇ -Amino acids also comprise a carbon atom to which is bonded an amino group, a carboxyl group, and two distinctive groups (which can be the same group or can be different groups) , in which case the amino acid has two side chains.
  • side chains of naturally occurring amino acids include, for example, hydrogen (eg., as in glycine) , alkyl (eg., as in alanine, valine, leucine, isoleucine) , substituted alkyl (eg., as in threonine, serine, methionine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, and lysine) , arylalkyl (eg., as in phenylalanine) , substituted arylalkyl (eg., as in tyrosine), selenocysteine, pyrolysine and heteroarylalkyl (eg., as in histidine and tryptophan) .
  • hydrogen eg., as in glycine
  • alkyl eg., as in alanine, valine, leucine, isoleucine
  • substituted alkyl eg.
  • amino acid also includes ⁇ - ⁇ -, ⁇ -, and ⁇ -amino acids, and the like, and ⁇ -imino acids such as proline.
  • amino acids includes proline.
  • Non-naturally occurring amino acids are also known in the art, as set forth in, for example, Williams (ed.), “Synthesis of Optically Active ⁇ -Amino Acids", Pergamon Press, 1989; Evans et al . (1990) J. Amer. Chem.
  • mutants or variants of the unmodified xylanase encompassed in the present invention may be prepared by introducing appropriate nucleotide changes into the DNA or cDNA of the unmodified xylanase and thereafter expressing the resulting modified DNA or cDNA in a host cell, or by in vi tro synthesis.
  • Such mutant and/or variants include, for example, deletions from, or insertions or substitutions of, amino acid residues within the amino acid sequence of the unmodified xylanase. Any combination of deletion, insertion, and substitution may be made to arrive at an amino acid sequence variant of the unmodified xylanase, provided that such variant possesses the desired characteristics described herein.
  • nucleotide sequence of nucleic acid molecules which encode xylanase that would be particularly useful in the present invention are part of the public domain. Nucleic acid molecules which encode xylanase may be found, for example, in the Genbank database
  • the nucleic acid sequence of ⁇ -xylosidase from Aspergillus niger can be found at accession number AF108944; Bacillus pumilus xylan 1, 4-beta-xylosidase can be found at accession number AF107211; Trichoderma viride mRNA for endo-1, 4-beta- xylanase has accession number AJ012718.1.
  • International patent application WO01/49859 also discloses a number of nucleic acid sequences for xylanase.
  • amino acid sequence variants of the unmodified xylanase There are two principal variables in the construction of amino acid sequence variants of the unmodified xylanase: the location of the mutation site and the nature of the mutation. These are variants from the amino acid sequence of the unmodified xylanase, and may represent naturally occurring allelic forms of the unmodified xylanase, or predetermined mutant forms of the unmodified xylanase made by mutating the unmodified xylanase DNA, either to arrive at an allele or a variant not found in nature. In general, the location and nature of the mutation chosen will depend upon the unmodified xylanase characteristic to be modified.
  • mutations can be made in the unmodified xylanase nucleotide sequence without affecting the amino acid sequence of the unmodified xylanase encoded thereby.
  • Other mutations can be made that will result in the unmodified xylanase having an amino acid sequence that is very different, but which is functionally active.
  • Such functionally active amino acid sequence variants of the unmodified xylanase are selected, for example, by substituting one or more amino acid residues with other amino acid residues of a similar or different polarity or charge .
  • Insertional, deletional, and substitutional changes in the amino acid sequence of the unmodified enzyme may be made to improve the stability of the unmodified enzyme before it is used in the present invention.
  • trypsin or other protease cleavage sites are identified by inspection of the encoded amino acid sequence for an arginyl or lysinyl residue. These are rendered inactive to protease by substituting the residue with another residue, preferably a basic residue such as glutamine or a hydrophobic residue such as serine; by deleting the residue; or by inserting a prolyl residue immediately after the residue.
  • any cysteine residues not involved in maintaining the proper conformation of the unmodified xylanase for functional activity may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking .
  • Cysteinyl residues most commonly are reacted with ⁇ - haloacetates (and corresponding amines) , such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives.
  • Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, ⁇ -bromo- ⁇ - (5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro- 2-pyridyl disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-l, 3-diazole.
  • Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
  • Para- bro ophenacyl bromide also is useful; the reaction is preferably performed in 0.1M sodium cacodylate at pH 6.0. Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides . Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
  • Suitable reagents for derivatizing ⁇ -amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridcxal; chloroborohydride; trinitrobenzenesulfonic acid; 0- methylisourea; 2, 4-pentanedione; and transaminase- catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal , 2 , 3-butanedione, 1, 2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK a of the guanidine functional group.
  • these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.
  • “Plasmids” are DNA molecules that are capable of replicating within a host cell, either extrachromosomally or as part of the host cell chromosome (s) , and are designated by a lower case “p” preceded and/or followed by capital letters and/or numbers .
  • Suitable vectors containing the nucleotide sequence encoding the mutant, variant or wild- type xylanase of interest and appropriate control sequences employs standard recombinant DNA methods. DNA is cleaved into fragments, tailored, and ligated together in the form desired to generate the vectors required. Normally it is desirable to add a signal sequence which provides for secretion of the xylanase.
  • Typical examples of useful genes are: 1) Signal sequence-- (pro-peptide) - - carbohydrate-binding domain- -linker- - xylanase sequence of interest, or 2) Signal sequence-- (pro-peptide)-- xylanase sequence of interest- -linker-- carbohydrate-binding domain, in which the pro-peptide sequence normally contains 5-100, eg. 5-25, amino acid residues.
  • Preparation of plasmids or vectors capable of expressing enzymes having the amino acid sequences derived from fragments of more than one polypeptide is well known in the art (see, for example, WO 90/00609 and WO 95/16782) .
  • the DNA of the xylanase of interest may be included within a replication system for episomal maintenance in an appropriate cellular host or may be provided without a replication system, where it may become integrated into the host genome.
  • the DNA may be introduced) into the host in accordance with known techniques such as transformation, transfection, microinjection or the like.
  • Host cells that are transformed or transfected with the above-described plasmids and expression vectors are cultured in conventional nutrient media modified as is appropriate for inducing promoters or selecting for drug resistance or some other selectable marker or phenotype.
  • the culture conditions such as temperature, pH, and the like, suitably are those previously used for culturing the host cell used for cloning or expression, as the case may be, and will be apparent to those skilled in the art.
  • Suitable host cells for cloning or expressing the vectors herein are prokaryotes, yeasts, and higher eukaryotes, including insect, vertebrate, and mammalian host cells.
  • Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, for example, E. coli , Bacillus species such as B . subtilis , Pseudomonas species such as P. aeruginosa , Salmonella typhimurium, or Serratia marcescens .
  • eukaryotic microbes such as filamentous fungi or yeast are suitable hosts for enzyme-encoding vectors. Saccharomyces cerevisiae , or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
  • Suitable host cells for the expression of mutant, variant or wild-type enzymes are also derived from multicellular organisms. Such host cells are capable of complex processing and glycosylation activities. In principle, any higher eukaryotic cell culture is useable, whether from vertebrate or invertebrate culture.
  • invertebrate cells include insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar) , Aedes aegypti (mosquito) , Aedes albopictus (mosquito) , Drosophila melanogaster (fruitfly) , and Bombyx mori host cells have been identified. Luckow, et al . , Bio/Technology 6:47-55 (1988); Miller, et al . , in Genetic Engineering, vol. 8, pp.277-279 (Plenum Publishing, 1986); Maeda, et al . , Nature 315:592-594 (1985).
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can be utilized as hosts.
  • plant cells are transfected by incubation with certain strains of the bacterium Agro acterium tumefaciens , which has been previously altered to contain mutant, variant or wild-type enzyme DNA.
  • Agro acterium tumefaciens the DNA encoding the mutant, variant or wild-type enzyme is transferred into cells, such that they become transfected, and will, under appropriate conditions, express the mutant, variant or wild-type enzyme.
  • regulatory and signal sequences compatible with plant cells are available, such as the nopaline synthase promoter and polyadenylation signal sequences, and the ribulose biphosphate carboxylase promoter.
  • Depicker et al . , J. Mol. Appl. Gen. 1:561-573 (1982).
  • Herrera- Estrella et al . , Nature 310:115-120 (1984).
  • D ⁇ A segments isolated from the upstream region of the T- D ⁇ A 780 gene are capable of activating or increasing transcription levels of plant-expressible genes in recombinant D ⁇ A-containing plant tissue.
  • European Pat. Pub. No. EP 321,196 published June 21, 1989).
  • monkey kidney cells (CV1, ATCC CCL 70) ; African green monkey kidney cells (VERO-76, ATCC CRL-1587) ; human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51) ; TRI cells (Mather, et al . , Annals N. Y. Acad. Sci . 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2) .
  • the host may be grown to express the xylanase.
  • One particularly preferred system of expression useful in this invention involves fermentation in which the mutant, variant or wild-type xylanase of interest is introduced into a bacterial or yeast host as described above and then cultured in the presence of nutrient media containing suitable carbon and nitrogen sources and inorganic salts, using procedures known in the art such as that described in Bennett, J.W. and LaSure(Eds.) "More Gene Manipulations in Fungi", Academic Press, CA, (1991) .
  • fermentation refers to any growth condition which results in production of an enzyme by an organism(s) .
  • fermentation can refer to small or large scale fermentation and includes, for example, shake- flask cultivation, continuous, batch, fed-batch and solid state fermentation in laboratory or industrial fermenters.
  • the mutant, variant or wild- type xylanase may be isolated by any method that is suitable for isolating active xylanase from organisms and/or growth media. Suitable methods known in the art include, for example, centrifugation, filtration, spray drying, evaporation, precipitation, ion exchange chromatography, gel filtration chromatography, hydrophobic- interaction chromatography (HIC) , affinity chromatography or the like, and combinations thereof.
  • An example of an isolation method is as follows: fermentation broth is separated from the culture medium by centrifugation at 8000rpm. Xylanase is precipitated from the supernatant using a 65% saturated solution of ammonium sulphate.
  • the precipitate is subsequently dissolved in 25mM phosphate buffer pH 7, 5mM EDTA.
  • the solution is then applied to a Q-Sepharose FF (diameter 5cm, length 10cm) Anion Exchange column.
  • the column is subsequently washed with 25mM phosphate buffer pH 7, 5mM EDTA until an absorbancy of 0.2 Absorbance Units at 280nm is attained.
  • a gradient of 0 to 0.5M NaCl in 25mM phosphate buffer pH 7, 5mM EDTA is applied to the column in 80 minutes followed by a gradient from 0.5 to 1M NaCl in 10 minutes. Elution may be performed in the first gradient.
  • the xylanase is an acidic xylanase, or in other words, a xylanase that is capable of cleaving the ⁇ - 1, 4-glycosidic bonds of xylan at a pH of between 3.0 and 6.5.
  • the xylanase for use in the method of the invention may be a single isolated xylanase or a mixture of xylanases from the same or different sources.
  • xylanases are commercially available such as Pulpzyme HB (Novo Nordisk) .
  • the xylanase is used as a single isolated xylanase.
  • the xylanase may represent part of a mixture of enzymes or other compounds.
  • the xylanase may be used in a crude form with contaminating compounds including other enzymes and proteins.
  • the xylanase may not be the only enzyme to which an uncharged or positively-charged molecule is linked; however, the resulting mixture will retain the ability to cleave xylan at elevated temperature because of the presence in the mixture of xylanase having an uncharged or positively-charged molecule linked to a side chain of an amino acid residue of the enzyme or to a carboxy-terminal amino acid residue of the enzyme.
  • an uncharged or positively-charged molecule is contacted with the amino acid side chain.
  • the term "contacted” refers to sufficient contact between the amino acid side chain and the uncharged or positively-charged molecule which permits the uncharged or positively-charged molecule to be linked to the amino acid side chain in conditions sufficient for linking the uncharged or positively-charged molecule to an amino acid side chain and/or a carboxy-terminal amino acid of the enzyme.
  • the term "uncharged or positively-charged molecule” means any compound that is uncharged or positively-charged and which improves the capacity of the enzyme to cleave ⁇ -1, 4-xylosidic bonds at elevated temperature and/or extends the half-life of the enzyme.
  • the uncharged or positively-charged molecule may be an aromatic molecule such as aniline or 2- guanidino-benzimidazole, a heterocyclic amine molecule such as cytosine, cytidine or pyridine, an amine- containing carbohydrate such as glucosamine or chitosan, or an aliphatic amine-containing molecule such as arginine methyl ester.
  • linked refers to any linkage formed between a portion of the amino acid side chain and the uncharged or positively-charged molecule. It will be appreciated by those skilled in the art that following linkage of the uncharged or positively-charged molecule to the amino acid side chain, the amino acid side chain to which the uncharged or positively-charged molecule is linked will be altered and will differ from the amino acid side chains common to many proteins owing to the presence of the uncharged or positively-charged molecule linked to the side chain of the amino acid.
  • amino acid side chains “common to many proteins” will be understood by those skilled in the art to mean the side chains belonging to the amino acids alanine, asparagine, aspartate, arginine, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, lysine, leucine, methionine, phenylalanine, proline, serine, tyrosine, tryptophan, threonine and valine.
  • the uncharged or positively-charged molecule may be linked to the amino acid side chain in any manner. In one embodiment, the uncharged or positively- charged molecule is linked to the amino acid side chain through one or more nitrogen atoms.
  • the uncharged or positively-charged molecule is linked to the amino acid side through an amide bond.
  • the uncharged or positively-charged molecule may be linked to the amino acid side chain through a linker.
  • a "linker" is a molecule which is not part of the uncharged or positively-charged molecule nor part of the amino acid side chain, but serves to link the uncharged or positively-charged molecule to the side chain of the amino acid.
  • the "conditions sufficient" for linking the uncharged or positively-charged molecule to a side chain of an amino acid residue or a terminal amino acid residue may be any conditions which allow a reaction to occur between the amino acid side chain and the uncharged or positively- charged molecule which results in linkage of the uncharged or positively-charged molecule to the amino acid side chain.
  • the conditions sufficient for linking the uncharged or positively charged molecule to a side chain of an amino acid residue and/or a terminal amino acid residue comprise activating the carboxyl groups of the xylanase at a temperature preferably between 18°C and 50°C, more preferably between 20°C and 40°C, even more preferably between 20°C and 28°C, and a pH preferably between 3.0 and 7.0, more preferably between 4.5 and pH7.0, and contacting the activated carboxyl groups with an uncharged or positively-charged molecule containing nucleophile.
  • the carboxyl groups are on the side chains of aspartate and/or glutamate residues and/or on the carboxy-terminal amino acid.
  • the term "activated" refers to a modification of an existing functional group to generate or introduce a new reactive functional group from the prior existing functional group, wherein the new reactive functional group is capable of undergoing reaction with another functional group to form a covalent bond.
  • a component containing carboxylic acid (-COOH) groups can be activated by reaction with N-hydroxy- succinimide or N-hydroxysulfosuccinimide using known procedures, to form an activated carboxylate (which is a reactive electrophilic group), ie., an N- hydroxysuccinimide ester or an N-hydroxysulfosuccinimide ester, respectively.
  • Activation of carboxylic acids may be accomplished in a variety of ways and by using a number of different reagents as described in Larock, "Comprehensive Organic Transformations", VCH Publishers, New York, 1989, all of which are incorporated herein by reference. However, activation often involves reaction with a suitable hydroxyl-containing compound in the presence of a dehydrating agent such as dicyclohexylcarbodiimide (DCC) or dicyclohexylurea (DHU) .
  • a dehydrating agent such as dicyclohexylcarbodiimide (DCC) or dicyclohexylurea (DHU) .
  • a carboxylic acid can be reacted with an alkoxy-substituted N-hydroxy- succinimide or N-hydroxysulfosuccinimide in the presence of DCC to form reactive electrophilic groups, the N- hydroxysuccinimide ester and the N-hydroxysulfosuccinimide ester, respectively.
  • Carboxylic acids may also be activated by reaction with an acyl halide such as an acyl chloride (eg., acetyl chloride), again using known procedures, to provide an activated electrophilic group in the form of a reactive anhydride group.
  • a carboxylic acid may be converted to an acid chloride group using, eg., thionyl chloride or an acyl chloride capable of an exchange reaction.
  • thionyl chloride or an acyl chloride capable of an exchange reaction Specific reagents and procedures used to carry out such activation reactions will be known to those of ordinary skill in the art and are described in the pertinent texts and literature.
  • activated carboxyl groups means the rendering of one or more the carboxyl groups of the side chains of an amino acid of an enzyme reactive with a nucleophile.
  • nucleophile and “nucleophilic” refer to a functional group that is electron rich, has an unshared pair of electrons acting as a reactive site, and reacts with a positively charged or electron-deficient site, generally present on another molecule.
  • Electrophilic groups herein are positively charged or electron-deficient, typically electron-deficient .
  • carboxyl groups of the enzyme are activated by incubating the enzyme with a carbodiimide, preferably utilising the carbodiimide condensation method described by Sheehan and Hess, and Khorana [Sheehan and Hess, J. Am. Chem. Soc. 77:1067, 1955; Khorana, Chem.Ind. 1087, 1995].
  • carboxyl groups of the xylanase are activated by incubating the enzyme with Woodwards reagent.
  • the difference between carbodiimide and Woodwards Reagent activation of carboxyl group is that in case of carbodiimide the carboxyl group must be protonated (COOH) , whereas in case of Woodwards reagent the carboxyl group may be ionised (COO " ) .
  • COOH protonated
  • Woodwards reagent the carboxyl group may be ionised (COO " ) .
  • Some enzymes are precipitated at low pH, therefore for these enzymes the Woodward chemistry is better.
  • the reaction is a condensation of the carboxyl with a substituted carbodiimide to form an active O-acylourea intermediate.
  • the carbodiimide may be, for example, l-ethyl-3 (3-dimethylaminopropyl) carbodiimide or 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide methiodide.
  • Methods for the use of carbodiimide in the activation of carboxyl groups are provided in, for example, Carraway, K.L. and Koshland, D.E. Jr, Carbodiimide modification of proteins. In: Methods in Enzy ology (Hirs, C.H.W.
  • uncharged or positively-charged molecule containing nucleophile refers to any nucleophile comprising an uncharged or positively-charged molecule.
  • Uncharged or positively-charged molecule containing nucleophiles may include, for example, aniline hydrochloride, pyridine hydrochloride, cytosine, glucosamine, chitosan, cytidine, arginine methyl ester dihydrochloride .
  • the carboxyl groups of the amino acid may be activated with carbodiimide prior to adding the uncharged or positively-charged molecule containing nucleophile to the reaction.
  • the carboxyl groups of the amino acid side chains are activated with carbodiimide in the presence of the uncharged or positively-charged molecule containing nucleophile.
  • the nucleophile is dissolved in an appropriate buffer such as, for example, K 2 HP0 4 /KH 2 P0 4 buffer at a pH of preferably between 3.0 and 7.0, more preferably between 4.0 and 6.0.
  • the buffer may optionally contain a xylanase inhibitor.
  • Suitable inhibitors may be xylobiose, xylotetriose, xylotriose, xylopentiose, or any other substrate of xylanase which is capable of protecting the active site of xylanase from linkage of an uncharged or positively- charged molecule to the active site residues.
  • Xylanase is added to the solution either as a dried preparation or as a solution.
  • the reaction is initiated by the addition of carbodiimide to a final concentration of preferably between 30mM and 200mM, more preferably between 40mM and l O OmM .
  • the enzyme is further purified using techniques known in the art such as, for example, dialysis, centrifugation, filtration, spray drying, evaporation, precipitation, ion exchange chromatography, gel filtration chromatography, hydrophobic- interaction chromatography, affinity chromatography or the like, or combinations thereof.
  • the modified xylanase comprises:
  • a pyridine molecule linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid of the enzyme (b) the amino acid sequence of a xylanase having a pyridine molecule linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid of the enzyme; (c) an aniline molecule linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid of the enzyme;
  • cytosine molecule linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid of the enzyme
  • an amino acid sequence of a xylanase having a cytosine molecule linked to the side chain of an aspartate. residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid of the enzyme (i) a glucosamine molecule linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid of the enzyme;
  • a 2-guanidino-benzimidazole molecule linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid of the enzyme (1) an amino acid sequence of a xylanase having a 2- guanidino-benzimidazole molecule linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid of the enzyme; (m) a arginine methyl ester molecule linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid of the enzyme; (n) an amino acid sequence of a xylanase having an arginine methyl ester molecule linked to the side chain of an aspartate residue, a glutamate residue, a tyrosine residue and/or to a carboxy terminal amino acid
  • enzymes comprising two or more different uncharged or positively-charged molecules linked to side chains of amino acids of the enzyme.
  • the uncharged or positively-charged molecule may be linked, for example, by incubating the enzyme with carbodiimide in the presence of two or more nucleophiles, each of the nucleophiles comprising a different uncharged or positively-charged molecule.
  • one type of uncharged or positively-charged molecule may be linked to the amino acid side chain of the enzyme, and a second type subsequently linked to a side chain of an amino acid of the enzyme.
  • the modified xylanase may be used directly after the uncharged or positively-charged molecule has been linked to the xylanase
  • the modified xylanase is purified using a conventional enzyme purification method.
  • the modified xylanase of the present invention may be purified by salting out with ammonium sulfate or other salts, gel filtration, dialysis, ion exchange chromatography, hydrophobic chromatography, crystallization, or by using a solvent such as acetone or an alcohol or the like. All of these methods are disclosed in well known literature such as Inman, "Methods in Enzymology", Vol.
  • uncharged or positively-charged molecules have been linked to the side chain of an amino acid residue and/or terminal amino acid of the enzyme
  • assays well known in the art may be employed.
  • the linking of uncharged or positively-charged molecules may be readily "observed” using techniques such as, for example, structures from X- ray crystallographic techniques, NMR techniques, de novo modelling, homology modelling, PAGE, amino acid analysis, et cetera.
  • compositions comprising the modified xylanase of the present invention.
  • the composition may comprise multiple enzymatic activities, such as an aminopeptidase, an amylase, a carbohydrase, a carboxypeptidase, a catalase, a chitinase, a cutinase, a deoxyribonuclease, an esterase, an ⁇ -galactosidase, a ⁇ -galactosidase, an ⁇ -glucosidase, a ⁇ -glucosidase, a haloperoxidase, an invertase, a laccase, a lipase, a mannosidase, a mutanase, an oxidase, a pectinolytic enzyme, a peroxidase, a phytase, a polyphenoloxidase, a proteolytic enzyme,
  • the composition may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition.
  • the composition may be in the form of a granulate or a microgranulate.
  • the additional enzymes to be included in the composition may be stabilized in accordance with methods known in the art. For example, see U.S. Pat. No. 4,238,345 issued Dec. 9,
  • the dosage of the enzyme composition of the invention and other conditions under which the composition is used may be determined on the basis of methods known in the art depending upon the application.
  • modified xylanase according to the present invention and compositions comprising the enzyme may be applied in industrial processes.
  • the modified xylanase of the present invention can be formulated into powdered or liquid detergents.
  • These detergent cleaning compositions or additives can also include other enzymes, such as known proteases, xylanases, cellulases, lipases, or endoglycosidases, as well as builders and stabilizers.
  • the modified xylanase of the present invention is useful in formulating various detergent compositions.
  • a number of known compounds are suitable surfactants useful in compositions comprising the modified xylanase of the present invention. These include non-ionic, anionic, cationic, anionic, or zwitterionic detergents, as disclosed in U.S. Pat. No. 4,404,128 to Anderson and U.S. Pat. No. 4,261,868 to Flora et al . , which are hereby incorporated by reference.
  • a suitable detergent formulation is that described in Example 7 of U.S. Pat. No. 5,204,015 to Caldwell et al . , which is hereby incorporated by reference.
  • the modified xylanase of the present invention may be used for any purpose that native or wild-type xylanases are used.
  • the modified xylanase can be used, for example, in bar or liquid soap applications, dish-care formulations, contact lens cleaning solutions or products, peptide synthesis, feed applications such as feed additives or preparation of feed additives, waste treatment, textile applications such as the treatment of fabrics, and as fusion-cleavage enzymes in protein production.
  • the modified xylanase of the present invention may achieve improved wash performance in a detergent composition (as compared to the unmodified enzyme) .
  • "improved wash performance" in a detergent is defined as increasing cleaning of certain enzyme- sensitive stains such as grass or blood, as determined by light reflectance evaluation after a standard wash cycle.
  • modified xylanase of the present invention does not create any special use limitation.
  • any temperature and pH suitable for the detergent is also suitable for the present compositions as long as the pH is within a suitable range and the temperature is below the described modified xylanase' s denaturing temperature.
  • the modified xylanase in accordance with the invention can be used in a cleaning composition without detergents, again either alone or in combination with builders and stabilizers.
  • the laundry detergent and/or fabric care compositions of the invention may also contain additional detergent and/or fabric care components.
  • additional components, and levels of incorporation thereof will depend on the physical form of the composition, and the nature of the cleaning operation for which it is to be used.
  • the laundry detergent and/or fabric care compositions of the present invention preferably further comprise a detergent ingredient selected from cationic surfactants, proteolytic enzymes, bleaching agents, builders-in particular zeolite A and sodium tripolyphosphate-and/or clays .
  • a detergent ingredient selected from cationic surfactants, proteolytic enzymes, bleaching agents, builders-in particular zeolite A and sodium tripolyphosphate-and/or clays .
  • These laundry detergent and/or fabric care compositions achieve improved overall cleaning including stain removal and whitening maintenance, while preventing any negative effect on the fabric.
  • These compositions further provide improved fabric care, including anti- bobbling, depilling, colour appearance, fabric softness and fabric anti-wear properties and benefits, while preventing any negative effect on the fabric.
  • the laundry detergent and/or fabric care compositions according to the invention can be liquid, paste, gels, bars, tablets, spray, foam, powder or granular forms.
  • Granular compositions can also be in "compact” form, the liquid compositions can also be in a "concentrated” form.
  • the compositions of the invention may for example, be formulated as hand and machine laundry detergent compositions including laundry additive compositions and compositions suitable for use in the soaking and/or pre- treatment of stained fabrics, rinse added fabric softener compositions. Pre-or post treatment of fabric include gel, spray and liquid fabric care compositions. A rinse cycle with or without the presence of softening agents is also contemplated.
  • compositions suitable for use in a laundry machine washing method preferably contain both a surfactant and a builder compound and addition one or more detergent components preferably selected from organic polymeric compounds, bleaching agents, additional enzymes, suds suppressors, dispersants, lime-soap dispersants, soil suspension and anti-redeposition agents and corrosion inhibitors .
  • Laundry compositions can also contain softening agents, as additional detergent components.
  • the laundry detergent and/or fabric care compositions according to the present invention comprise a surfactant system wherein the surfactant can be selected from non- ionic and/or anionic and/or cationic and/or ampholytic and/or zwitterionic and/or semi-polar surfactants.
  • the surfactant is typically present at a level of from 0.1% to 60% by weight. More preferred levels of incorporation are 1% to 35% by weight, most preferably from 1% to 30% by weight of laundry detergent and/or fabric care compositions in accord with the invention.
  • the surfactant is preferably formulated to be compatible with enzyme components present in the composition.
  • the surfactant is most preferably formulated such that it promotes, or at least does not degrade, the stability of any enzyme in these compositions.
  • Cationic detersive surfactants suitable for use in the laundry detergent and/or fabric care compositions of the present invention are those having one long-chain hydrocarbyl group.
  • cationic surfactants include the ammonium surfactants such as alkyltrimethylammonium nalogenides and quaternary ammonium surfactants such as coconut trimethyl ammonium chloride or bromide; coconut methyl dihydroxyethyl ammonium chloride or bromide; decyl triethyl ammonium chloride; decyl dimethyl hydroxyethyl ammonium chloride or bromide; C ⁇ 2 -i 5 dimethyl hydroxyethyl ammonium chloride or bromide; coconut dimethyl hydroxyethyl ammonium chloride or bromide; myristyl trimethyl ammonium methyl sulphate; lauryl dimethyl benzyl ammonium chloride or bromide; lauryl dimethyl (ethenoxy) 4 ammonium chloride or bromide
  • Typical cationic fabric softening components include the water-insoluble quaternary-ammonium fabric softening actives or their corresponding amine precursor, the most commonly used having been di-long alkyl chain ammonium chloride or methyl sulfate.
  • Preferred cationic softeners among these include the following: 1) ditallow dimethylammonium chloride (DTDMAC) ; 2) dihydrogenated tallow dimethylammonium chloride; 3) dihydrogenated tallow dimethylammonium methylsulfate; 4) distearyl dimethylammonium chloride; 5) dioleyl dimethylammonium chloride; 6) dipalmityl hydroxyethyl methylammonium chloride; 7) stearyl benzyl dimethylammonium chloride; 8) tallow trimethylammonium chloride; 9) hydrogenated tallow trimethylammonium chloride; 10) C 12 - 14 alkyl hydroxyethyl dimethylammonium chloride; 11) C ⁇ 2 - ⁇ 8 alkyl dihydroxyethyl methylammonium chloride; 12) di (stearoyloxyethyl) dimethylammonium chloride (DSOEDMAC) ; 13) di ( tallow-oxy-e
  • Biodegradable quaternary ammonium compounds have been presented as alternatives to the traditionally used di- long alkyl chain ammonium chlorides and methyl sulfates. Such quaternary ammonium compounds contain long chain alkyl, alkenyl, alkyne or groups interrupted by functional groups such as carboxy groups. Said materials and fabric softening compositions containing them are disclosed in numerous publications such as EP-A-0, 040, 562 , and EP-A- 0,239,910. Polyethylene, polypropylene, and polybutylene oxide condensates of alkyl phenols are suitable for use as the nonionic surfactant of the surfactant systems of the present invention, with the polyethylene oxide condensates being preferred.
  • These compounds include the condensation products of alkyl phenols having an alkyl group containing from about 6 to about 14 carbon atoms, preferably from about 8 to about 14 carbon atoms, in either a straight- chain or branched-chain configuration with the alkylene oxide.
  • the ethylene oxide is present in an amount equal to from about 2 to about 25 moles, more preferably from about 3 to about 15 moles, of ethylene oxide per mole of alkyl phenol.
  • nonionic surfactants of this type include IgepalTM CO-630, marketed by the GAF Corporation; and TritonTM X-45, X-114, X-100 and X-102, all marketed by the Rohm & Haas Company. These surfactants are commonly referred to as alkylphenol alkoxylates (eg., alkyl phenol ethoxylates) .
  • the condensation products of primary and secondary aliphatic alcohols with from about 1 to about 25 moles of ethylene oxide are suitable for use as the non-ionic surfactant of the non-ionic surfactant systems of the present invention.
  • the alkyl chain of the aliphatic alcohol can either be straight or branched, primary or secondary, and generally contains from about 8 to about 22 carbon atoms .
  • About 2 to about 7 moles of ethylene oxide and most preferably from 2 to 5 moles of ethylene oxide per mole of alcohol are present in said condensation products.
  • non-ionic surfactants of this type include TergitolTM 15-S-9 (the condensation product of Cn-C ⁇ 5 linear alcohol with 9 moles ethylene oxide) , TergitolTM 24- L-6 NMW (the condensation product of C 12 -C 14 primary alcohol with 6 moles ethylene oxide with a narrow molecular weight distribution) , both marketed by Union Carbide Corporation; NeodolTM 45-9 (the condensation product of C14-Cls linear alcohol with 9 moles of ethylene oxide), NeodolTM 23-3 (the condensation product of C ⁇ 2 -C ⁇ 3 linear alcohol with 3.0 moles of ethylene oxide) , NeodolTM 45-7 (the condensation product of C14-Cls linear alcohol with 7 moles of ethylene oxide) , NeodolTM 45-5 (the condensation product of C14-Cls linear alcohol with 5 moles of ethylene oxide) marketed by Shell Chemical Company, KyroTM EOB (the condensation product of C ⁇ 3 -C ⁇ alcohol with 9 moles ethylene
  • non-ionic surfactant of the surfactant systems of the present invention are the alkylpolysaccharides disclosed in US Patent 4,565,647, Llenado, issued January 21,1986, having a hydrophobic group containing from about 6 to about 30 carbon atoms, preferably from about 10 to about 16 carbon atoms and a polysaccharide, eg. a polyglycoside, hydrophilic group containing from about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7 saccharide units.
  • Any reducing saccharide containing 5 or 6 carbon atoms can be used, eg., glucose, galactose and galactosyl moieties can be substituted for the glucosyl moieties (optionally the hydrophobic group is attached at the 2-, 3-, 4-, etc. positions thus giving a glucose or galactose as opposed to a glucoside or galactoside) .
  • the intersaccharide bonds can be, eg., between the one position of the additional saccharide units and the 2-, 3-, 4-, and/or 6-positions on the preceding saccharide units.
  • the laundry detergent and/or fabric care compositions of the present invention may also contain ampholytic, zwitterionic, and semi-polar surfactants, as well as the non-ionic and/or anionic surfactants other than those already described herein.
  • Ampholytic surfactants are also suitable for use in the laundry detergent and/or fabric care compositions of the present invention. These surfactants can be broadly described as aliphatic derivatives of secondary or tertiary amines, or aliphatic derivatives of heterocyclic secondary and tertiary amines in which the aliphatic radical can be straight-or branched-chain.
  • One of the aliphatic substituents contains at least about 8 carbon atoms, typically from about 8 to about 18 carbon atoms, and at least one contains an anionic water-solubilizing group, e. g. carboxy, sulfonate, sulfate. See U. S. Patent No. 3,929,678 to Laughlin et al . , issued December 30,1975 at column 19, lines 18-35, for examples of ampholytic surfactants.
  • the laundry detergent and/or fabric care compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such ampholytic surfactants .
  • Zwitterionic surfactants are also suitable for use in laundry detergent and/or fabric care compositions. These surfactants can be broadly described as derivatives of secondary and tertiary amines, derivatives of heterocyclic secondary and tertiary amines, or derivatives of quaternary ammonium, quaternary phosphonium or tertiary sulfonium compounds. See US Patent No. 3,929,678 to Laughlin et al . , issued December 30,1975 at column 19, line 38 through column 22, line 48, for examples of zwitterionic surfactants.
  • the laundry detergent and/or fabric care compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1 % to about 10% by weight of such zwitterionic surfactants.
  • Semi-polar non-ionic surfactants are a special category of non-ionic surfactants which include water- soluble amine oxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from about 1 to about 3 carbon atoms; water-soluble phosphine oxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from about 1 to about 3 carbon atoms; and water-soluble sulfoxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and a moiety selected from the group consisting of alkyl and hydroxyalkyl moieties of from about 1 to about 3 carbon atoms .
  • the modified xylanase is used in the preparation of an animal feed, for example, a cereal-based feed.
  • the cereal can be at least one of wheat, barley, maize, sorghum, rye, oats, triticale, and rice.
  • the cereal component of a cereal-based feed constitutes a source of protein, it is usually necessary to include sources of supplementary protein in the feed such as those derived from fish-meal, meat-meat, or vegetables.
  • Sources of vegetable proteins include at least one of full fat soybeans, rapeseeds, canola, soybean-meal, rapeseed-meal, and canola-meal.
  • a modified enzyme of the present invention in an animal feed can enable the crude protein value and/or digestibility and/or amino acid content and/or digestibility coefficients of the feed to be increased, which permits a reduction in the amounts of alternative protein sources and/or amino acids supplements which had previously been necessary ingredients of animal feeds .
  • the feed provided by the present invention may also include other enzyme supplements such as one or more of ⁇ - glucanase, glucoamylase, mannanase, ⁇ -galactosidase, phytase, lipase, ⁇ -arabinofuranosidase, ⁇ -amylase, esterase, oxidase, oxido-reductase, and pectinase.
  • enzyme supplements such as one or more of ⁇ - glucanase, glucoamylase, mannanase, ⁇ -galactosidase, phytase, lipase, ⁇ -arabinofuranosidase, ⁇ -amylase, esterase, oxidase, oxido-reductase, and pectinase.
  • Example 1 Chemical modification of xylanase from Trichoderma longibrachiatum .
  • Nucleophile (amount as indicated in Table 1) ) was added to 40 mM K 2 HP0 4 /KH 2 P0 4 (pH as indicated in table 1) buffer and the pH was readjusted with 2 M NaOH.
  • Xylanase was purchased from Megazyme . Dialyzed and concentrated xylanase solution was added in the above mentioned nucleophilic solution at a concentration of 10-150 ⁇ g ml "1
  • the enzyme activity is determined by Reducing Sugar Assay using dinitrosalicylic acid Reagent. Appropriate amounts of xylanase solution (50-200 ⁇ l) were added to 1 ml of 1% (w/v) xylan solution in 50 mM Na 2 HP0 4 /citric acid, pH 5 buffer and incubated at 45 °C. After 15 min the reaction was stopped by adding 1 ml of Dinitrosalicylic acid reagent and boiled for 5 min. The mixture is cooled and A 5 4 0 is determined against reagent blank.
  • Half-lives (irreversible thermal denaturation) were determined by heating (100-200 ⁇ l) of xylanases at a certain temperature (55 and/or 60 °C) . Aiiquots were taken at various time intervals, cooled in ice and residual activity determined by assaying the enzyme at 45 °C for 15 min. The results of assays are summarised in Table 1.
  • Linking of an uncharged or positively charged molecule to the carboxyl group of a side chain of an amino acid of xylanase resulted in up to a 5- fold increase in half-life at 55°C and up to a 10-fold increase in half-life at 60°C compared to unmodified (native) xylanase from Tric ode ⁇ na longibrachiatum.
  • the T opt of the unmodified enzyme was increased by 5°C.

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Abstract

Cette invention porte sur une enzyme et sur des procédés de production d'une enzyme pouvant effectuer le clivage de liaisons β-1,4-xylosidiques de xylane. Cette enzyme comprend une xylanase qui a été modifiée par l'introduction d'une molécule neutre ou chargée positivement liée à une chaîne latérale d'un acide aminé de la xylanase et/ou à un acide aminé C-terminal de la xylanase. L'enzyme agit à une température élevée et/ou présente une demi-vie prolongée par rapport à la xylanase non modifiée correspondante.
PCT/AU2002/001487 2001-12-21 2002-11-01 Amelioration de la stabilite d'enzymes WO2003056003A1 (fr)

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PCT/AU2002/001486 WO2003056002A1 (fr) 2001-12-21 2002-11-01 Stabilite d'amylase amelioree
PCT/AU2002/001487 WO2003056003A1 (fr) 2001-12-21 2002-11-01 Amelioration de la stabilite d'enzymes
PCT/AU2002/001484 WO2003056000A1 (fr) 2001-12-21 2002-11-01 Enzyme modifiee et procede de modification
PCT/AU2002/001488 WO2003056004A1 (fr) 2001-12-21 2002-11-01 Ameliorations de la thermolabilite d'enzymes
PCT/AU2002/001485 WO2003056001A1 (fr) 2001-12-21 2002-11-01 Enzymes modifiees et procedes de modification
PCT/AU2002/001483 WO2003055999A1 (fr) 2001-12-21 2002-11-01 Enzyme modifie et son procede de modification

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PCT/AU2002/001486 WO2003056002A1 (fr) 2001-12-21 2002-11-01 Stabilite d'amylase amelioree

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PCT/AU2002/001484 WO2003056000A1 (fr) 2001-12-21 2002-11-01 Enzyme modifiee et procede de modification
PCT/AU2002/001488 WO2003056004A1 (fr) 2001-12-21 2002-11-01 Ameliorations de la thermolabilite d'enzymes
PCT/AU2002/001485 WO2003056001A1 (fr) 2001-12-21 2002-11-01 Enzymes modifiees et procedes de modification
PCT/AU2002/001483 WO2003055999A1 (fr) 2001-12-21 2002-11-01 Enzyme modifie et son procede de modification

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US8841091B2 (en) 2004-12-22 2014-09-23 Novozymes Als Enzymes for starch processing
CN106244645B (zh) * 2016-08-31 2020-07-10 保龄宝生物股份有限公司 一种循环利用米曲霉菌体生产低聚果糖的方法
US11932860B2 (en) 2017-10-03 2024-03-19 Kikkoman Corporation Method for producing alkaline phosphatase, alkaline phosphatase obtained using said method, and vector and transformant for production thereof

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WO2003056002A1 (fr) 2003-07-10
WO2003055999A1 (fr) 2003-07-10
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AUPR968801A0 (en) 2002-01-24
WO2003056000A1 (fr) 2003-07-10

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