WO2015120314A1 - Polypeptides possédant une activité nitrile hydratase et polynucléotides codant pour ceux-ci - Google Patents

Polypeptides possédant une activité nitrile hydratase et polynucléotides codant pour ceux-ci Download PDF

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Publication number
WO2015120314A1
WO2015120314A1 PCT/US2015/014889 US2015014889W WO2015120314A1 WO 2015120314 A1 WO2015120314 A1 WO 2015120314A1 US 2015014889 W US2015014889 W US 2015014889W WO 2015120314 A1 WO2015120314 A1 WO 2015120314A1
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polypeptide
nitrile
seq
functional group
compound
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PCT/US2015/014889
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English (en)
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Vahe BANDARIAN
Micah NELP
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Arizona Board Of Regents For The University Of Arizona
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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
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/38—Nucleosides
    • C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01084—Nitrile hydratase (4.2.1.84)

Definitions

  • the present invention relates to isolated polypeptides having nitrile hydratase activity and isolated polynucleotides encoding said polypeptides.
  • the invention also relates to nucleic acid constructs, vectors, and host cells comprising said polynucleotides as well as methods of producing and using said polypeptides.
  • One particular embodiment of the invention relates to a method for converting a nitrile functional group in a compound to an amide functional group.
  • Nitrile hydratases catalyze the industrially important conversion of nitriles to amides under mild conditions and are a rare example of metalloenzymes that ligate trivalent cobalt or iron in non-corrin and non-heme environments.
  • NHases Nitrile hydratases
  • the active site complexes of both metal forms are similar in many respects: each is low spin with low redox potentials, and they share the same set of ligands, including three cysteine sulfurs, two of which are post- translationally oxidized, one to cysteine-sulfenic acid and the other to cysteine sulfuric acid, both of which are necessary for activity.
  • 3 Along with the closely related thiocyanate hydrolase, NHases are currently the only known enzymes to employ these unusual modifications in metal binding and possibly catalysis. 4
  • polypeptides having nitrile hydratase-activity are selected from the group consisting of: (a) a polypeptide comprising, often essentially of, an amino acid sequence having at least about 75%, typically at least 85%, and often at least 95% sequence identity to the mature polypeptide of SEQ ID NO:2; (b) a polypeptide encoded by a polynucleotide that hybridizes under at least high stringency conditions, often under at least very high stringency conditions with (i) the mature polypeptide coding sequence consisting essentially of of SEQ ID NO: l, (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence consisting essentially of of SEQ ID NO: l, or (iii) the full-length complement of (i) or (ii), wherein very high stringency conditions are defined as prehybridization and hybridization at 42 °C in 5 x SSPE, 0.3% SDS, 200
  • polypeptide SEQ ID NO:2 includes histidine tag, which is optional.
  • the histidine tag is the first 20 amino acid sequences in SEQ ID NO:2, in particular amino acid sequence of MGS SHHHHHHS SGLVPRGSH .
  • polynucleotide SEQ ID NO: l includes coding sequence for this amino acid sequence, in particular the first 60 nucleotide sequences which corresponds to "ATGGGCAGCA GCCATCATCA TCATCATCAC
  • AGCAGCGGCC TGGTGCCGCG CGGCAGCCAT represents the histidine tag portion.
  • nucleotide or the polypeptide (not including the histidine tag portion) has about no more or less than 10%, typically, no more or less than 5%, and often no more or less than 1% additional nucleotide or amino acid residue.
  • a polypeptide consists essentially of amino acid SEQ ID NO:2, which has 195 amino acids (not including the histidine tag)
  • a polynucleotide consists essentially of nucleotide of SEQ ID NO: l, which has 588 nucleotides (again not including histidine tag), it means such a polynucleotide has 588 ⁇ 10% (i.e., 59) nucleotides, typically 588 ⁇ 5% (i.e., 29) nucleotides, and often 588 ⁇ 1% (i.e., 6) nucleotides.
  • polypeptides of the present invention provide isolated polynucleotides encoding the polypeptides of the present invention; nucleic acid constructs, recombinant expression vectors, and recombinant host cells comprising the polynucleotides; and methods of producing the polypeptides.
  • Still other aspects of the invention provide methods for converting a nitrile compound to an amide compound, said methods comprising: treating a nitrile compound with a polypeptide disclosed herein having nitrile hydratase activity under conditions sufficient to produce an amide compound.
  • any nitrile compounds can be converted to a corresponding amide compound using methods of the invention.
  • the nitrile compound is toyocamycin. In such instances, the resulting amide compound is sangivamycin.
  • the nitrile compound is 3-cyanopyridine,
  • the polypeptide comprises the amino acid sequence of
  • SEQ ID NO: 2 or the mature polypeptide thereof; or a fragment thereof having nitrile hydratase activity.
  • said polypeptide is encoded by the polynucleotide contained in plasmid pET28a in E. coli strain BL21(DE3).
  • nucleic acid construct comprising an isolated polynucleotide encoding the polypeptide described herein, wherein said polynucleotide is operably linked to one or more heterologous control sequences that direct the production of the polypeptide in an expression host.
  • Still other aspects of the invention provide a recombinant host cell comprising a polynucleotide described herein.
  • the polynucleotide comprises a sequence that encodes the polypeptide described herein.
  • Yet other aspects of the invention provide a recombinant host cell comprising within its genome (or inserted within its genome) a nucleotide sequence having at least 75%, typically at least 85%, and often at least 95% sequence identity to sequence of SEQ ID NO: 1 or a cDNA sequence thereof.
  • Still another aspect of the invention provides a method for converting a nitrile functional group in a compound to an amide functional group.
  • Such a method generally includes contacting a compound having a nitrile functional group (i.e., -CN) to an amide functional group (i.e., -CONH 2 ) in the presence of any of the polypeptides and/or recombinant hose cells of the invention.
  • Figure 1 Analytical size exclusion with ToyJ and standards.
  • Figure 2 UV-visible comparison of ToyJ and ToyJKL
  • Figure 3 ESI-MS of ToyJ showing full envelope. The +27, +25, and +23 charge state of the fully modified ToyJ species are highlighted.
  • Orbitrap Velos with a mass width of 1.6 and resolution of 100,000.
  • FIG. 5 EPR of ToyJKL. Black trace, EPR spectrum of ToyJKL (496 ⁇ ) chemically reduced to the Co(II) state with sodium dithionite (10 mM). Red trace that does not show any discernible EPR signals is the spectrum of ToyJKL (220 ⁇ ) not treated with reductant. Experimental conditions: microwave frequency, 9.335 GHz; microwave power, 2 mW; magnetic field modulation amplitude, 0.5 mT; temperature, 30 K.
  • Figure 6 ToyJ activity at various enzyme concentrations.
  • Figure 7 HPLC trace of the product sangivamycin and substrate toyocamycin.
  • Figure 8 Michaelis-Menten plots of ToyJKL and ToyJ catalyzed conversion of toyocamycin to its amide form, sangivamycin.
  • Figure 9 Comparison of activity with ToyJ and heat denatured ToyJ. All assays contained 0.5 mM toyocamycin and 10 ⁇ protein ToyJ.
  • Figure 10 ToyJ activity in hydrating 3-cyanopyridine.
  • Figure 11 ToyJKL activity in hydrating 3-cyanopyridine.
  • Isolated polypeptides of the present invention have at least about 50%>, at least about 60%, at least about 70%, at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, and often at least 100% of the nitrile hydratase activity of the mature polypeptide of SEQ ID NO: 2.
  • the term "about” refers to ⁇ 20%, typically ⁇ 10%, and often ⁇ 5% of the numeric value.
  • nitrile hydratase enzyme activity is or can be determined by measuring the conversion of toyocamycin to sangivamycin by nitrile hydratase enzyme(s) under the following conditions: 10 ⁇ of nitrile hydratase enzyme protein/ 50 ⁇ -5 mM of toyocamycin in 0.1 mL of 0.05 M potassium phosphate (pH 6.5) for 3 min at a suitable temperature, e.g., 10-50 °C, compared to a control without addition of nitrile hydratase enzyme protein.
  • Product is detected by reverse-phase HPLC analysis using a Zorbax C-18 column with 50 mM triethylammonium acetate (pH 6.8) as the mobile phase.
  • Nitrile hydratases possess a mononuclear iron or cobalt cofactor whose coordination environment includes rare post-translationally oxidized cysteine sulfenic and sulfuric acid ligands. This cofactor is located in the a- subunit at the interfacial active site of the heterodimeric enzyme.
  • toyocamycin nitrile hydratase is a unique three-subunit member of this family involved in the metabolism of pyrrolopyrimidine antibiotics. The three subunits of TNHase are homologous to the a- and ⁇ -subunits of prototypical NHases.
  • Some aspects of the invention provide the active site complex containing a- subunit, optionally with a histidine tag, that has been expressed apart from the other two subunits.
  • the UV-visible and mass spectra of the protein provide evidence that this subunit alone is capable of synthesizing the active site complex with full post-translational
  • the a- subunit is also catalytically active with the natural substrate, toyocamycin, as well as the niacin precursor 3-cyanopyridine. Comparisons of the steady state kinetic parameters of the single subunit variant to the heterotrimeric protein show that the additional subunits impart additional substrate specificity and catalytic efficiency.
  • the present inventors have discovered that the -subunit is the minimal sequence needed for nitrile hydration. Moreover, it was discovered that the a-subunit provides a simplified scaffold for conversion of a nitrile compound to an amide compound under a mild conditions as well as allowing one to study the mechanism and post-translational modification of this important class of catalysts.
  • TNHase toyocamycin nitrile hydratase
  • ToyJ is homologous to the NHase a- subunit and ToyK and ToyL to the C- and N-terminal halves, respectively, of the NHase ⁇ - subunit.
  • the Streptomyces protein is heterotrimeric.
  • ToyJKL is also homologous to thiocyanate hydrolase, which shares with ToyJKL a composition of three different subunits. 11
  • ToyJKL with ToyL N-terminally His 6 -tagged was purified from E. coli grown in media supplemented with CoCL (50 ⁇ ). ToyJKL is orange and exhibits a UV- visible spectrum that is typical of other cobalt containing nitrile hydratases, which exhibits low intensity shoulders at 314 nm and 429 nm (blue trace in Figure l). 12 The protein concentration was determined for several preparations by amino acid analysis and metal content by ICP-OES, the results of which revealed there are 0.6 ⁇ 0.2 mol cobalt atoms per heterotrimer in the recombinant heterologously expressed protein.
  • ESI-MS was used to further confirm the presence of the post-translational modification, which was inferred from the similarities between the UV-visible spectra of ToyJ and ToyJKL.
  • an Orbitrap Velos mass spectrometer was used because it affords the sensitivity and resolution to clearly differentiate between protein that is fully or partially modified. Deconvolution of the mass envelope (Fig. 3) showed that the most abundant species was consistent with the mass of ToyJ that lacks the starting methionine, as expected for a bacterial recombinant protein.
  • EPR electron paramagnetic resonance
  • This disclosure provides insight into the vast differences in the catalytic efficiency between model complexes and NHases.
  • the activity of ToyJ demonstrates what is possible in a single molecule with a significantly truncated hydrogen bonding network relative to the complete enzyme.
  • polypeptide having nitrile hydratase activity is selected from the group consisting of: (a) a polypeptide comprising, often consisting essentially of, an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 2; (b) a polypeptide encoded by a
  • polypeptide that hybridizes under at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 , (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1 , or (iii) the full-length complement of (i) or (ii), wherein very high stringency conditions are defined as prehybridization and hybridization at 42 °C in 5 x SSPE, 0.3% SDS, 200 g/mL sheared and denatured salmon sperm DNA, and 50%) formamide, and washing three times each for 15 minutes using 2 x SSC, 0.2%> SDS at 70 °C; and (c) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having, often consisting essentially of, at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or a cDNA sequence thereof; wherein said polypeptide has the
  • the polypeptide consists essentially of amino acid shown in SEQ ID NO: 2 or the mature polypeptide thereof; or a fragment thereof having nitrile hydratase activity.
  • fragment thereof refers to a polypeptide consisting of about 60% or more, typically about 70% or more, often about 80% or more, more often about 90%) of more of the reference polypeptide or polynucleotide.
  • the polypeptide is encoded by the polynucleotide contained in plasmid pET28a in E. coli strain BL21(DE3).
  • Another aspect of the invention provides a nucleic acid construct comprising an isolated polynucleotide encoding the polypeptide described herein.
  • the polynucleotide is operably linked to one or more heterologous control sequences that direct the production of the polypeptide in an expression host.
  • Still another aspect of the invention provides a recombinant host cell comprising a polynucleotide comprising a nucleotide sequence that encodes a polypeptide consisting essentially of SEQ ID NO:2.
  • a recombinant host cell comprising a polynucleotide comprising a nucleotide sequence that encodes a polypeptide consisting essentially of SEQ ID NO:2.
  • any microorganism e.g., bacteria, that can be used to express (e.g., produce) the polypeptide of the invention can be used as the host cell.
  • Yet another aspect of the invention provides a method for producing an amide compound from a nitrile compound.
  • Such a method comprises contacting a nitrile compound with a polypeptide of the invention or a recombinant host cell of the invention, both of which are disclosed herein, or a combination thereof under conditions sufficient to produce an amide compound.
  • polypeptide consisting essentially of an a-subunit of toyocamycin nitrile hydratase (TNHase), wherein said polypeptide comprises an active site for converting a nitrile functional group to an amide functional group.
  • TNFase toyocamycin nitrile hydratase
  • the polypeptide has an activity for converting toyocamycin to
  • Still yet another aspect of the invention provides a metal-polypeptide complex.
  • the metal-polypeptide complex comprises a polypeptide unit and a metal.
  • the polypeptide unit consists essentially of SEQ ID NO:2 or a fragment thereof, and a transition metal ion.
  • the metal-polypeptide complex is capable of converting a nitrile functional group of a compound to an amide functional group.
  • the transition metal ion comprise cobalt.
  • Another aspect of the invention provides a method for converting a nitrile functional group to an amide functional group in a compound.
  • Such a method comprises contacting a compound comprising a nitrile functional group with a metal-polypeptide complex of the invention under conditions sufficient to convert said nitrile functional group to an amide functional group.
  • ToyJ and ToyJKL Plasmids containing all three subunits were prepared from those constructed and described previously (McCarty et ah, 2008). For expression of His 6 -ToyJ, toyJ was cloned into the Ndel and Xhol sites of pET28a. ToyJKL, which was His 6 -tagged on the g subunit was prepared by co-expression of toyJ, toyK, and toyL in E. coli as follows. ToyJ was expressed by cloning toy J into the Ndel/Xhol sites of pAC YCDuet- 1. ToyK was expressed by introducing toyK into the Ndel/Xhol sites of pCDFDuet-1. ToyL was expressed from a plasmid containing toyL in Ndel/Xhol sites of pET28a.
  • Protein was eluted with a linear gradient from 0.05 M potassium phosphate buffer containing 0.05 M imidazole at pH 7.4 to one containing 0.5 M imidazole over 25 mL.
  • Orange fractions containing TNHase were identified by SDS-PAGE, pooled, and concentrated using an Amicon Vivaspin Turbo centrifugal concentrator (PES 10K membrane).
  • the protein was buffer exchanged into 0.05 M HEPES'NaOH (pH 7.5) using a Bio-Rad 10DG column. Protein aliquots were frozen in liquid nitrogen and stored at - 80 °C.
  • Protein concentration was determined using the Bradford method (BioRad) with BSA as a standard. Garratt Callahan (Burlingame, CA) performed ICP-OES to determine metal content. Amino acid analysis was conducted by the Molecular Structure Facility at the University of California, Davis. Empirically determined correction factors of 0.3 for ToyJ and 0.55 for ToyJKL were found to correlate the Bradford assay results to the amino acid analysis.
  • Activity assays Enzyme concentrations used are that of cobalt concentration as determined by ICP-OES. Reactions to determine protein activity contained 0.05 M potassium phosphate (pH 6.5), varying concentrations of toyocamycin (Berry and Associates) from 0.1 to 2.5 mM and varying concentrations of 3-cyanopyridine from 0.05 to 0.75 M. The reactions were initiated by addition of 10 ⁇ ToyJ and quenched at various times by addition of 30% (w/v) trichloroacetic acid to a final concentration of 1.5% (w/v).
  • the products sangivamycin or nicotinamide and residual toyocamycin or 3-cyanopyridine were quantified by HPLC using an Agilent Zorbax Eclipse XDB-C-18 (4.6 x 50 mm) with an isocratic elution of 80% 0.1 M triethyl ammonium acetate (pH 6.8) and 20% methanol at a flow rate of 0.75 mL/min.
  • Sangivamycin elutes at 2.3 min
  • 3- cyanopyridine eluted at 2.3 min.
  • UV-visible spectroscopy UV-visible spectra were obtained using an Agilent 8453
  • Mass spectrometry of ToyJ Mass spectra were obtained using an LTQ Velos
  • ToyJ was prepared by exchanging 0.1 mL aliquots into deionized water using a nick column (GE Healthcare). This was lyophilized and resuspended in a 7:3 solution of 0.1% trifluoroacetic acid and methanol to a final concentration of 0.1 mM. This was directly infused using an Advion Nanomate in LC Coupler at a flow rate of 200 nL/min with an applied voltage of 2.0 kV. Resolution was set to 100,000 from 400 to 1800 m/z with 4.361 msec ion injection time with two microscans.
  • ToyJKL in 0.05 M KPi pH 7.4 was lyophilized and resuspended in anaerobic water and 20% glycerol in a Coy anaerobic glove. It was reduced by addition of sodium dithionite to a final concentration of 10 mM; the final concentration of cobalt was 0.5 mM as determined by ICP-OES. EPR samples of this were frozen in liquid nitrogen anaerobically.
  • HEPES pH 7.4 0.05 M HEPES pH 7.4 were brought to 20% glycerol; the final concentrations of cobalt in each were 0.15 and 0.22 mM, respectively, as determined by ICP-OES.
  • EPR spectra were obtained using a Bruker Elexsys E500 spectrometer at the microwave frequency of 9.335 GHz, microwave power of 2 mW, and the magnetic field modulation amplitude of 0.5 mT.
  • the measurement temperature was 30 K.
  • oligomeric state with size exclusion chromatography The oligomeric states of ToyJ and ToyJKL were determined using analytical size exclusion chromatography with a HiPrep 16/60 Sephacryl S200 column (GE Healthcare) and gel filtration protein standard (Biorad 151-1901) as indicated in the figure. The enzymes and standards were injected into the column pre-equilibrated with 0.02 M HEPES pH 7.4 and 0.15 M NaCl and eluted over 150 mL at 0.1 mL/min. Elution volumes were determined using UV traces and SDS- PAGE analysis.
  • some of the compounds whose nitrile functional group was converted to an amide functional group include: (PreQO) ; cyanopyridine); ' (acrylamide); (butyronitrile); and (benzylcyanide).

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Abstract

La présente invention concerne des polypeptides isolés possédant une activité nitrile hydratase et des polynucléotides isolés codant pour lesdits polypeptides. L'invention concerne également des constructions d'acides nucléiques, des vecteurs et des cellules hôtes comprenant lesdits polynucléotides ainsi que des procédés de production et d'utilisation desdits polypeptides.
PCT/US2015/014889 2014-02-07 2015-02-06 Polypeptides possédant une activité nitrile hydratase et polynucléotides codant pour ceux-ci WO2015120314A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5789211A (en) * 1991-05-02 1998-08-04 Nitto Chemical Industry Co., Ltd. Gene encoding a polypeptide having nitrile hydratase activity, a transformant containing the gene and a process for the production of amides using the transformant
US20020127633A1 (en) * 1999-10-06 2002-09-12 Board Of Trustees Operating Michigan State University Modified synthetases to produce penicillins and cephalosporins under the control of bicarbonate
US20070154997A1 (en) * 2003-05-08 2007-07-05 Payne Mark S Nucleic acid fragments encoding nitrile hydratase and amidase enzymes from comamonas testosteroni 5-MGAM-4D and recombinant organisms expressing those enzymes useful for the production of amides and acids
WO2014009473A1 (fr) * 2012-07-12 2014-01-16 Novozymes A/S Polypeptides ayant une activité lipase et polynucléotides codant pour ceux-ci

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5789211A (en) * 1991-05-02 1998-08-04 Nitto Chemical Industry Co., Ltd. Gene encoding a polypeptide having nitrile hydratase activity, a transformant containing the gene and a process for the production of amides using the transformant
US20020127633A1 (en) * 1999-10-06 2002-09-12 Board Of Trustees Operating Michigan State University Modified synthetases to produce penicillins and cephalosporins under the control of bicarbonate
US20070154997A1 (en) * 2003-05-08 2007-07-05 Payne Mark S Nucleic acid fragments encoding nitrile hydratase and amidase enzymes from comamonas testosteroni 5-MGAM-4D and recombinant organisms expressing those enzymes useful for the production of amides and acids
WO2014009473A1 (fr) * 2012-07-12 2014-01-16 Novozymes A/S Polypeptides ayant une activité lipase et polynucléotides codant pour ceux-ci

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MCCARTY, R ET AL.: "Rosetta Stone For Deciphering Deazapurine Biosynthesis: Pathway For Pyrrolopyrimidine Nucleosides Toyocamycin And Sangivamycin.", CHEM BIOL., vol. 15, no. 8, 25 August 2008 (2008-08-25), pages 1 - 17, XP025533985 *

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