WO2006063587A1 - Tester l’activité d’une enzyme en utilisant l’absorbance - Google Patents

Tester l’activité d’une enzyme en utilisant l’absorbance Download PDF

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Publication number
WO2006063587A1
WO2006063587A1 PCT/DK2005/000786 DK2005000786W WO2006063587A1 WO 2006063587 A1 WO2006063587 A1 WO 2006063587A1 DK 2005000786 W DK2005000786 W DK 2005000786W WO 2006063587 A1 WO2006063587 A1 WO 2006063587A1
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Prior art keywords
enzyme
polypeptides
library
substrate
solid
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PCT/DK2005/000786
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English (en)
Inventor
Gernot J. Abel
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Novozymes A/S
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Publication of WO2006063587A1 publication Critical patent/WO2006063587A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry

Definitions

  • the present invention relates to a method of testing the activity of an enzyme and to screening a library of polypeptides for an enzyme of interest.
  • Enzymes are used within a number of different industries, such as the detergent, baking and paper industry.
  • the finding of new and/ the improvement of known enzymes often involves screening of host cells expressing a library of different enzymes or variants of a known enzyme for such new and/or improved enzymes.
  • the screening process generally involves screening a large number of polypeptides on a laboratory scale and then a few enzymes which appear of interest are then further tested in a real application (or in a set-up similar to the real application).
  • the large numbers of polypeptides are screened in an assay in which the activity of the enzyme can be tested.
  • the choice of assay to use in this initial screen or test is very important as it is an advantage if it is inexpensive, suitable for handling a large number of samples and that the enzymes identified in this assay are also suitable for use in the real application for which they are intended.
  • the present invention provides a method for measuring the activity of an enzyme comprising measuring the absorbance of a liquid phase in which the enzyme has been contacted with a substrate on a solid.
  • the present invention provides a method of screening a library of polypeptides for an enzyme of interest comprising measuring the absorbance of a liquid phase in which the polypeptides of the library of polypeptides have been contacted with a substrate on a solid and selecting an enzyme of interest.
  • the enzyme/enzyme of interest may belong to a known class of enzymes, or it may be of an unknown enzyme class, e.g. an enzyme having a desired functional activity but not necessarily belonging to a known enzyme class.
  • enzyme class E. C. refers to the internationally recognized enzyme classification system, Recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, Academic Press, Inc., 1992.
  • the enzyme/enzyme of interest may belong to one of the following classes: oxidoreductases (EC 1.-.-.-), transferases (EC 2.-.-.-), hydrolases (EC 3.-.-.-), lyases (EC 4.-.- .-), isomerases (EC 5.-.-.-) and ligases (EC 6.-.-.-).
  • oxidoreductases EC 1.-.-.-
  • transferases EC 2.-.-.-
  • hydrolases EC 3.-.-.-
  • lyases EC 4.-.- .-
  • isomerases EC 5.-.-.-
  • ligases EC 6.-.-.-.-
  • Oxidoreductases examples include peroxidases (EC 1.11.1), laccases (EC 1.10.3.2) and glucose oxidases (EC 1.1.3.4).
  • transferases may be transferases belonging to any of the following sub-classes: a) Transferases transferring one-carbon groups (EC 2.1); b) Transferases transferring aldehyde or ketone residues (EC 2.2); acyltransferases (EC 2.3); c) Glycosyltransferases (EC 2.4); d) Transferases transferring alkyl or aryl groups, other than methyl groups (EC 2.5); and e) Transferases transferring nitrogeneous groups (EC 2.6).
  • the transferase may be a transglutaminase (protein-glutamine gamma- glutamyltransferase; EC 2.3.2.13).
  • hydrolases include: Carboxylic ester hydrolases (EC 3.1.1.-). In particular it may be a lipolytic enzyme, i.e. an enzyme which can hydrolyze an ester bond.
  • lipases such as triacyl-glycerol lipase (EC 3.1.1.3), lipoprotein lipase (EC 3.1.1.34), monoglyceride lipase (EC 3.1.1.23), lysophospholipase, ferulic acid esterase and esterase (EC 3.1.1.1 , EC 3.1.1.2).
  • the numbers in parentheses are the systematic numbers assigned by the Enzyme Commission of the International Union of Biochemistry in accordance with the type of the enzymatic reactivity of the enzyme.
  • the lipolytic enzyme may be prokaryotic, particularly a bacterial enzyme, e.g. from Pseudomonas.
  • Pseudomonas lipases e.g. from P. cepacia (US 5,290,694, pdb file 1OIL), P. glumae (N Frenken et al. (1992), Appl. En-vir. Microbiol. 58 3787-3791 , pdb files 1TAH and 1QGE), P. pseudoalcaligenes (EP 334 462) and Pseudomonas sp.
  • strain SD 705 (FERM BP-4772) (WO 95/06720, EP 721 981 , WO 96/27002, EP 812 910).
  • the P. glumae lipase sequence is identical to the amino acid sequence of Chromobactehum viscosum (DE 3908131 A1).
  • Other examples are bacterial cutinases, e.g. from Pseudomonas such as P. mendocina (US 5,389,536) or P. putida (WO 88/09367).
  • the lipolytic enzyme may be eukaryotic, e.g. a fungal lipolytic enzyme such as lipolytic enzymes of the Humicola family and the Zygomycetes family and fungal cutinases.
  • fungal cutinases are the cutinases of Fusarium solani pisi (S. Longhi et al., Journal of Molecular Biology, 268 (4), 779-799 (1997)) and Humicola insolens (US 5,827,719).
  • the Humicola family of lipolytic enzymes consists of the lipase from H. lanuginosa strain DSM 4109 and lipases having more than 50 % homology with said lipase.
  • the lipase from H. lanuginosa (synonym Thermomyces lanuginosus) is described in EP 258 068 and EP
  • the Humicola family also includes the following lipolytic enzymes: lipase from Penicillium camembertii (P25234), lipase/phospholipase from Fusarium oxysporum (EP25234), lipase/phospholipase from Fusarium oxysporum (EP25234), lipase/phospholipase from Fusarium oxysporum (EP25234), lipase/phospholipase from Fusarium oxysporum (EP25234), lipase/phospholipase from Fusarium oxysporum (EP25234), lipase/phospholipase from Fusarium oxysporum (EP25234), lipase/phospholipase from Fusarium oxysporum (EP25234), lipase/phospholipase from Fusarium oxysporum (EP25234), lipase/phospholipase from Fusarium oxysporum
  • Aspergillus foetidus W33009
  • phospholipase A1 from A. oryzae JP-A 10-155493
  • lipase from A. oryzae D85895
  • lipase/ferulic acid esterase from A. niger Y09330
  • lipase/ferulic acid esterase from A. tubingensis Y09331
  • lipase from A. tubingensis WO 98/45453
  • lysophospholipase from A. niger WO 98/31790
  • lipase from F. solanii having an isoelectric point of 6.9 and an apparent molecular weight of 30 kDa (WO 96/18729).
  • the Zygomycetes family comprises lipases having at least 50 % homology with the lipase of Rhizomucor miehei (P19515). This family also includes the lipases from Absidia reflexa, A. sporophora, A. corymbifera, A. blakesleeana, A. ghseola (all described in WO 96/13578 and WO 97/27276) and Rhizopus oryzae (P21811). Numbers in parentheses indicate publication or accession to the EMBL, GenBank, GeneSeqp or Swiss-Prot databases.
  • hydrolases include but are not limited to phytases (EC 3.1.3.-), e.g. 3- phytases (EC 3.1.3.8) and 6-phytases (EC 3.1.3.26); glycosidases (EC 3.2, which fall within a group denoted herein as "carbohydrases"), such as alpha-amylases (EC 3.2.1.1); peptidases (EC 3.4, also known as proteases); and other carbonyl hydrolases.
  • Other hydrolases include xyloglucanase, arabinase, rhamno-galactoronase, pectinases, ligninases (for example polyphenol hydrolase).
  • proteases include but are not limited to those of animal, vegetable or microbial origin or chemically modified or protein engineered mutants.
  • the protease may be a serine protease or a metallo protease, particularly an alkaline microbial protease or a trypsin-like protease.
  • alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279).
  • trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.
  • protease enzymes examples include AlcalaseTM, SavinaseTM, PrimaseTM, DuralaseTM, EsperaseTM, and KannaseTM (Novozymes A/S), MaxataseTM, MaxacalTM, MaxapemTM, ProperaseTM, PurafectTM, Purafect OxPTM, FN2TM, and FN3TM (Genencor International Inc.).
  • carbohydrase is used to denote not only enzymes capable of breaking down carbohydrate chains (e.g. starches) of especially five- and six- membered ring structures (i.e. glycosidases, EC 3.2), but also enzymes capable of isomerizing carbohydrates, e.g. six-membered ring structures such as D-glucose to five- membered ring structures such as D-fructose.
  • Carbohydrases of relevance include the following (EC numbers in parentheses): alpha-amylases (3.2.1.1), beta-amylases (3.2.1.2), glucan 1 ,4-alpha-glucosidases (3.2.1.3), cellulases (3.2.1.4), endo-1 ,3(4)-beta-glucanases (3.2.1.6), endo-1 ,4-beta-xylanases (3.2.1.8), dextranases (3.2.1.11), chitinases (3.2.1.14), polygalacturonases (3.2.1.15), lysozymes (3.2.1.17), beta-glucosidases (3.2.1.21), alpha-galactosidases (3.2.1.22), beta-galactosidases (3.2.1.23), mannanase (3.2.1.25), amylo-1 ,6-glucosidases (3.2.1.33), xylan 1 ,
  • enzymes not yet classified may also be relevant for the present invention.
  • the enzyme/enzyme of interest may also be a variant of a known enzyme, wherein the term "variant" is to be understood as an enzyme which differs from another enzyme, typically a known enzyme generally called a parent enzyme, with regard to at least one amino acid position.
  • the present invention also relates to a method for screening a library of polypeptides for an enzyme of interest.
  • library of polypeptides is to be understood as a collection of at least two different polypeptides; i.e. at least two polypeptides which differ at one or more amino acid positions, e.g. the number of amino acids in the polypeptides may be different and/or the amino acid(s) at a particular position may be different.
  • the library of polypeptides may be prepared by introducing a library of nucleic acid sequences encoding the library of polypeptides into a host cell capable of expressing the polypeptides.
  • said library of nucleic acid sequences may encode variants of a parent enzyme; i.e. polypeptides which differ at, at least one amino acid position compared to a parent enzyme.
  • the screening method may be used to screen for variants of a parent enzyme.
  • variants may be produced by e.g. random mutagenesis or site-directed mutagenesis of a parent enzyme or by other methods known to a person skilled in the art.
  • the library of polypeptides may be a library of variants of a parent enzyme.
  • parent enzymes examples include but are not limited to those mentioned above in the section of enzymes.
  • the parent enzyme may be a lipolytic enzyme e.g. a lipase from Humicola, e.g. H. lanuginosa, or Pseudomonas or Bacillus.
  • said library of nucleic acid sequences may encode polypeptides derived from one or a number of different organisms.
  • the method may be used to screen one or a number of different organisms for expression of an enzyme activity of interest.
  • the library of polypeptides may be prepared by synthesizing the polypeptides.
  • Methods for preparing a library of nucleic acid sequences, introducing it into a host cell, expressing the polypeptides encoded by said library of polypeptides in the host cells and methods for synthesizing polypeptides are well known to a person skilled in the art and may e.g. be found in "Molecular cloning: A laboratory manual", Sambrook et al. (1989), Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds.); "Current protocols in Molecular Biology", John Wiley and Sons, (1995); Harwood, C. R., and Cutting, S. M.
  • the substrate of the present invention may be any compound which is a substrate for the enzyme or enzyme of interest to be tested or screened for in a method of the present invention.
  • a new enzyme or a variant of known enzyme Before a new enzyme or a variant of known enzyme are used in an industrial application their activity is usually tested in an assay in the laboratory. Often a large number of new enzymes or variants of known enzymes are tested or screened in such an assay to find an enzyme which is most suitable for a particular use.
  • the ability of such an assay to indicate whether or not a particular enzyme will be useful in a given application depends on a number of parameters, such as the similarity between the substrate used in the assay and the substrate which the enzyme is supposed to function against in its real application.
  • an advantage of the present invention is that it is possible to use the substrate in the methods which the enzyme/enzyme of interest is intended to function against in its real application. Another advantage is that such substrates are generally cheaper than if one has to use synthetically or synthetically modified substrates. The choice of substrate depends on the enzyme/enzyme of interest. Examples of substrates for different enzymes are well known to a person skilled in the art.
  • the substrate may be comprised in a mixture of compounds. This may for example be relevant if a method of the present invention is used to test the activity of an enzyme or screen for an enzyme of interest which is capable of (or has an improved capability) of catalyzing the degradation of stains on clothes during washing.
  • the substrate should be a compound comprising an ester bond or a composition comprising a compound which comprises an ester bond.
  • suitable compounds or compositions include lard or butter.
  • the substrate should be a protein or polypeptide or a composition comprising a protein or polyeptide, such as blood, milk, grass egg, beef (meat), tomato, cacao, chocolate.
  • the amount of substrate on the solid is generally adapted so that the reaction between the substrate and the enzyme/enzyme of interest may show a relation between the amount of enzyme/enzyme of interest and the response.
  • the amount of substrate and/or enzyme generally depends on the particular enzyme and/or substrate and it is well known to a person skilled in the art to adjust these amounts so that there is a correlation between the amount of enzyme/enzyme of interest and the response.
  • a solid is to be understood as a compound which is in its solid phase at standard ambient temperature and pressure, i.e. at 25 0 C and 1 bar.
  • the solid of the present invention may be any solid compound, such as textile, plastic, metal, ceramic or rock.
  • the solid of the present invention is a textile.
  • textile includes fabrics, garments, and yarns.
  • Fabric can be constructed from fibers by weaving, knitting or non-woven operations. Weaving and knitting require yarn as the input whereas the non-woven fabric is the result of random bonding of fibers (paper can be thought of as non-woven).
  • the term "fabric” is also intended to include fibers and other types of processed fabrics.
  • Woven fabric is constructed by weaving "filling" or weft yarns between wrap yarns stretched in the longitudinal direction on the loom. The wrap yarns must typically be sized before weaving in order to lubricate and protect them from abrasion at the high speed insertion of the filling yarns during weaving.
  • the filling yarn can be woven through the warp yarns in a "over one - under the next" fashion (plain weave) or by "over one - under two" (twill) or any other myriad of permutations.
  • Strength, texture and pattern are related not only to the type/quality of the yarn but also the type of weave. Generally, dresses, shirts, pants, sheeting's, towels, draperies, etc. are produced from woven fabric.
  • Knitting is forming a fabric by joining together interlocking loops of yarn. As opposed to weaving which is constructed from two types of yarn and has many "ends", knitted fabric is produced from a single continuous strand of yarn. As with weaving, there are many different ways to loop yarn together and the final fabric properties are dependent both upon the yarn and the type of knit. Underwear, sweaters, socks, sport shirts, sweat shirts, etc. are generally derived from knit fabrics.
  • Non-woven fabrics are sheets of fabric made by bonding and/or interlocking fibers and filaments by mechanical, thermal, chemical or solvent mediated processes.
  • the resultant fabric can be in the form of web-like structures, laminates or films. Typical examples are disposable baby diapers, towels, wipes, surgical gowns, fibers for the "environmental friendly” fashion, filter media, bedding, roofing materials, backing for two-dimensional fabrics and many others.
  • the textile used in the present invention may be any known textile (woven, knitted, or non-woven).
  • the textile may be a cellulose-containing or cellulosic textile, such as cotton, viscose, rayon, ramie, linen, lyocell (e.g., Tencel, produced by Courtaulds Fibers), or mixtures thereof, or it may be a synthetic textile such as one of polyester, polyamic or nylon or mixtures of these or a mixture of cellulose-containing or cellulosic fibres and synthetic fibres.
  • a suitable textile is one comprising other natural fibers such as wool and silk or mixtures of these or mixtures of these and one or more of the above mentioned fibres.
  • mixtures of fibres include but are not limited to viscose/cotton blends, lyocell/cotton blends, viscose/wool blends, lyocell/wool blends, cotton/wool blends; flax (linen), ramie and other fabrics based on cellulose fibers, including all blends of cellulosic fibers with other fibers such as wool, polyamide, acrylic and polyester fibers, e.g. cotton/polyester blends, viscose/cotton/polyester blends, wool/cotton/polyester blends, flax/cotton blends etc.
  • wool means any commercially useful animal hair product, for example, wool from sheep, camel, rabbit, goat, llama, and known as merino wool, Shetland wool, cashmere wool, alpaca wool, mohair, etc. and includes wool fiber and animal hair.
  • the textile may be bleached, dyed or undyed.
  • poly(ethylene terephthalate) which is synthesized by condensation, drawn into fibers from a melt, possibly cut to stables, possibly mixed with other fiber types, and spun to yarn. The yarn is dyed and knitted into cloth or made into carpets, or the yarn is woven into fabric and dyed.
  • an enzyme to bind or adhere to a textile depends both on the particular enzyme but also on the type of textile.
  • lipolytic enzymes appear to be more difficult to remove from a polyester-textile than from a cotton-textile during rinsing, in general the adherence or binding of lipolytic enzymes to hydrophobic materials may be stronger than to more hydrophilic materials.
  • the enzyme or library of polypeptides are in a liquid phase when it or they are contacted with a substrate on a solid.
  • liquid phase is to be understood as including a solution, i.e. a true solution, a suspension, a dispersion and a colloidal solution, where these terms may be understood as e.g. described in Hawley's Condensed Chemical Dictionary, John Wiley and
  • liquid refers to phases being liquid at standard ambient temperature and pressure, i.e. at 25°C and 1 bar.
  • the term "substrate on a solid” is to be understood as the substrate is bound to the solid by any type of chemical and/or physical bond, such as a covalent bond, a hydrogen bond, an electrostatic bond or ionic bond, or the attraction of molecules to each other by van der Waals forces.
  • the solid is a textile the substrate may be embedded into the textile fibres so as to resemble a stain on dirty clothes.
  • the enzyme and the library of polypeptides may be contacted with the substrate on a solid by adding a liquid phase (typically a solution) of the enzyme or library of polypeptides to the substrate on a solid or the enzyme or library of polypeptides may be added to a liquid phase comprising the substrate on a solid.
  • the enzyme of library of polypeptides may be in a liquid phase, such as solution, or in solid form when added to a liquid phase comprising the substrate on a solid. If the enzyme or library of polypeptides is in liquid phase said liquid may be the same as the liquid in which the substrate on the solid is it may be a different liquid. For example if the substrate on a solid is present in a detergent-solution a solution of the enzyme or library of polypeptides may be added to the detergent-solution. In this case the solution of the enzyme or library of polypeptides may typically be different from the detergent-solution as the conditions suitable for storage of an enzyme or library of polypeptides may typically be different from those of a detergent-solution.
  • the liquid phase may in particular comprise at least 30v/v% water, or at least 40v/v% water, or at least 50v/v% water, or at least 60v/v% water, or at least 70v/v% water, or at least 80v/v% water, or at least 90v/v% water, or at least 95v/v% water, or 100v/v% water.
  • the supernatant from the host cells may be added to the substrate on the solid or if it or they are expressed inside the host, e.g. in inclusion bodies, the host cells may be lysed and the lysate or a fraction thereof may be added to the substrate on the solid.
  • the enzyme or library of polypeptides may also be purified before contacting them with the textile.
  • purified refers to that the enzyme/library of polypeptides has been removed from their native environment. If the enzyme/library of polypeptides has been expressed by a host cell the native environment refers to the host cells and compounds different from the enzyme/library of polypeptides secreted by the host cell.
  • enzymes or polypeptides which have been prepared synthetic this may refer to the removal of other components which have been present during the synthesising process.
  • the enzyme or library of polypeptides may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
  • the enzyme or library of polypeptides may be contacted with the substrate on the solid by adding a detergent-solution comprising the enzyme or library of polypeptides to the substrate on the solid.
  • a detergent-solution is in the context of the present invention to be understood as a liquid phase comprising one or more surfactants, which may be non-ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic.
  • the surfactants are typically present at a level of from 0.1% to 60% by weight.
  • the detergent-solution may contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
  • an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
  • the detergent-solution may usually contain from about 0.2% to about 40% of a non- ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides").
  • a non- ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides”).
  • glucamides N-acyl N-alkyl derivatives of glucosamine
  • the detergent-solution may further contain 0-65 % of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
  • a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
  • the detergent-solution may further comprise one or more polymers.
  • polymers include but are not limited to carboxymethylcellulose, poly(vinylpyrrolidone), poly(ethylene glycol), polyvinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
  • the detergent-solution may further contain a bleaching system which may comprise a
  • H2O2 source such as perborate or percarbonate which may be combined with a peracid- forming bleach activator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate.
  • a peracid- forming bleach activator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate.
  • the bleaching system may comprise peroxyacids of e.g. the amide, imide, or sulfone type.
  • the detergent-solution may further comprise conventional enzyme-stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in e.g. WO 92/19709 and WO 92/19708.
  • enzyme-stabilizing agents e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in e.g. WO 92/19709 and WO 92/197
  • the detergent-solution may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
  • fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
  • the detergent-solution may comprise one or more other enzyme than the enzyme or enzyme of interest of the present invention, such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., a laccase, and/or a peroxidase.
  • enzymes generally used in detergents are well-known to a person skilled in the art.
  • mechanical stress may be used when the enzyme or library of polypeptides is contacted with the substrate on the solid.
  • the solid is a textile contacting the enzyme or library of polypeptides with the substrate on the solid may be performed as described in WO 02/42740 which discloses a method for testing the cleaning effect of a compound or composition thereof.
  • the activity of an enzyme is measured by measuring the absorbance of a liquid phase in which the enzyme has been contacted with a substrate on a solid.
  • Methods for measuring the absorbance of a liquid phase are well known to a person skilled in the art and the present invention is not limited to the use of a particular method.
  • the present invention relates in one embodiment to a method for measuring the activity of an enzyme comprising measuring the absorbance of a liquid phase in which the enzyme has been contacted with a substrate on a solid.
  • This method may comprise the steps of:
  • the present invention relates to a method of screening a library of polypeptides for an enzyme of interest comprising measuring the absorbance of a liquid phase in which the polypeptides of the library of polypeptides have been contacted with a substrate on a solid and selecting an enzyme of interest.
  • This method may comprise the steps:
  • Measuring the absorbance is an indirect measure of the activity of the enzyme/enzyme of interest. As it is well known that assay-to-assay variations may occur it may be an advantage to correlate the measured absorbance to the absorbance measured for a control. By comparing with a control the activity of the enzyme may be expressed as a relative and/or an actual enzyme activity depending on the choice of control.
  • An example of a suitable control which the absorbance of the liquid phase in which the enzyme or library of polypeptides have been contacted with a substrate on a solid may be compared with is the absorbance of the same liquid phase comprising the same substrate on a solid but without the enzyme.
  • Another suitable example is the absorbance measured for a known enzyme using the same method and under the same conditions. For example if the screening method of the present invention is used to screen a library of variants of a parent polypeptide the absorbance measured for the polypeptides of the library may be compared to that measured for the parent polypeptide.
  • a control is the use of a so-called internal standard which enables one to correct for assay-assay variations.
  • a well known enzyme showing low activity in the assay and a well known enzyme showing high activity in the assay may be used as internal standards and these are then included in every assay; the variations in enzymatic activity this shows may then be used as an indicator of the assay-assay variations.
  • the methods of the present invention may in particular be carried out at 5-95 0 C, e.g. 10-80 0 C, 20-70 0 C 1 20-60 0 C, 20-50 0 C.
  • microtiter plates with e.g. 24 wells/plate, 96 wells/plate, 384 wells/plate, 1536 wells/plate or a higher number of wells per plate, or nanoliter well-less compartments.
  • An advantage of using a microtiter plate is that it is generally easy to automate the detection procedure which is particularly useful when a library of polypeptides is screened.
  • said textile may have the form of a small patch with a size suitable for placement at the bottom of the well in a plate.
  • the methods of the present invention may be used to test an enzyme or screen for an enzyme which has a particular characteristic.
  • they may be used to test the wash performance of an enzyme or screen for an enzyme which has a particular wash performance, e.g. an improved wash performance as compared to some given standard.
  • wash performance is to be understood as the ability of an enzyme to remove stains from clothes.
  • the negative control used in example 1 is Subtilisin 309.
  • the positive control used in example 2 is Subtilisin 309 with a S99AD modification (a)
  • S99AD modification means that at position 99 of the amino acid sequence of Subtilisin 309 the S has been modified to an A and then a D has been inserted after position 99).
  • Swatches of wfk10N were punched into wells of a standard 96 well microtiterplate.
  • 150 ⁇ l of automated dishwashing detergent (5g/L 16 0 DH) was added to each well and 10 ⁇ l of a purified protease variant, negative or positive control (3-30 nM) was dispensed into every well.
  • the plate was incubated at 30 0 C and 900 rpm for 20 minutes. Wash water was transferred to a new microtiter plate and absorbance (optical density OD) of the wash water was measured at 405 nm.
  • Table 1 shows the optical density (OD) measured for each of the different proteases/protease variants and different protease concentrations. Stain removal was shown to correlate to an increase in absorbance.
  • Swatches of wfkiON (wfk-Cleaning Technology Research Institute) were punched into wells of a standard 96 well microtiterplate.
  • 150 ⁇ l of automated dishwashing detergent (5g/L 16 0 DH) was added to each well and 10 ⁇ l of the supernatant of Bacillus cells expressing the protease/protease variants was dispensed into every well.
  • the plate was incubated at 30 0 C and 900 rpm for 20 minutes. Wash water was transferred to a new microtiter plate and absorbance (optical density OD) of the wash water was measured at 405 nm (the results are shown in table 2). Stain removal was shown to correlate to an increase in absorbance.
  • results in table 2 show that the method may also be used to measure the protease activity of the supernatant of Bacillus cells expressing the protease/protease variant. Furthermore, the results show that both Variant 1 and 2 are capable of removing more stain than Subtilisin 309.

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Abstract

La présente invention concerne un procédé de mesure de l’activité d’une enzyme et de criblage d’une bibliothèque de polypeptides pour une enzyme intéressante, les deux procédés comprenant la mesure de l’absorbance d’une phase liquide dans laquelle l’enzyme ou la bibliothèque de polypeptides a été mise en contact avec un substrat sur un solide, où le procédé de criblage d'une bibliothèque de polypeptides comprend en outre une étape de sélection d'une enzyme intéressante.
PCT/DK2005/000786 2004-12-14 2005-12-13 Tester l’activité d’une enzyme en utilisant l’absorbance WO2006063587A1 (fr)

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DKPA200401927 2004-12-14

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12031273B2 (en) 2021-12-07 2024-07-09 Buckman Laboratories International, Inc. System and method of dynamic corrective enzyme selection and formulation for pulp and paper production

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999034011A2 (fr) * 1997-12-24 1999-07-08 Genencor International, Inc. Methode amelioree pour tester une enzyme preferee et/ou une composition detergente preferee
US6379942B1 (en) * 1998-12-21 2002-04-30 Genencor International, Inc. Chemically modified enzymes with multiple charged variants
US6586224B1 (en) * 1999-07-22 2003-07-01 The Procter & Gamble Company Subtilisin protease variants having amino acid deletions and substitutions in defined epitope regions

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999034011A2 (fr) * 1997-12-24 1999-07-08 Genencor International, Inc. Methode amelioree pour tester une enzyme preferee et/ou une composition detergente preferee
US6379942B1 (en) * 1998-12-21 2002-04-30 Genencor International, Inc. Chemically modified enzymes with multiple charged variants
US6586224B1 (en) * 1999-07-22 2003-07-01 The Procter & Gamble Company Subtilisin protease variants having amino acid deletions and substitutions in defined epitope regions

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12031273B2 (en) 2021-12-07 2024-07-09 Buckman Laboratories International, Inc. System and method of dynamic corrective enzyme selection and formulation for pulp and paper production

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