WO2010097619A1 - Enzymes et cofacteurs immobilisés - Google Patents

Enzymes et cofacteurs immobilisés Download PDF

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Publication number
WO2010097619A1
WO2010097619A1 PCT/GB2010/050315 GB2010050315W WO2010097619A1 WO 2010097619 A1 WO2010097619 A1 WO 2010097619A1 GB 2010050315 W GB2010050315 W GB 2010050315W WO 2010097619 A1 WO2010097619 A1 WO 2010097619A1
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Prior art keywords
enzyme
composite
factor
carrier material
dehydrogenase
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PCT/GB2010/050315
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English (en)
Inventor
Anne-Marie Hickey
Charlotte Wiles
Bongkot Ngamsom
Paul Watts
Gillian M Greenway
Jenny Littlechild
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The University Of Hull
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Publication of WO2010097619A1 publication Critical patent/WO2010097619A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • 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
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • 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
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/002—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by oxidation/reduction reactions
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic

Definitions

  • the present invention relates to the immobilization of an enzyme and a corresponding co-factor in such a way so as to provide regeneration means for the co-factor.
  • an immobilized enzyme composite which comprises an enzyme, a co-factor for the enzyme and a carrier material.
  • the composite may be electrically conductive.
  • methods of making an immobilized enzyme composite, methods of catalyzing a reaction and apparatus for carrying out such methods are also included in the present disclosure, inter alia, are methods of making an immobilized enzyme composite, methods of catalyzing a reaction and apparatus for carrying out such methods.
  • a co-factor is a non-protein chemical compound that acts as a stoichiometric agent in biotransformation reactions.
  • the most commonly used co-factors include organic compounds such as nicotinamide adenine dinucelotide NAD(P) + /NAD(P)H, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and adenosine trisphosphate (ATP).
  • Co-factors are modified during catalysis and therefore, the enzyme cannot catalyze further reactions until the co-factor is replaced or regenerated back to its original state. Often the co-factor is much more expensive than the desired reaction product and so efficient regeneration of co-factors is essential for large-scale synthetic applications. A high reaction rate is often difficult to achieve if the products are allowed to accumulate in the reaction media and it is therefore advantageous to allow continuous feeding of substrates and removal of products in large-scale synthetic applications. Continuous flow reactors have been applied successfully with enzymes that do not require co- factors. However, when continuous flow reactions are used in conjunction with co- factor-associated reactions, the co-factors leave the reaction media along with the products, thus leading to the need to replace the co-factors which as indicated is expensive.
  • TTN Total turnover number
  • TTNs of 10 3 - 10 5 may be sufficient to make a process economically viable, depending on the value of the target material. Electrochemical regeneration methods typically have TTNs of less than 10 3 (Faber 1997. Biotransformations in Organic Chemistry, 3 rd ed. Springer: Berlin, pp 160).
  • the TTN of electrochemical regeneration techniques can be improved by increasing the electrochemical surface area by using a conducting material.
  • Siu et al Solid State Phe., 1087, 1124
  • VOTP vanadium
  • TMOS tetramethly orthosilicate
  • Siu et al utilized a solution of NAD + and thus required separation of NAD + from the end product. Siu et al did not consider a system in which an enzyme is co-immobilized with a co-factor. In addition, the vanadium is believed to leach out of the matrix over time, causing regeneration to reduce and eventually stop.
  • an immobilized enzyme composite which comprises (a) an enzyme, (b) a co-factor for said enzyme and (c) a carrier material, wherein the composite is electrically conductive.
  • the composite is electrically conductive.
  • at least one of the enzyme and the co-factor is immobilized on the carrier material or at least one of the enzyme and the co-factor is encapsulated within the carrier material.
  • an immobilized enzyme composite which comprises (a) an enzyme, (b) a co-factor for said enzyme, (c) an electrically conductive material; and (d) a carrier material, wherein the carrier material comprises a porous material.
  • the carrier material comprises pores, opening or channels or a combination thereof.
  • the carrier material may be a solid support and may be selected from a silica-based material e.g. glass, sol-gel, a membrane, a silica gel, agarose, a ceramic and a polymer which comprises a carboxygroup and amine reactive group.
  • the carrier material is porous.
  • the carrier material is a glass matrix e.g. a controlled pore glass (CPG) matrix. Further details of the carrier material are provided below.
  • the carrier material is functionalized to provide a reactive linking group.
  • the composite comprises an electrically conducting material.
  • the electrically conducting material is an electrically conductive polymer immobilized on the carrier material.
  • the electrically conductive polymer may be immobilized on the carrier material via a linkage selected from an amide linkage, an epoxide linkage, an alkylbromide linkage, an imide linkage, an aldehyde linkage and an alcohol linkage.
  • the electrically conductive polymer is a non-swellable polymer.
  • the polymer is selected from poly 3,4-ethylenedioxythiophene (PEDOT), polypyrrole, polyanilines, polyacetylenes, polythiophenes, and blends thereof and derivatives thereof e.g. substituted polypyrrole, polyaniline and polythiophene and combinations thereof.
  • the polymer is polypyrrole.
  • the enzyme is an oxidoreductase. In one embodiment, the enzyme is a dehydrogenase. In an embodiment, the enzyme is selected from an alcohol dehydrogenase, a homoserine dehydrogenase, an aminopropanol oxidoreductase, diacetyl reductase, glycerol dehydrogenase, propanediol phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, D-xylulose reductase, L-xylulose reductase, lactate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, HMG-CoA dehydrogenase, biliverdin reductase, dihydrofolate reductase, glutathione reductase, thioredoxin reductase, horse liver alcohol dehydrogenase,
  • the co-factor is selected from co-enzyme Q, glutathione, co-enzyme B, nicotinamide adenine dinucleotide (NAD + /NADH), nicotinamide adenine dinucleotide phosphate (NADP + /NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and an analog thereof.
  • the co-factor may be modified as compared to a naturally occurring co-factor.
  • the co-factor may comprise a modifying group, e.g. PEI, dextran, PEG, polylysine or poly(acrylic acid) (PAA).
  • the co-factor may further comprise a tag molecule e.g. a poly histidine tag.
  • a support comprising; (a) an enzyme and (b) a co-factor of said enzyme each immobilized thereon, wherein the support is electrically conductive.
  • the support may comprise an electrically conducting material.
  • a support comprising; (a) an enzyme and (b) a co-factor of said enzyme each immobilized on the support, wherein the support is electrically conductive and wherein the support comprises a porous material.
  • the support may comprise an electrically conducting material.
  • the support comprises a glass matrix, a membrane, a silica gel, a sol-gel derived matrix or agarose. In one embodiment, the support comprises a derivatized glass matrix.
  • the support comprises a controlled pore glass (CPG) matrix.
  • the support comprises an electrically conducting material.
  • the electrically conducting material may be an electrically conductive polymer.
  • the electrically conductive polymer may be a polymer selected from poly 3,4-ethylenedioxythiophene (PEDOT), polypyrrole, polyanilines, polyacetylenes, polythiophenes, and blends thereof and derivatives thereof e.g. substituted polypyrrole, polyaniline and polythiophene and combinations thereof.
  • the polymer is polypyrrole.
  • the support comprises an enzyme which is an oxidoreductase e.g. a dehydrogenase.
  • the enzyme is selected from an alcohol dehydrogenase, a homoserine dehydrogenase, an aminopropanol oxidoreductase, diacetyl reductase, glycerol dehydrogenase, propanediol phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, D-xylulose reductase, L-xylulose reductase, lactate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, HMG-CoA dehydrogenase, biliverdin reductase, dihydrofolate reductase, glutathione reductase, thioredoxin reductase, horse liver alcohol dehydrogenase
  • the support comprises a co-factor is selected from co-enzyme Q, glutathione, co-enzyme B, nicotinamide adenine dinucleotide (NAD + /NADH), nicotinamide adenine dinucleotide phosphate (NADP + /NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and analogs thereof.
  • the co-factor is modified and for example comprises a modifying group selected from PEI, dextran, PEG, polylysine and poly(acrylic acid) (PAA).
  • an apparatus comprising a composite as described herein or a support as described herein.
  • the apparatus further comprises a first electrode member and a second electrode member in electrical communication with the composite or support.
  • the electrode members may be platinum electrodes.
  • the apparatus may further comprise a voltage source.
  • the apparatus may comprise a reaction chamber for housing the electrode members and the composite or the support.
  • the apparatus may also comprise an inlet and an outlet in communication with said reaction chamber.
  • the apparatus is a microfluidic flow reactor. In one embodiment, the apparatus is for use as a biosensor.
  • a method of preparing an immobilized enzyme composite which comprises: a) providing a first carrier material, b) binding a material which is capable of regenerating a co-factor to the first carrier material to form a second carrier material; c) immobilizing an enzyme on the second carrier material; d) immobilizing a co-factor to the second carrier material, to form a composite which comprises an immobilized enzyme, an immobilized co-factor and a material which is capable of regenerating the co-factor.
  • the first carrier material is porous.
  • a method of catalyzing a redox reaction which comprises contacting the composite or the support described herein with a substrate for the enzyme.
  • the method comprises providing a continuous source of the substrate to the composite or support.
  • the method comprises providing an electrical current to the reaction.
  • the method comprises applying a voltage between a first electrode member and a second electrode member which are in electrical communication with the composite or support. The voltage may be applied continuously or discontinuously to the electrode members.
  • the method is for selective conversion of one enantiomer in an enantiomeric mixture, wherein the enzyme selectively reacts with one enantiomer to form a derivative of said enantiomer.
  • the method is for the synthesis of a chiral compound e.g. a chiral alcohol.
  • the method is for the conversion of racemic 2-phenylpropionaldehyde to (SJ-2-phenyl-1-propanol.
  • FIG. 1 illustrates the production of an electrically conductive matrix.
  • CPG 120-200 mesh, 500 A
  • the aminopropyl-functionalized CPG was derivatized with pyrrole-2-carboxylic acid to afford a pale yellow powder.
  • the material was non-conducting however upon treatment with Na 2 S 2 O 8 , in the presence of pyrrole, the immobilized pyrrole moieties polymerized to afford a dark grey powder (indicative of poly(pyrrole) formation) and was found to conduct.
  • Figure 2 shows NADH generated from 2.5 mM NAD + in phosphate buffer pH 7.0 (a) without CPG-PPy matrix (triangle) and (b) with CPG-PPy matrix (square), illustrating a 40% enhancement of NADH generation in the presence of the conducting CPG- PPy matrix.
  • Figure 3 illustrates the production (S)-2-Phenyl-1-propanol by the enzymatic reduction of racemic 2-phenylpropionaldehyde, catalysed by horse liver alcohol dehydrogenase, in the presence of NADH.
  • Figure 4 illustrates, with a continuous supply of NADH, an average conversion of 43 % was obtained over a 20 h period. The experiments were carried out at room temperature with a flow rate 2 ⁇ l min "1 .
  • Figure 5 is a schematic representation of the flow reactor set-up used to evaluate the in-situ electrochemical regeneration of NADH.
  • Figure 6 illustrates in situ electrochemical regeneration of NADH using the a composite according to the invention.
  • a voltage of 12V was applied to the reactor for ⁇ hours and the reaction products analysed every 2 hours.
  • Figure 7 illustrates the effect of reducing the voltage supplied to the reaction.
  • the system was run continuously until the NADH was exhausted, prior to the application of either 3, 6, 8 or 12 V for 6 h.
  • [substrate] 2.6 x 10 ⁇ 3 mM, pH 7.5, reaction carried out at room temperature, flow rate 2 ⁇ l min 1 .
  • a composite comprising an immobilized enzyme.
  • an immobilized enzyme composite which comprises (a) an enzyme, (b) a co-factor for said enzyme, (c) an electrically conductive material; and (d) a carrier material, wherein the carrier material comprises a porous material.
  • the term "enzyme” is used to describe any protein which catalyzes a desired reaction.
  • the enzyme of the present invention is any enzyme which requires a co-factor for catalysis of a reaction.
  • Co-factors act as stoichiometric agents and undergo chemical reactions with substrates. Co-factors may be (1) tightly bound to the protein portion of an enzyme and therefore an integral part of its functional structure or (2) may be loosely associated.
  • the co-factors of the first class include metal atoms e.g. iron, copper or magnesium and moderately sized organic molecules called prosthetic groups, many of which contain a metal atom, often in a coordination complex.
  • the second class of co-factors includes small organic molecules.
  • the small organic molecules are often referred to as "coenzymes".
  • co-factor includes the class of co-enzymes.
  • the present invention is concerned with co-factors which can be electrochemically regenerated.
  • the co-factor is a molecule which is loosely associated with the enzyme and which, rather than directly contributing to the catalytic ability of an enzyme, participates with the enzyme in the catalytic reaction.
  • Enzymes which require co-factors and which therefore form part of the present invention include, for example, oxidoreductases. Included in this class of enzymes are dehydrogenases.
  • the composite comprises an enzyme selected from a class of oxidoreductases, e.g. a dehydrogenase.
  • the enzyme is selected from an aldehyde dehydrogenase, an acetaldehyde dehydrogenase, an alcohol dehydrogenase, a glutamate dehydrogenase, lactate dehydrogenase, pyruvate dehydrogenase, a glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase.
  • the enzyme is selected from alcohol dehydrogenase, a homoserine dehydrogenase, an aminopropanol oxidoreductase, diacetyl reductase, glycerol dehydrogenase, propanediol phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, D-xylulose reductase, L-xylulose reductase, lactate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, HMG-CoA dehydrogenase, biliverdin reductase, dihydrofolate reductase, glutathione reductase, thioredoxin reductase, horse liver alcohol dehydrogenase and glucose dehydrogenase, succinate dehydrogenase, Baeyer-Villiger monoxygenase, a lac
  • the enzyme is a naturally-occurring enzyme.
  • the enzyme is a recombinant enzyme, an artificial enzyme or a modified naturally occurring enzyme.
  • the enzyme is a truncated enzyme.
  • the enzyme is an organic or inorganic molecule that mimics an enzyme's properties.
  • the present invention includes the use of a co-factor which is immobilized e.g. on a carrier material.
  • the immobilization of the co-factor may enable the enzyme and the co- factor to be in close proximity to each other, thus providing a larger surface area for the reaction catalyzed by the enzyme to take place.
  • the present invention includes the use of an enzyme and its corresponding co-factor in the composite and/or support. Therefore, the present invention provides a co-factor which is required for the catalytic activity of the enzyme.
  • the co-factor is selected from co-enzyme Q, glutathione, co-enzyme B, nicotinamide adenine dinucleotide (NAD + ), the reduced form of NAD - NADH, nicotinamide adenine dinucleotide phosphate (NADP + ) , the reduced form of NADP - NADPH, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN)
  • the co-factor is NADH.
  • the co-factor is NAD + .
  • the co-factor is an analogue of a co-factor.
  • the co-factor is a biomimetic analogue of a co-factor such as those disclosed in Ansell and Lowe, Applied Microbiology and Biotechnology, 1999, vol. 51 , no. 6 pp 703-710.
  • the co-factor is an analog of NAD + , NADH, NADP + Or NADPH.
  • the composite comprises an oxidoreductase and NADH or NAD + or an analogue thereof.
  • the composite comprises an alcohol dehydrogenase e.g. horse liver alcohol dehydrogenase and NADH.
  • the composite comprises an alcohol dehydrogenase and NAD + .
  • the composite comprises NADPH, NADP + or an analogue thereof and an enzyme selected from a dehydrogenase, a monooxygenase, hydroxylase and a reductase.
  • the present invention provides a combination of an immobilized enzyme and co-factor, wherein at least the co-factor is immobilized in contact with a material which is capable of regenerating the co-factor ("regenerating material").
  • the enzyme, co-factor and regeneration material are immobilized on a support or carrier material.
  • the enzyme, co-factor and regeneration means are immobilized on the same carrier or support material.
  • support may be considered to be relate to the same entity and their use is interchangeable.
  • the carrier material is capable of having an enzyme, a co-factor and a regeneration material immobilized thereon.
  • immobilizing enzymes There are four principal methods known in the art of immobilizing enzymes: (a) adsorption, (b) covalent binding; (c) entrapment and (d) membrane confinement.
  • immobilization and “immobilize” encompasses all of these methods.
  • the carrier material of the present invention may be any material to which an enzyme may be immobilized and retain its catalytic activity using such methods. Characteristics to be considered when considering a suitable carrier material include for example pH, ionic strength, surface area and porosity.
  • the carrier material can be prepared in a manner such that it contains internal pores, channels, openings or a combination thereof, which allow the movement of the compound throughout the immobilization material, but constrain the enzyme to substantially the same space within the immobilization material.
  • the carrier material may be porous.
  • the enzyme is cross-linked to the carrier material.
  • Cross-linking is the intermolecular cross-linking of enzymes by bifunctional or multifunctional reagents.
  • the enzyme is entrapped in the carrier material, i.e. is incorporated into the lattices of a semipermeable carrier material.
  • the enzyme can be incorporated into a semipermeable gel or enclosed in a semipermeable polymer membrane.
  • the carrier material may be permeable to a compound that is smaller than an enzyme. Stated another way, the carrier material may allow the movement of a compound that is smaller than an enzyme through it so the compound can contact the enzyme immobilized on or within the carrier material.
  • the carrier material contains pores, channels, openings or a combination thereof, where the pores, channels, openings or combination thereof, do not allow the enzyme to move substantially from its space, but they do allow a compound smaller than the enzyme to move through the carrier material and contact the enzyme.
  • the pores, channels or openings have physical dimensions that satisfy the above requirements and depend on the size and shape of the specific enzyme to be immobilized.
  • aspects of the present invention further provide an immobilized co-factor e.g. a co-factor which is immobilized on a carrier material.
  • the co-factor may be absorbed onto an outer surface of the carrier material.
  • the co-factor may be entrapped inside the carrier material e.g. if the carrier material is porous or contained in cross-linked polymer networks.
  • the co-factor is covalently bonded to a surface of the carrier material.
  • the carrier material may be functionalized to enable immobilization of the enzyme and the co-factor.
  • the carrier material may be functionalized from its original state to provide groups which can be used to covalently bind the enzyme and/or co-factor.
  • Reagents which can be used to functionalize the carrier material to provide coupling means include e.g. carbodiimides, glutaraldehyde, cyanogen bromide, 3-aminopropyltriethoxysilane and 3-glycidoxypropyltrimethoxysilane. Details of coupling conditions which can be used in the present invention are included in for example J. Krenkova and F. Foret, Electrophoresis, 2004, 25, 3550-3563.
  • the enzyme is covalently bound to the carrier material.
  • the active site of the enzyme is not involved in the binding to the carrier material to ensure it can be presented to the enzyme's substrate.
  • the enzyme is covalently bound to the surface or surfaces of the carrier material via an epoxide group.
  • the enzyme and/or co-factor may be independently immobilized on a carrier material surface by diazotization, amide bond formation, alkylation, arylation and/or amidation.
  • the carrier material is selected from glass, ceramics, nitrocellulose, a polymer, a silica-based material, amorphous silicon carbide, castable oxides, polyimides, polymethylmethacrylates, polystyrenes, or silicone elastomers.
  • the carrier material is substantially resistance to swellage e.g. a silica- based material.
  • the carrier material is a monolithic material. These may be useful in embodiments in which the composite is for use in microfluidic applications and/or devices.
  • the carrier material is a glass matrix.
  • the carrier material e.g. glass, may be derivatized to enable covalent binding of the enzyme and/or co-factor.
  • the carrier material is a Controlled Pore Glass matrix.
  • Controlled pore glass having any of a variety of particle sizes and pore sizes can be used in the invention including, for example, CPG with a median particle size falling within about 37 to 200 microns and a median pore size falling within about 65 to 3300 Angstroms.
  • the carrier material is a CPG has a median particle size and pore size of about 120 to 200 microns and about 500 Angstroms, respectively.
  • CPG is typically obtained with a median particle size specification and there can be variation within a lot.
  • a lot of 100-micron CPG can include members be as small as about 75 or as large as about 125 microns.
  • Controlled Pore Glass media are commercially available from e.g. Millipore Corporation.
  • the carrier material is a CPG matrix which has been derivatized to enable covalent bonding of an enzyme and a co-factor. In one embodiment, the carrier material is derivatized to enable bonding of a material which is capable of regenerating the co-factor.
  • the carrier material is a CPG matrix which has been derivatized to provide an amino group for binding of an electrically conducting polymer as a regenerating material. Further details of the regenerating material are provided below.
  • the CPG matrix is aminopropyl-derivatized. In one embodiment, this amino derivatized CPG matrix is further derivatized to provide an epoxy linkage. In one embodiment, the CPG matrix is an aminopropyl-derivatized CPG matrix, which has been further derivatized with 3-glycidoxypropyl triethoxysilane on which an enzyme and co-factor are immobilized. Regenerating Material
  • the present invention provides a composite which comprises a material capable of regenerating the co-factor.
  • a material capable of regenerating the co-factor are more expensive than the desired products of a process and therefore embodiments of the present invention are advantageous since the co-factor is regenerated, thus avoiding or reducing the requirement to replace the co-factor.
  • the composite is electrically conductive and therefore is capable of electrochemical regeneration of the co-factor.
  • the composite comprises an electrically conductive polymer which is capable of regenerating the co- factor.
  • the electrically conductive (which is interchangeable herein with the term “electrically conducting”) includes polymers which can be reversibly oxidized and reduced.
  • conducting polymer refers to an organic polymer- containing material which is capable of electronic conduction.
  • Electrically conductive polymers are known in the art and include, for example, polyacetylene and polyaniline, polyquinoline, polyquinoxaline, poly(p-phenylene sulfite), poly(phenylquinoxaline), poly(p-phenylene), polypyrrole, and polyphthalocyaninesiloxane.
  • Certain chemically synthesized conjugated polymers are initially insulators (i.e. in a neutral state) and it is only through oxidation (p-doping) and less frequently reduction (n-doping) by chemical or electrochemical means, that the necessary mobile charge carriers for conductivity are formed.
  • An electrically conductive polymer which may be used in the present invention may be produced by interacting a polymer with a dopant (oxidation or reduction). The polymer is reacted with an electron donor dopant or an electron acceptor dopant to modify its room temperature conductivity.
  • the electron donor or acceptor is known in the art as n-type and p-type dopants, respectively
  • the electrically conductive polymer is a non-swellable polymer i.e. a polymer which does not swell or exhibits limited swelling. This may be advantageous in apparatus such as flow reactors since, if the polymer swells, it can block the flow of fluid between the composite, thus preventing the immobilized enzyme contacting its substrate and catalyzing a reaction.
  • non-swellable is used herein to describe electrically conductive polymers that are substantially incapable of imbibing fluid and expanding when in contact with fluid present in the environment of use, e.g. when brought into contact with a fluid comprising the enzyme substrate.
  • the term “swellable,” as used herein refers to materials i.e. polymers that are capable of imbibing fluid and expanding when in contact with fluid present in the environment of use. These polymers may be useful in embodiments in which the composite is for use in microfluidic devices and/or applications.
  • the electrically conductive polymer is selected from poly 3,4- ethylenedioxythiophene (PEDOT), polypyrrole, polyanilines, polyacetylenes, polythiophenes, poly(phenylenesulfide), and poly(phenylenevinylene), and blends thereof and derivatives thereof e.g. substituted polypyrrole, polyaniline and polythiophene and combinations thereof, which can be made using methods available in the art.
  • the electrically conductive polymer comprises polypyrrole. Derivatives which can be used include substituted polyanilines, polypyrroles and polythiophenes, such as N-substituted polypyrroles.
  • the electrically conductive polymer is applied as a single layer coating of a single polymer or as a multilayered film to alter the properties of the applied polymers.
  • the electrically conductive polymer is immobilized e.g. on or within a carrier material or support.
  • the polymer may be covalently bonded to the carrier material or support.
  • the polymer is bound to the carrier material via an amide linkage which may be provided as a result of derivitization of the carrier material.
  • the polymer is attached to the carrier material via an imide linkage.
  • the polymer is attached to the carrier material via a reactive alcohol group.
  • the polymer is covalently bonded to the carrier material through an alkyl group.
  • the electrically conductive polymer may be immobilized on the carrier material via a linkage selected from an epoxy linkage, an alkylbromide linkage or an aldehyde linkage.
  • the present invention provides an immobilized enzyme composite which includes an enzyme, a co-factor which is required for the enzyme to catalyze a reaction and a carrier material.
  • the composite is preferably electrically conductive and may in certain embodiments comprise an electrically conductive polymer.
  • each of the enzyme, the co-factor and the electrically conductive polymer are independently covalently attached to the carrier material.
  • the carrier material is derivatized to provide linking groups for each of the enzyme, the co-factor and the electrically conductive polymer.
  • the composite of the present invention may therefore provide an improved enzyme system in which means for regenerating the co-factor, e.g.
  • an electrically conductive polymer in the form of an electrically conductive polymer, is immobilized in close proximity to the co-factor and optionally also the enzyme.
  • the means for regenerating the co-factor e.g. in the form of an electrically conductive polymer, is immobilized on a porous material together with a co-factor and an enzyme.
  • Embodiments of the present invention may provide a large surface area on which a reaction catalyzed by the enzyme can take place, which may result in an increased reaction rate and improved efficiency.
  • the present invention may have utility in industrial scale reactions catalyzed by enzymes which require co-factors as described herein. Further uses of the composites and apparatus of the present invention are described in more detail below.
  • an immobilized enzyme composite which comprises: a) providing a first carrier material, b) binding a material which is capable of regenerating a co-factor to the first carrier material to form a second carrier material; c) immobilizing an enzyme on the second carrier material; d) immobilizing a co-factor to the second carrier material, to form a composite which comprises an immobilized enzyme, an immobilized co-factor and a material which is capable of regenerating the co-factor.
  • the material which is capable of regenerating a co-factor is electrically conductive and step (b) of the method comprising forming an electrically conductive carrier material.
  • step (d) is carried out prior to step (c).
  • the first carrier material is porous.
  • the first carrier material is glass, e.g. Controlled Pore Glass (CPG).
  • the carrier material is a ceramic, or a silica-based material e.g. a sol-gel.
  • the method comprises derivatizing the first carrier material prior to step (b) to provide a linking group for binding the electrically conductive material to the first carrier material.
  • the linking group may be for example an inorganic or organic molecule.
  • the linker may be a silane, e.g., sianosilane, thiosilane, aminosilane, etc.
  • the linking group is an amine group.
  • the method may comprise reacting the first carrier material with 3-aminopropyl triethoxysilane to form an aminopropyl linking group.
  • the regenerating material is an electrically conductive polymer and the method comprises derivatizing the first carrier material to provide a derivatized first carrier material comprising a linking group for binding the polymer.
  • step (b) of the method may comprise (i) contacting the derivatized first carrier material with a solution comprising a precursor monomer of the polymer so as to immobilize the precursor monomer on the carrier material.
  • Step (b) of the method may further comprise (ii) polymerizing the immobilized precursor monomer to form the second carrier material.
  • the first precursor monomer is pyrrole-2-carboxylic acid. In an alternative embodiment, the first precursor monomer is a 2 amino pyrrole carboxy derivative of aniline.
  • the method comprises contacting the first precursor monomer in a solvent, e.g. a halogenated solvent e.g. dichloromethane.
  • the solution further comprises a coupling agent e.g. a carbodiimide e.g. N, N' dicyclohexylcarbodiimide or N, N'-diisopropylcarbodiimide.
  • a coupling agent e.g. a carbodiimide e.g. N, N' dicyclohexylcarbodiimide or N, N'-diisopropylcarbodiimide.
  • Other coupling agents which may be used in the present invention include e.g.
  • step (ii) comprises contacting the carrier material with a catalyst and a second monomer under conditions which permit polymerization of the immobilized monomer.
  • the second monomer is pyrrole.
  • the catalyst is sodium persulphate.
  • catalysts which may be used are generally known and can be chosen, for example, from the group of inorganic acids, such as hydrochloric acid, sulphuric acid, chlorosulphonic acid and nitric acid; Lewis acids such as compounds containing positive ions of iron, aluminium, tin, titanium, zirconium, chromium, manganese, cobalt, copper, molybdenum, tungsten, ruthenium, nickel, palladium and/or platinum; and a halogen, a sulphate, a nitrate, a sulphonate and/or an acetyl acetonate.
  • suitable catalysts are ozone, diazonium salts, organic catalysts, for example benzoquinone, and other persulphates, e.g. sodium persulphate, ammonium persulphate and potassium persulphate.
  • the method comprises contacting the carrier material with the catalyst and second precursor monomer for at least 24 hours, e.g. 48 hours, 50 hours, 60 hours or 70 hours. In one embodiment, the method comprises contacting the carrier material with the catalyst and second precursor monomer for approximately 72 hours.
  • the method further comprises e.g., following step (b), derivatizing the second carrier material to provide a linking group for use in immobilizing the enzyme and/or the co-factor.
  • the method comprises derivatizing the second carrier material to form an epoxide linkage.
  • the second carrier material is derivatized by contacting the second carrier material with 3-glycidoxypropyl triethoxysilane under conditions which permit derivatization of the second carrier material.
  • the second carrier material is derivatized to provide an epoxy linkage.
  • step (c) comprises contacting the second carrier material with a solution comprising an enzyme under conditions which permit immobilization of the enzyme on the carrier material.
  • step (d) comprises contacting the second carrier material with a solution comprising a co-factor under conditions which permit immobilization of the enzyme on the carrier material. It will be apparent that in alternative embodiments, the order of these steps can be reversed such that the second carrier material is contacted with a solution comprising a co-factor prior to contacting the second carrier material with a solution comprising the enzyme.
  • the method comprises contacting the second carrier material with a solution comprising an enzyme and a co-factor and thus step (c) and (d) are combined into a single step.
  • the method comprises contacting the second carrier material by pumping.
  • the solution comprising the co-factor further comprises a buffer e.g. a carbonate buffer.
  • the solution comprising the enzyme further comprises a buffer, e.g. a phosphate buffer for example a potassium phosphate buffer.
  • the composites and supports of the present invention may be included in an apparatus.
  • An example of an apparatus which may be used in the present invention is disclosed in EP Patent Publication No.1780412.
  • the apparatus comprises a reaction chamber which houses the composite or support during use. In use, the reaction chamber may be packed with the composite or support described herein.
  • the apparatus typically comprises an inlet which permits flow of a reaction mixture which may comprise a substrate or substrates of the enzyme into the reaction chamber and thus enabling the substrate to contact the immobilized enzyme and co-factor.
  • the apparatus may be provided with pumps and the like to facilitate flow of the reaction mixture into and through the reaction chamber.
  • the apparatus may further comprise an outlet to enable a reaction mixture comprising the product of the enzyme catalyzed reaction to be removed from the reaction chamber.
  • the outlet may be sealable to permit a batch process to take place.
  • the apparatus is a continuous flow reactor. In one embodiment, the apparatus is a biosensor.
  • the apparatus includes a first electrode member and a second electrode member each of which are in electrical communication with the composite e.g. when in the reaction chamber.
  • the first electrode member and the second electrode member may each be in electrical communication with regeneration material of the composite.
  • the electrode members are in electrical communication therewith.
  • the first electrode member and the second electrode member may be in electrical communication with the electrically conductive polymer only when in fluid communication e.g. when contacted by a fluid which may containing a substrate for the enzyme.
  • the electrode members are electrodes.
  • the electrode members may be any electrically conductive material including metals, metal oxides, conductive polymers, and conductive carbon.
  • conductive materials include a thin layer of a metal such as gold, silver, platinum, palladium, copper, tungsten, ruthenium or alloys of these metals with each other or with other metals, as well as a thin layer of conductive carbon powder.
  • the electrode members may be made of or coated with conducting or semi-conducting materials, for example, gold, platinum, palladium, silver, carbon, etc.
  • Semi-conducting materials used as electrode members in the present invention may be selected, for example, from Group IN-V, Group Hl-V alloys, Group H-Vl, Group I- VII, and Group IV semiconductors.
  • the first electrode member is an electrode comprising a metal selected from gold, silver and platinum.
  • the second electrode member is an electrode comprising a metal selected from gold, silver and platinum.
  • the first and/or second electrode may comprise a conducting polymer composition coating.
  • Such a coating may include any compatible conductive polymer, and preferably a polymer composition selected from the group consisting of polyaniline, polyacetylene, polyquinoline, polyquinoxaline, poly(p-phenylene sulfite), polyphenylquinoxaline), (poly(p-phenylene), polypyrrole, and polyphthalocyaninesiloxane.
  • the apparatus is a microfluidic device.
  • a "microfluidic device” or “microfluidic chip” refers to a system of microscale fluid control components, such as channels, reservoirs, junctions such as T-junctions, and the like. Typically, these components are incorporated into a single solid substrate, for example, by chemically etching channels and reservoirs into the surface of a chip, e.g., a glass microscope slide, a polymer slab or a silicon wafer. The silicon wafer can be patterned and etched (see, e.g., G. Kovacs, 1998, Micromachined Transducers Sourcebook, Academic Press; M. Madou, 1997, Fundamentals of Microfabrication, CRC Press).
  • the carrier material is an electrically conductive microfluidic chip and the enzyme and co-factor are immobilized thereon.
  • the microfluidic chip further includes an electrically conductive polymer.
  • the microfluidic device comprises a CPG matrix microchannel on which the enzyme and co-factor have been immobilized.
  • the device may be configured to allow the manipulation of liquids, including reagents and solvents, to be transferred or conveyed within the micro channels and reaction chamber using mechanical or non-mechanical pumps.
  • Such device may comprise columns, pumps, mixers, valves and the like.
  • the microfluidic channels or tubes (referred to as micro-channels or capillaries herein) have at least one cross- sectional dimension (e.g., height, width, depth, diameter) from about 1 to about 1 ,000 ⁇ m, alternately from about 1 to about 500 ⁇ m, or even from about 10 to about 500 ⁇ m.
  • the micro-channels make it possible to manipulate extremely small volumes of liquid on the order of nl_ to ⁇ l_.
  • the micro reactors may also comprise one or more reservoirs in fluid communication with one or more of the micro-channels, each reservoir typically having a volume of about 5 to about 1 ,000 ⁇ l_.
  • reaction chamber or “reactor” or “micro-reactor” refers to a feature on the microfluidic chip where the reactions may take place.
  • the reaction chamber has one or more micro-channels connected to it that deliver reagents and/or solvents or are designed for product removal (controlled by on-chip valves).
  • the reaction chamber has a height diameter to height with a ratio of greater than about 3, greater than about 5, greater than about 10 or more.
  • the reactor height may be about 25 micrometer to about 1 ,000 micrometers.
  • the reactor may have a diameter from about 1 ,000 to about 20,000 micrometers.
  • the micro- reactor may be packed with the composite or support as described herein. Such a device may result in a high surface to volume ration of the enzyme and co-factor, thus enhancing the rate of a reaction the enzyme catalyzes.
  • the use of microfluidic devices also allows for reduced amounts of enzyme, substrates, co-factors and other reagents, thus reducing costs.
  • the carrier material when the composite is for use in a microfluidic device, is substantially resistant to swell, thus reducing the likelihood of back pressure.
  • the composite comprises a silica-based carrier material e.g. glass.
  • the composite comprises a monolithic carrier material. In one embodiment, the composite is covalently bonded to walls of the channels within the device so as not to be pumped out of the device during a flow reaction.
  • the present invention provides for immobilization of an enzyme and its co-factor and therefore may provide a system which can be run as a continuous flow reaction.
  • the apparatus is adapted for continuous flow.
  • An exemplary flow reactor is shown in Figure 5.
  • the flow reactor 1 comprises a reaction chamber 3 in which the composite or support 5 is packed.
  • the reaction chamber comprises an inlet 7 and an outlet 9.
  • the reaction chamber also comprises a first Pt electrode 11 and a second Pt electrode 13 which are separated by approximately 1cm.
  • the apparatus also includes means for providing a voltage to the electrodes.
  • the apparatus includes means 15 for placing a reaction mixture which includes a substrate for the enzyme in the reaction chamber 3.
  • the present invention provides a composite and support which include at least one enzyme and its corresponding co-factor.
  • the composite is for use in methods for catalyzing reactions involving a substrate of the enzyme.
  • Substrates of the enzymes used in the present invention are well-known in the art.
  • the substrate is an alcohol e.g. when the enzyme is an alcohol dehydrogenase.
  • the substrate may be a primary or secondary alcohol.
  • the enzyme may therefore catalyze the oxidation of a primary or secondary alcohol to an aldehyde or a ketone respectively.
  • a method of catalysing a reaction comprising use of a composite as described herein.
  • the substance is a known substrate of the enzymatic reaction to be tested.
  • the method comprises contacting the composite with a substrate of the enzyme under conditions suitable for catalysis of the reaction by the enzyme to take place.
  • the substrate and composite are incubated in a reaction mixture that provides conditions conducive to the occurrence of the enzymatic reaction.
  • the method comprises contacting the composite with the substrate under flow conditions.
  • the present invention also provides methods for identifying substrates of enzymes and modulators of enzymatic activities.
  • a method for assaying the effect of a substance on activity of an enzyme comprising:
  • the determining step can comprise detecting whether a change in the amount of said enzymatic reaction occurs relative to the amount of said enzymatic reaction in the absence of the substance. In one embodiment, the determining step comprises detecting a decrease in the amount of said enzymatic reaction relative to the amount of said enzymatic reaction in the absence of the substance, thereby identifying the substance as an inhibitor of said enzymatic reaction. In other embodiments, said determining step can comprise detecting an increase in the amount of said enzymatic reaction relative to the amount of said enzymatic reaction in the absence of the substance, thereby identifying the substance as an activator of said enzymatic reaction.
  • the methods of the invention can be used to determine whether a substance is a substrate of an enzymatic reaction of interest.
  • the composite comprises an enzyme known to catalyze the reaction of interest and substrate and composite are incubated in a reaction mixture that provides conditions conducive to the occurrence of the enzymatic reaction.
  • the method comprises regenerating the co-factor of the composite.
  • the method comprises applying a voltage to the reaction.
  • the provision of an electrical charge may enable the regenerating material to regenerate the co-factor.
  • the method may not require replacement of the co- factor as often as prior art methods.
  • the composite is for use in the synthesis of enantiomerically pure stereoisomers of chiral alcohols.
  • a method of selectively reacting one enantiomer in an enantiomeric mixture comprising contacting the enantiomeric mixture with a composite as described herein.
  • the composite may be included in an apparatus described herein.
  • the method may further comprise applying a voltage to the composite e.g. via a first electrode member and second electrode member which are in electrical communication with the composite.
  • the application of the voltage may then result in regeneration of the co-factor by the composite e.g. via the electrically conductive polymer.
  • the composite comprises an enzyme which selectively reacts with one enantiomer, called the "reactive enantiomer", in the enantiomeric mixture to form a derivative of the reactive enantiomer.
  • the immobilized enzyme and enantiomeric mixture are contacted under conditions suitable for reacting the immobilized enzyme and the reactive enantiomer, thereby forming a product mixture comprising the unreactive enantiomer and a derivative of the reactive enantiomer.
  • Enantiomers are asymmetric molecules that can exist in two different isomeric forms which have different configurations in space. Because they do not have a plane of symmetry, enantiomers are not identical with their mirror images; molecules which exist in two enantiomeric forms are chiral, which means that they can be regarded as occurring in "left" and "right” handed forms.
  • the most common cause of chirality in organic molecules is the presence of a tetrahedral carbon bonded to four different substituents or groups. Such a carbon is referred to as a chiral center, or stereogenic center.
  • a method for indicating the three-dimensional arrangement of atoms (or the configuration) at a stereogenic center is to refer to the arrangement of the priority of the groups when the lowest priority group is oriented away from a hypothetical observer: If the arrangement of the remaining three groups from the higher to the lower priority is clockwise, the stereogenic center has an "R" (or "D") configuration; if the arrangement is counterclockwise, the stereogenic center has an "S" (or "L”) configuration.
  • Enantiomers have the same empirical chemical formula, and are generally chemically identical in their reactions. However, enantiomers show different chemical reactivity toward other asymmetric compounds, and respond differently toward asymmetric physical disturbances. The most common asymmetric disturbance is polarized light.
  • An enantiomer can rotate plane-polarized light; thus, the enantiomer is optically active.
  • Two different enantiomers of the same compound will rotate plane-polarized light in the opposite direction; thus, the light can be rotated to the left or counterclockwise for a hypothetical observer (this is levarotatory or "1", or minus or "-”) or it can be rotated to the right or clockwise (this is dextrorotatory or "d” or plus or "+”).
  • the sign of optical rotation (+) or (-) is not related to the R, S designation).
  • racemic mixture A mixture of equal amounts of two chiral enantiomers is called a racemic mixture, or racemate, and is denoted either by the symbol (+/-) or by the prefix "d, I" to indicate a mixture of dextrorotatory and levorotatory forms. Racemic mixtures show zero optical rotation because equal amounts of the (+) and (-) forms are present. But generally the presence of a single enantiomer rotates the light in only one direction; thus, a single enantiomer is referred to as optically pure.
  • Optically pure compounds are of interest as chiral synthons.
  • One reason is that asymmetric molecules in living organisms are usually present in only one of their possible chiral forms.
  • the synthetic reactions in the absence of asymmetric catalysts
  • the synthetic reactions in the absence of asymmetric catalysts
  • the synthetic reactions generally produce both chiral forms at an equal rate, leading to an equimolar, or racemic, mixture of the product isomers.
  • the separation of a racemic mixture into its two constituent enantiomers is called resolution, but it is difficult to separate a racemic mixture
  • the three-dimensional shape, or stereochemistry, of biomolecules is extremely important to their biological function.
  • enantiomers of the same structure may have very different biological effects.
  • the drug thalidomide was synthesized and administered as a racemate; only one enantiomer was an effective antinausea drug, whereas the other enantiomer was an effective teratogen, which was tragically discovered after administration of the racemate to pregnant women.
  • chiral 1 ,2-propanediols are useful in the preparation of cardiovascular drugs, anti viral drugs, and enantiomerically pure crown ethers (Hoff et al. (1996) Tetrahedron: Asymmetry 7:3181-3186).
  • These and related chiral compounds may also serve as synthons for chiral polymers, chromatography matrices, or as derivatization reagents for stereochemical analysis of chiral acids by LC or NMR.
  • optically active secondary alcohols particularly those with asymmetric carbon containing fluoroalkyl groups (e.g., trifluormethyl-), are a material of interest in ferroelectric and anti-ferroelectric liquid crystals (U.S. Pat. No. 6,239,316).
  • the composite comprises horse liver alcohol dehydrogenase and NADH.
  • the composite may further comprise polypyrrole and may be used to catalyse the reduction of ( ⁇ )-2-phenylpropionaldehyde to (SJ-2-phenyl-propanol.
  • Figure 2 shows NADH generated from 2.5 mM NAD + in phosphate buffer pH 7.0 and compares the two systems, (a) without CPG-PPy matrix and (b) with CPG-PPy matrix, illustrating a 40% enhancement of NADH generation in the presence of the conducting CPG-PPy matrix.
  • HLADH immobilisation was achieved by pumping a solution of HLADH (2 mg) in 0.2 M potassium phosphate buffer (2.0 ml, pH 7.0) through the reactor at 1 ⁇ l min 1 for 24 h. After this time, any unbound enzyme was washed from the system using potassium phosphate buffer.
  • CPG-PPy (0.08 g) was stirred with 3-glycidoxypropyl triethoxysilane (0.32 mmol) in ethanol (20 ml) for 3 h, to afford the epoxide derived CPG-PPy.
  • ethanol 20 ml
  • the epoxide functionalized CPG-PPy was subsequently packed into a polymeric flow reactor (5 mm i.d. x 5 cm length), retained using silanized glass wool and prior to enzyme immobilisation, the material was washed with carbonate buffer (pH 10.0).
  • NADH immobilisation was achieved by pumping a solution of NADH (0.08 M) in 0.2 M carbonate buffer through the flow reactor at 1 ⁇ l min "1 for 3 days. After this time, any unbound co-factor was washed from the reactor using 0.2 M carbonate buffer.
  • the final step was to co-immobilize the enzyme and co-factor in order to develop a system that would enable the continuous reduction of 2-phenylpropionaldehyde and regeneration of NADH.
  • the aforementioned procedures were performed in series, ensuring that between enzyme and co-factor immobilisation, the functionalized CPG was washed with 0.2 M carbonate buffer.
  • Example 5 Enzymatic reduction of ( ⁇ )-2-phenylpropionaldehyde to (S)-2-phenyl-1- propanol.
  • the feedstock was pumped through the flow reactor at a flow rate of 2 ⁇ l min "1 and the reaction products collected at the reactor outlet.
  • the reaction products were collected and analysed off-line by HPLC using a BDS Hypersil C18 column (Phenomenex), with a flow rate of 1 ml min "1 , 35 % acetonitrile in water as the mobile phase and detected at 215 nm; 2.5 mM ⁇ /-benzoyl-L- phenylalanine was employed as the internal standard.
  • a conducting CPG-PPy material was successfully synthesised and utilised as a solid- support for the immobilisation of HLADH and NADH. Enzymatic reduction of racemic 2-phenylpropionaldehyde to (S)-2-phenyl-1-propanol was subsequently achieved under continuous flow, enabling excellent conversions to be obtained for upto 20 h. Upon exhaustion of the immobilized NADH, in-situ electrochemical regeneration was found to restore enzyme activity, with repetition of this cycle enabling continued operation for in excess of 100 h.
  • the CPG-PPy matrix described herein may provide a cost effective solution to the challenge of immobilising enzymes that require a co-factor.

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Abstract

La présente invention concerne l'immobilisation d'enzymes et de leurs cofacteurs dans un système qui permet la régénération des cofacteurs. La présente invention concerne en outre des procédés et des dispositifs qui utilisent de tels systèmes.
PCT/GB2010/050315 2009-02-25 2010-02-24 Enzymes et cofacteurs immobilisés WO2010097619A1 (fr)

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