WO2008075325A2 - Polypeptides de peroxydase hématique - Google Patents

Polypeptides de peroxydase hématique Download PDF

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WO2008075325A2
WO2008075325A2 PCT/IE2007/000124 IE2007000124W WO2008075325A2 WO 2008075325 A2 WO2008075325 A2 WO 2008075325A2 IE 2007000124 W IE2007000124 W IE 2007000124W WO 2008075325 A2 WO2008075325 A2 WO 2008075325A2
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protein
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ser
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PCT/IE2007/000124
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WO2008075325A3 (fr
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Ciaran Fagan
Barry Ryan
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Dublin City University
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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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)

Definitions

  • the invention relates to a method of generating a mutant heme peroxidase protein suitable for immobilisation to a support.
  • the invention also relates to mutant horseradish peroxidase proteins.
  • HRP and other peroxidase enzymes are inactivated by high concentrations of hydrogen peroxide (H 2 O 2 ) , despite its being a primary substrate at lower concentrations. This means that (i) only low H 2 O 2 concentrations can be used with these enzymes, (ii) that H 2 O 2 levels must be carefully dosed and monitored or (iii) it may be necessary to use a helper enzyme such as glucose oxidase to generate moderate H 2 O 2 concentrations in situ from glucose and O 2 (Biotech Bioeng 2000 69: 286-291.). This seriously limits HRP' s usefulness in biocatalysis or other situations. Our HRP mutants help overcome these problems.
  • H 2 O 2 hydrogen peroxide
  • Immobilization of enzymes onto solid surfaces or particles usually takes place in a random orientation.
  • the enzyme's active site may face the solid phase or be otherwise hindered from optimal reaction with substrates in bulk solution, whether in a biosensor, flow-through bioreactor or other arrangement.
  • the performance of the device may be sub-optimal.
  • Our HRP mutants can be immobilized in a directional fashion, such that the active site always faces out from the solid phase to bulk solution, the better to improved performance.
  • 00/06718 describe a polynucleotide coding horseradish peroxidase which has one or more mutations at an amino acid position selected from 255, 37, 131 and 223. They show improved tolerance of 25 mM peroxide over 30 min by mutant
  • a method of generating a heme peroxidase protein suitable for immobilisation to a support comprising the step of introducing a reactive residue at at least one tethering position in the protein, wherein the tethering position is such that, when the protein is immobilised to the support through the at least one reactive residue, the protein is oriented with the active site substantially facing away from the support.
  • the tethering position will be located on a plane of the protein opposite to the active site of the protein, or opposite to an entrance of the active site of the protein.
  • the protein when the protein is immobilised to a support through the reactive residue (s) at the tethering position (s), the protein will be disposed on the support with its active site (or the entrance to the active site) oriented outwardly, i.e. facing away from the support, thus facilitating access of substrate to the active site.
  • a reactive residue is introduced in at least two tethering positions in the protein.
  • the protein will be fixed to the support at at least two points, thereby minimising movement of the protein away from the desired orientation.
  • the protein is selected from the group comprising: horseradish peroxidase (HRP) and soyabean peroxidase (SBP) .
  • HRP horseradish peroxidase
  • SBP soyabean peroxidase
  • HRP horseradish peroxidase
  • SBP soyabean peroxidase
  • HRP horseradish peroxidase
  • SBP soyabean peroxidase
  • HRP horseradish peroxidase
  • SBP soyabean peroxidase
  • the tethering position is selected from the group comprising: R118; R159; and R283, wherein the residue position corresponds to the numbering system used in SEQUENCE ID NO: 1.
  • a reactive residue is introduced into the protein at two tethering positions, reactive residue is introduced into the protein at the three tethering positions indicated above.
  • the reactive residue may be introduced into the protein by any means available to the skilled person, such as, for example, recombinant DNA technology. Typically, the reactive residue is introduced by means of a conservative substitution of the native residue at the tethering position.
  • the reactive residue is a polar amino acid.
  • the reactive residue is selected from the group comprising: lysine; cysteine; glutamic acid, aspartic acid; and a reactive amino acid analogue.
  • the reactive residue is lysine.
  • the method further includes a step of modifying at least one unwanted reactive residue located in the protein adjacent to the active site, wherein the modification renders the residue unreactive.
  • reactive residues would be, for example, lysine, cysteine, glutamic acid and aspartic acid.
  • the unwanted reactive residue will generally comprise a lysine residue.
  • K232, K241, and K174 using the numbering system of SEQUENCE ID NO: 1) .
  • at least two unwanted reactive residues are modified, these residues typically being K232 and K241.
  • the three unwanted reactive residues K232, K241 and K174 are modified.
  • the unwanted reactive residue is replaced with an amino acid selected from the group comprising: valine; isoleucine; leucine; phenylalanine; and asparagine.
  • the lysine residue at position 232 is replaced with a phenylalanine replaced with an asparagine (N) .
  • the protein is generated using recombinant DNA technology.
  • the method further includes a step of introducing a purification tag into the protein.
  • a purification tag will be known to those skilled in the art, for example, poly histidine tags, poly lysine tags, etc.
  • the tags will be introduced into the molecule at the N or C terminal of the protein.
  • the support may be a solid phase support such as a polyether sulphone membrane, or a particle-type support such as CNBr-activated beads.
  • the invention also relates to a method of generating a support comprising a heme peroxidase enzyme immobilised on the support, the method comprising the steps of providing a heme peroxidase protein employing the methods of the invention, and attaching the heme peroxidase protein to the support.
  • the heme peroxidase protein is covalently attached to the support.
  • the invention also relates to an isolated heme peroxidase protein having a reactive residue introduced into the protein at at least one tethering position in the protein sufficiently distant from the active site of the protein such that, when the protein is immobilised to the support through the at least one reactive residue, the protein is from the support.
  • the tethering position is located on a plane of the protein opposite to the active site of the protein, or opposite to an entrance of the active site of the protein.
  • a reactive residue is introduced in at least two tethering positions in the protein.
  • the isolated heme peroxidase protein is horseradish peroxidase (HRP) or soya bean peroxidase (SBP) . Ideally, it is HRP.
  • the tethering position is selected from the group comprising: R118; R159; and R283.
  • the protein comprises a reactive residue at two tethering positions, generally at positions R118 and R159.
  • the protein comprises a reactive residue at the three tethering positions indicated above.
  • the reactive residue is a polar, uncharged or electrically charged, amino acid.
  • the reactive residue is selected from the group comprising: lysine; cysteine; glutamic acid, aspartic acid; and a reactive amino acid analogue.
  • the reactive residue is lysine.
  • the proteins of the invention will not include reactive residues at a position in the protein adjacent to the active site in the protein.
  • at least one of these residues will be modified to render the residue unreactive, typically by substitution with an unreactive residue or by chemical modification.
  • the protein is HRP
  • the unwanted reactive residue is replaced with an amino acid selected from the group comprising: valine; isoleucine; leucine; phenylalanine; and asparagine.
  • the lysine residue at position 232 is replaced with a phenylalanine (F) and the lysine residue located at position 241 is replaced with an asparagine (N) .
  • the protein is a recombinant protein, ideally recombinant plant protein.
  • the protein is recombinant plant HRP.
  • the protein comprises a suitable purification tag.
  • the invention therefore provides an isolated polypeptide comprising an amino acid sequence of SEQUENCE ID NO: 1, or a chemically active variant thereof, wherein at least one of the arginine residues located at positions 118, 159, and 283 is replaced with a reactive residue, ideally lysine. Typically, at least two of the arginine residues located at positions 118, 159, or 283 are replaced with a reactive residue. Ideally, each of the arginine residues located at positions 118, 159, or 283 is replaced with a reactive residue.
  • the reactive residue is selected from the group comprising: lysine; cysteine; glutamic acid, aspartic acid; and a reactive amino acid analogue.
  • At least one of the lysine residues located at positions 232, 241 and 174 is replaced with an unreactive residue.
  • two of the residues are replaced, these lysine residues preferably being those located at positions 232 and 241.
  • all of the lysine residues are replaced.
  • the lysine residue at position 232 is located at position 241 is replaced with an asparagine (N) .
  • isolated polypeptide of the invention are described in Table 1 in which Mutants 1, 2, 3 and 4 correspond with SEQUENCE ID NO'S: 2, 3, 4 and 5, respectively.
  • the invention therefore provides an isolated polypeptide comprising, or consisting essentially of, an amino acid sequence selected from the group comprising: SEQUENCE ID NO's: 2 to 5, or a chemically active variant thereof.
  • the invention also provides a support comprising an isolated heme peroxidase protein, or an isolated polypeptide, of the invention covalently attached thereto.
  • the invention also relates to mutant horseradish peroxidase proteins having improved thermal and solvent characteristics, and especially mutant horseradish peroxidase proteins having improved hydrogen peroxide stability.
  • the invention also provides an isolated polypeptide comprising an amino acid sequence of SEQUENCE ID NO: 1, or a chemically active variant thereof, and further comprising mutations selected from the group comprising: TIlOV; K232N; K241F; K232F; K232N/K241N; K232F/K241N.
  • the sequences of these mutant proteins are provided in SEQUENCE ID NO's: 6 to 11, respectively.
  • the invention also provides an isolated polypeptide comprising, or consisting essentially of, an amino acid sequence selected from the group comprising: SEQUENCE ID NO's: 6 to 11, or a chemically active variant thereof.
  • the isolated polypeptide comprises, or consists essentially of, a sequence selected from the group comprising: SEQUENCE ID NO: 6, 7 and 8. i _ molecule encoding an isolated polypeptide of the invention.
  • the invention also relates to the use of the isolated polypeptides of the invention in biocatalysis, biosensor, waste treatment, and therapeutic, applications.
  • heme peroxidase protein should be understood as meaning a peroxidase having a ferriprotoporphyrin IX prosthetic group located at the active site.
  • the plant enzymes horseradish peroxidase (HRP) and plant soyabean peroxidase (SBP) are examples of plant heme peroxidases.
  • support should be understood as meaning any type of solid phase or particle based support upon which the protein may be immobilised by means of any suitable means such as, for example, adsorption, entrapment or chemical attachment.
  • suitable supports include polyether sulphone membranes, particle-type support such as CNBr-activated beads, glyoxyl agarose, controlled pore glass, and chitosan. Other types of suitable supports will be known to those skilled in the art.
  • reactive residue should be understood as meaning any natural or un-natural amino acid, or amino acid analogue, or chemical derivative thereof, that is capable of reacting with a support, either on its own, or via a linker group.
  • the reactive residue is generally lysine, or another polar amino acid such as, for example, cysteine, aspartic acid, glutamic acid, and the like.
  • the structure and chemical properties of the active site allow the recognition and binding of the substrate.
  • the active site is usually a small pocket at the surface of, or within, the enzyme that contains residues responsible for the substrate specificity (charge, hydrophobicity, steric hindrance) and catalytic residues which often act as proton donors or acceptors or are responsible for binding a cofactor such as Pyridoxal, Thiamine or NAD.
  • the term "chemically active variant” should be taken to mean a variant or fragment of wild-type HRP that retains the enzymatic functionality of the parent HRP protein.
  • a "variant" of the HRP protein shall be taken to mean proteins having amino acid sequences which are substantially identical to wild-type HRP protein, especially plant wild-type HRP.
  • the term should be taken to include proteins or polypeptides that are altered in respect of one or more amino acid residues.
  • the term variant should be taken to exclude any polypeptides in which the residues at important positions in the protein are modified, i.e at the active site or at the tethering position.
  • such alterations involve the insertion, addition, deletion and/or substitution of 5 or fewer amino acids, more preferably of 4 or fewer, even more preferably of 3 or fewer, most preferably of 1 or 2 amino acids only. Insertion, addition and substitution with natural and modified amino acids is envisaged.
  • the variant may have conservative amino acid changes, wherein the amino acid being introduced is similar structurally, chemically, or functionally to that being substituted.
  • HRP proteins which have been altered by substitution or deletion of catalytically-important residues will be excluded from the term "variant".
  • f of residues in the active site is not envisaged.
  • the variant will have at least 60% amino acid sequence homology, preferably at least 70% or 80% sequence homology, more preferably at least 90% sequence homology, and ideally at least 95%, 96%, 97%, 98% or 99% sequence homology with wild-type plant HRP.
  • sequence homology comprises both sequence identity and similarity, i.e. a polypeptide sequence that shares 70% amino acid homology with wild-type plant HRP is one in which any 70% of aligned residues are either identical to, or conservative substitutions of, the corresponding residues in wild-type plant HRP.
  • variant is also intended to include chemically active isoforms of HRP, especially isoforms of plant HRP.
  • variant is also intended to include chemical derivatives of HRP protein, i.e. where one or more residues of HRP is chemically derivatized by reaction of a functional side group. Also included within the term variant are HRP molecules in which naturally occurring amino acid residues are replaced with amino acid analogues.
  • Proteins and polypeptides (including variants and fragments thereof) of and for use in the invention may be generated wholly or partly by chemical synthesis or by expression from nucleic acid.
  • the proteins and peptides of and for use in the present invention can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods known in the art (see, for example, J. M. Stewart and J. D. Young,
  • Fig. 1 is an illustration of wild-type HRP protein in which the arrow A points towards the active site and in which the three tethering positions (arginines) are identified by arrows B.
  • Fig. 2A is an illustration of mutant horseradish peroxidase according to the invention immobilised to a support in an orientated manner (with the arrow pointing towards the active site) .
  • Fig. 2B is an illustration of wild-type horseradish peroxidase immobilised to a support in an non-orientated manner (with the arrow pointing to the active site) .
  • Fig. 3 shows scanned images of DAB-stained, spotted immobilised rHRP activity. Time points indicated on the left are in hours. DAB coloration indicates peroxidase activity; this was noted within minutes for the mutant HRP polypeptides of the invention, whereas plant and wild-type recombinant HRP required a longer time to develop any coloration.
  • FIG. 1 there is illustrated a wild-type horseradish peroxidase protein molecule having the active site located on one plane of the molecule (indicated with the arrow A) and the tethering positions (represented by n ft niTM the arginine residues, Arg 118, 283 and P59A? 4 with arrows B) located on an opposite plane of the molecule, spatially separated from the active site.
  • the method of the invention one or more of the highlighted arginine residues is replaced with a reactive lysine residue, thereby providing one or more reactive residues at the tethering position (s) through which the molecule may be immobilised to a support in a desired orientation.
  • FIG. 2A there is illustrated a recombinant mutant horseradish peroxidase molecule according to the invention having two of the arginine residues shown in Fig. 1 substituted with reactive lysine residues.
  • the molecule is immobilised to the support through the reactive lysine residues thereby positioning the molecule in the correct orientation with the active site (arrow) facing away from the support.
  • wild- type recombinant horseradish peroxidase without the reactive residues located at the tethering position (s) is shown immobilised to the support in a random , non- orientated, fashion, with the active site facing into, or parallel to, the support.
  • access to the active site is easier when the molecule is immobilised in an orientated fashion as shown in Fig. 2A, and this has the effect of improving the efficiency of the immobilised enzyme .
  • Pall UltraBindTM US450 modified polyethersulfone affinity membranes The pQE60 vector was purchased from Qiagen (Valencia, CA) ; XL 10 Gold cells and QuickChangeTM Mutagenesis Kit were purchased from Stratagene (La Jolla, CA) .
  • the HRP gene was a generous gift from Prof. Frances H. Arnold (Caltech, CA, USA) .
  • the HRP gene was directionally cloned into the pQE60 vector as a fusion with the pectate lyase (PeIB) leader sequence (Lei S. P, Lin H. C, Wang S. S, Callaway J, Wilcox G. (1987). "Characterisation of the Erwinia carotovora pelB gene and its product pectate lyase.” Journal of Bacteriology, 169, 9, 4379-4383.) and a hexa- histidine purification tag, to generate pBR_I .
  • PeIB pectate lyase
  • E.coli XL 10 Gold was used as host strain to express the HRP protein.
  • a single cell transformed with pBR_I (or mutant derivative) was grown in LB medium containing 100 ⁇ g/mL ampicillin, 125 ⁇ g/mL tetracycline, 125 ⁇ g/mL chloramphenicol and 2% w/v glucose until the OD ⁇ oonm reached 0.4; the cells were removed via centrifugation at 2,000 x g for 5 min and resuspended in fresh LB supplemented with 100 ⁇ g/mL ampicillin, ImM ⁇ -ALA and 2mM CaCl 2 . The cells were then allowed to grow at 30 0 C, 220 rpm for 16 h.
  • the cells were centrifuged at 2,000 x g for 5 min and the supernatant was treated with 50% w/v (original volume, not % saturation) ammonium sulphate for 2 h at room temperature.
  • the cells were periplasmically lysed (French C, Keshavarz- Moore E, Ward J. M. (1996). Development of a simple method for the recovery of recombinant proteins from the E.coli periplasm. Enzyme Microbial Technology, 19, 332-338.) and the periplasmic contents were similarly treated with 50% w/v ammonium sulphate.
  • Proteins precipitated by 50% w/v ammonium sulphate were collected via centrifugation, resuspended in 5OmM phosphate buffer pH 7.5 and dialysed versus the same buffer overnight at 4°C.
  • Sodium chloride (IM) and GnCl (20OmM) were added to the dialysed fractions, and these latter were purified via nickel affinity chromatography at room temperature.
  • Sodium acetate (25mM, pH 4.5) was utilised to elute the bound HRP.
  • the eluted HRP was again dialysed versus 5OmM phosphate buffer pH 7.5 overnight at 4°C, after which the protein was concentrated (Amicon-Plus 20 concentrator tubes) , filter sterilised and stored at 4°C.
  • thermoinactivation time courses used 50 0 C.
  • the substrate ABTS (2, 2' -azino-bis ( 3-ethylbenzthiazoline-6- sulfonic acid) ) gives steady-state kinetics, permitting estimation of the apparent catalytic constant, k 3 app (Dunford HB. (1999) . "Heme Peroxidases”. VoI I, Wiley and Sons, New York, pp 1-23, 92-111) . 3, 3', 5,5'- Tetramethylbenzidine dihydrochloride (TMB; Josephy P. D, Eling T. and Mason R. P. (1982).
  • TMB 1 mg TMB was dissolved in 200 ⁇ L DMSO. This TMB solution was added to 9.8 mL of 10OmM Citric Acid buffer, pH5.5, and mixed to homogeneity (final DMSO and TMB concentrations 2% v/v and 32mM respectively) . To this solution, immediately prior to assay, 3 ⁇ L of H 2 O 2
  • TMB assay assay for remaining activity
  • Protein concentration was equalised in all experiments, to allow for the stabilising effect of protein-protein interactions.
  • One large sample was heated for fixed short time at progressively increasing temperatures, with transfers between water baths over relatively small temperature increments.
  • Initial temperature was 20 °C; the temperature was then increased in 10 °C increments over the range 20-40 °C and 5 °C increments over the range 40-80 0 C. Inspections were carried out on the resulting catalytic data to obtain the T 5 o, the temperature at which 50% catalytic activity was noted. All experiments were carried out in triplicate, with the average value used in T 50 inspections .
  • T 50 50 0 C
  • 50 ⁇ L aliquots were removed to a microplate on ice; however, in this procedure, time is varied whilst temperature remains constant. Aliquots were taken every minute for 10 min. The microplate containing the aliquots was then warmed to room temperature and assayed for catalytic activity (TMB assay) .
  • TMB assay catalytic activity
  • the results were expressed as a percent residual activity with respect to the recombinant enzyme in aqueous solution. All experiments were carried out in triplicate, with the average value used in C 50 calculations.
  • the C 50 value is the % v/v of organic solvent that reduces catalytic activity to half the initial value in aqueous solution.
  • the solvents used were dimethyl sulfoxide, methanol and dimethylformamide .
  • Recombinant HRP was prepared to a concentration of 360 nM in 50 mM phosphate buffer, pH 7.0. Reaction mixtures were set up with increasing concentrations of H 2 O 2 in 50 mM phosphate buffer, pH 7.0, in the range 0-50 mM. H 2 O 2 concentrations were determined spectrophotometrically at 240nm using 43.6 M -1 Cm "1 as the extinction coefficient (Hernandez-Ruiz J, Arnao M. B, Hiner A.N. P, Garcia-Canovas F. and Acosta M. (2001). Catalase- enzyme inactivation by H 2 O 2 . Biochemical Journal, 354,
  • the C 5 o value is the % v/v of H 2 O 2 that reduces catalytic activity to half the initial value in aqueous solution.
  • ABTS 2,2' -azino-bis (3-ethyl-benzthiazoline-6-sulphonic acid)
  • the present method was adapted from Childs and Bardsley (Childs R. E. and Bardsley W. G. (1975) .
  • a 20 mM stock solution of ABTS was prepared in 200 mM Na 2 HPO 4 / 100 mM citric acid pH 4.5 buffer.
  • a 100 mM H 2 O 2 stock solution was also prepared separately in the same.
  • a range of ABTS from 1.0 mM to 0.05 mM was prepared in buffer by serial dilution of the ABTS stock.
  • Recombinant HRP was prepared to 65 pM in 50 mM phosphate buffer, pH 7.0. This concentration of recombinant HRP yielded absorbance readings within the desired range of 0.1-0.9 at 405 nm.
  • 2.5 ⁇ L of 100 mM H 2 O 2 and 222.5 ⁇ L of the desired ABTS substrate dilution were aliquoted, followed by 25 ⁇ L of recombinant HRP to initiate the reaction.
  • Pall UltraBindTM Affinity Membrane Immobilisation Pall UltraBindTM affinity membranes comprise a polyethersulfone membrane modified with aldehyde surface chemistry, allowing direct protein immobilisation via free amine groups.
  • HRP (30 pM) , plant and recombinant, was immobilised onto the activated membrane as outlined in the manufacturer's instructions. The protein was resuspended in 50 mM sodium phosphate buffer, pH 7.5, and directly spotted onto the activated membrane; the latter was allowed to air-dry completely at room temperature for 10 min.
  • mutant HRP proteins suitable for "directional" immobilization to a support were generated employing the techniques described above.
  • the mutants may be identified as follows:
  • the wild type recombinant (SEQUENCE ID NO: 1) can bind effectively but not necessarily in optimal orientation.
  • SEQUENCE ID NO: 2 mutant which contains lysines substituted into the tethering positions 118, 159 and 283.
  • SEQUENCE ID NO: 4 mutant contains only the newly-substituted lysines at the tethering positions; the lysines close to the active site, Lys 232 and 241, have been substituted by other residues.
  • This mutant can immobilize only in the optimal orientation, i.e. with the active site facing out from the solid phase to the bulk liquid. Its greater effectiveness is shown in Figs 3.
  • T 50 value modelled lvalue, apparent half-life at 5O 0 C (t m ), calculated solvent C 50 , and apparent A 3 and K' m for each mutant. Values are the mean of three independent experiments in all cases. For all solvents studied, standard errors were ⁇ 5%.
  • Cys Asp Ala Ser lie Leu Leu Asp Asn Thr Thr Ser Phe Arg Thr Glu 50 55 60
  • Cys Arg Phe lie Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly Leu 180 185 190
  • Thr Pro Thr lie Phe Asp Asn Lys Tyr Tyr VaI Asn Leu Gl u Gl u Gin 225 230 235 240
  • Lys Gly Leu lie Gin ser Asp Gin Glu Leu Phe Ser Ser Pro Asn Ala 245 250 255
  • Cys Asp Ala Ser lie Leu Leu Asp Asn Thr Thr Ser Phe Arg Thr Glu 50 55 60
  • Cys Arg Phe lie Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly Leu 180 185 190
  • Lys Gly Leu lie Gin Ser Asp Gin Glu Leu Phe Ser Ser Pro Asn Ala 245 250 255
  • Cys Asp Ala Ser lie Leu Leu Asp Asn Thr Thr Ser Phe Arg Thr Glu 50 55 60
  • Cys Arg Phe lie Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly Leu f
  • Lys Gly Leu lie Gin Ser Asp Gin Glu Leu Phe ser Ser Pro Asn Ala 245 250 255
  • Cys Asp Ala ser lie Leu Leu Asp Asn Thr Thr ser Phe Arg Thr Glu 50 55 60
  • Cys Arg Phe lie Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly Leu 180 185 190
  • Cys Asp Ala Ser lie Leu Leu Asp Asn Thr Thr Ser Phe Arg Thr Glu 50 55 60
  • Cys Arg Phe lie Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly Leu 180 185 190
  • Page 8 10107wo.ST25 lie val Arg Asp Thr lie val Asn Glu Leu Arg ser Asp Pro Arg lie 20 25 30
  • Cys Asp Ala ser lie Leu Leu Asp Asn Thr Thr Ser Phe Arg Thr Glu 50 55 60
  • Cys Arg Phe lie Met Asp Arg Leu Tyr Asn Phe ser Asn Thr Gly Leu 180 185 190
  • Lys Gly Leu lie Gin Ser Asp Gin Glu Leu Phe Ser Ser Pro Asn Ala 245 250 255
  • Ala Ala ser lie Leu Arg Leu His Phe His Asp Cys Phe val Asn Gly 35 40 45
  • Cys Asp Ala Ser lie Leu Leu Asp Asn Thr Thr Ser Phe Arg Thr Glu 50 55 60
  • Cys Arg Phe lie Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly Leu
  • Lys Gly Leu lie Gin Ser Asp Gin Glu Leu Phe Ser Ser Pro Asn Ala 245 250 255
  • Cys Asp Ala Ser lie Leu Leu Asp Asn Thr Thr Ser Phe Arg Thr Glu 50 55 60
  • Cys Arg Phe lie Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly Leu 180 185 190
  • Cys Asp Ala Ser lie Leu Leu Asp Asn Thr Thr Ser Phe Arg Thr Glu 50 55 60
  • Cys Arg Phe lie Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly Leu 180 185 190
  • Cys Asp Ala Ser lie Leu Leu Asp Asn Thr Thr Ser Phe Arg Thr Glu 50 55 60
  • Cys Arg phe lie Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly Leu 180 185 190
  • Thr Pro Thr lie Phe Asp Asn Asn Tyr Tyr val Asn Leu Gl u Gl u Gin 225 230 235 240
  • Ala Ala ser lie Leu Arg Leu His Phe His Asp Cys Phe VaI Asn Gl y 35 40 45
  • Cys Asp Ala Ser lie Leu Leu Asp Asn Thr Thr ser Phe Arg Thr Gl u 50 55 60
  • Cys Arg Phe lie Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly Leu 180 185 190

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Abstract

La présente invention concerne un polypeptide isolé comprenant la séquence d'acides aminés de SEQUENCE ID NO: 1 ou un variant chimiquement actif de ce dernier, ledit polypeptide comprenant une mutation sélectionnée dans le groupe formé par: TIlOV; K232F/N et K241F/N. Cette invention concerne également un procédé de production d'une protéine de peroxydase hématique convenant pour l'immobilisation sur un support qui comprend les étapes suivantes: l'introduction d'un résidu réactif dans la protéine au niveau d'au moins une position de liaison dans la protéine- la position de liaison est suffisamment distante du site actif de la protéine pour que, lorsque la protéine est immobilisée sur le support par l'intermédiaire du ou des résidus réactifs, la protéine soit orientée de sorte que le site actif soit sensiblement situé loin du support. Lorsque la protéine est une protéine de peroxydase de raifort, la position de liaison est sélectionnée dans le groupe formé par: R118; R159 et R283. La présente invention concerne également un polypeptide isolé comprenant une séquence d'acides aminés de SEQUENCE ID NO: 1 ou un fragment biologiquement actif de ce dernier ou encore un variant de ce dernier, dans lequel au moins un des résidus arginine situés aux positions 118, 159 et 283 est remplacé par un résidu réactif.
PCT/IE2007/000124 2006-12-19 2007-12-18 Polypeptides de peroxydase hématique WO2008075325A2 (fr)

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EP06394027 2006-12-19
EP06394027.4 2006-12-19

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EP2768953A1 (fr) * 2011-10-19 2014-08-27 Technische Universität Graz Isoenzymes de la peroxydase du raifort
CN108239626A (zh) * 2016-12-27 2018-07-03 丰益(上海)生物技术研发中心有限公司 一种高酯化活力的脂肪酶突变体
EP3885436A1 (fr) * 2020-03-24 2021-09-29 Technische Universität Wien Polypeptides ayant une activité de peroxydase
WO2021191245A1 (fr) * 2020-03-24 2021-09-30 Technische Universität Wien Polypeptides à activité de peroxydase
CN117887681A (zh) * 2024-03-13 2024-04-16 潍坊新希望六和饲料科技有限公司 一种辣根过氧化物酶在制备脱霉饲料中的应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2768953A1 (fr) * 2011-10-19 2014-08-27 Technische Universität Graz Isoenzymes de la peroxydase du raifort
CN108239626A (zh) * 2016-12-27 2018-07-03 丰益(上海)生物技术研发中心有限公司 一种高酯化活力的脂肪酶突变体
CN108239626B (zh) * 2016-12-27 2022-10-04 丰益(上海)生物技术研发中心有限公司 一种高酯化活力的脂肪酶突变体
EP3885436A1 (fr) * 2020-03-24 2021-09-29 Technische Universität Wien Polypeptides ayant une activité de peroxydase
WO2021191245A1 (fr) * 2020-03-24 2021-09-30 Technische Universität Wien Polypeptides à activité de peroxydase
CN117887681A (zh) * 2024-03-13 2024-04-16 潍坊新希望六和饲料科技有限公司 一种辣根过氧化物酶在制备脱霉饲料中的应用
CN117887681B (zh) * 2024-03-13 2024-05-24 潍坊新希望六和饲料科技有限公司 一种辣根过氧化物酶在制备脱霉饲料中的应用

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