Classifications

• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
  • C12M21/18—Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
  • C12M25/16—Particles; Beads; Granular material; Encapsulation

Definitions

• This invention relates generally to methods and apparatus for conducting enzymatic reactions.
• the invention relates to methods and
• IMER Immobilized enzyme reactors
• Particularly useful matrices comprise porous particles which provide the desired high surface area-to-volume ratio.
• HPLC high performance liquid chromatography
• divinylbenzene Because the dense packing of these smaller beads creates a high resistance to liquid flow, the equipment is designed to operate at high pressures, which allows rapid fluid transfer.
• the particle size may be enlarged to increase capacity, but this generally only results in longer diffusional path lengths within the particle. Reducing or eliminating the dependence of immobilized enzyme reactors on the diffusive flow rate of solutes would enhance their productivity.
• Perfusive matrices capable of immobilizing enzymes and to the use of such matrices to perform enzymatic reactions.
• Perfusive matrices comprise rigid, porous, high surface area materials such as particles which may be of the same mean diameter as are employed in conventional
• chromatography matrices The geometry of perfusive matrices are configured to allow convective fluid transfer both within and between the particles.
• 10-20 ⁇ m diameter particles of perfusive matrices have throughpores of relatively large mean diameter (e.g., 6,000 to 8,000 A) and a high surface area network of internal, blind subpores of smaller mean diameter (500 to 1500 A) within the. throughpores.
• the enzymes can be immobilized on all available surface areas, including within the throughpores and the subpores.
• the perfusion matrix comprises packed particles
• the diameter of the particles determines the mean diameter of the interparticle spaces in a packed bed.
• intraparticle throughpores is less than 70, most preferably less than 50.
• Preferred subpore diameters are within the range of about 300-700 A.
• the low ratio (and correspondingly larger intraparticle pore size) substantially reduces particle pore effects.
• Preferred ratios of convective flow velocities through the interparticle and intraparticle pores are between about 10:1 to 100:1.
• the velocity of a mobile phase can be
• perfusive matrix material in chromatographic contexts is provided in co-pending U.S. Application No. 376,885, filed July 6, 1989, now U.S. Patent No. , and in Afeyan et al., (1990) Bio/Technology 8:203-206, the disclosures of which are incorporated herein by
• Perfusive matrix materials are available commercially from PerSeptive Biosystems, Inc. of
• this invention is a method for conducting an enzymatic reaction using a perfusive matrix formed by packing a multiplicity of particles defining therewithin throughpores and substrate
• a solution of enzyme substrate is passed through the matrix at a velocity sufficient to cause convective fluid flow in the throughpores at a rate greater than the rate of substrate diffusion through the throughpores.
• a matrix is provided defining inter-connected first and second throughpore sets wherein the members of the first throughpore set have a greater mean diameter than the members of the second throughpore set.
• Enzymes are immobilized, using chemistries known per se, or novel methods, on all surfaces of the matrix, including on surface regions in fluid communication with the members of the second throughpore set.
• a solution of enzyme substrate is passed through the matrix at a rate sufficient to induce convective flow through both throughpore sets, and to induce a rate of convective flow through the second throughpore set that is greater than the rate of diffusion of the substrate within that set.
• the invention provides a method for conducting an enzymatic reaction utilizing a matrix defining interconnected first and second
• Both throughpore sets comprise a multiplicity of throughpores for channeling through the matrix an enzyme solution and a substrate solution reactive with that enzyme.
• the matrix also includes interactive surface regions capable of immobilizing enzymes and which are in fluid communication with the members of the second pore set.
• Enzymes preferably are immobilized on the surfaces of the matrix by passing a solution containing the enzyme through the matrix.
• the fluid mixture is passed through the matrix at a rate sufficient to produce convective fluid flow through both pore sets, the velocity through the first set being greater than the velocity through the second set, and the convective fluid flow velocity through the second pore set being greater than the diffusive flow rate of the enzyme within the second pore set.
• the dimensions of the members of the second pore set and the interactive surface regions permit flow through the members of the second pore set at a rate such that the time for a solute to diffuse to and from the interactive surface regions is comparable to or shorter than the time for the solute to flow convectively past the region.
• the substrate solution then is passed through the matrix at a fluid flow rate sufficient to allow catalysis of the
• this fluid flow rate is sufficient to produce convective fluid flow through both pore sets. This allows the enzymatic reaction to take place under kinetically very favorable conditions. Specific dimensions and flow velocities are described in greater detail below.
• the invention provides a method for performing a series of enzyme reactions in successive zones within the matrix. In this method each enzyme is loaded at a perfusive fluid flow
• the enzymes are loaded in the order in which they are to be used. Because the perfusive flow rate allows the enzyme solution to encounter all available binding sites it flows past, the first enzyme added will saturate the available binding sites it first encounters, forming a discrete zone of immobilized enzyme. The second enzyme, added subsequently, then will flow past this zone of saturated binding sites containing the immobilized first enzyme and will begin binding at the first available binding sites
• the IMER may be constructed to mimic the actions of an enzyme complex found in nature.
• multiple different reactions may be required to obtain a product of interest. In these cases the product formed by
• reaction of a substrate with the first enzyme becomes the substrate for the reaction with the immobilized enzyme in the zone below.
• optimal kinetics for all reactions in the system can be achieved with one given flow rate.
• the multiple enzyme system may be used to perform catalytic reactions on different substrates.
• a particularly useful application of this method is in clinical analyses, to assay the presence and/or concentration of different solutes in a body fluid sample.
• the different enzymes need not be segregated into discrete zones.
• the invention provides a novel enzyme reactor including a rigid matrix defining interconnected first and second pore sets dimensioned to allow convective fluid flow through both throughpore sets, each pore set comprising a multiplicity of pores for channeling through the matrix a solution of enzyme or enzyme substrate.
• the matrix further defines surface regions comprising immobilized enzyme and in fluid communication with the members of the second throughpore set.
• plural different enzymes may be immobilized on the matrix surfaces.
• the different enzymes further may be immobilized on the matrix in successive zones.
• the rigid matrix comprises a multiplicity of interfacing particles defining an interstitial volume which constitutes the first throughpore set.
• Each individual particle defines a plurality of
• throughpores constituting the second pore set, and a plurality of blind pores in fluid communication with the throughpores, within which enzymatically active surface regions are located.
• throughpores, subpores, and any interconnecting pores are anisotropic.
• preferred particles have a mean diameter greater than about 50 ⁇ m, most preferably greater than about 100 ⁇ m, and have a ratio of mean particle diameter to mean intraparticle throughpore diameter of less than 70.
• a preferred geometry of the particles comprises adhered clusters of smaller similar clusters made up of small interadhered particles called porons.
• the preferred material for manufacture of the particles is polystyrene divinyl benzene copolyraer.
• FIGURE 1 is a schematic representation of a particle suitable for use in forming a perfusive matrix containing immobilized enzymes in accordance with the teachings of the present invention
• FIGURE 2 is a schematic representation of an apparatus embodying the present invention.
• FIGURE 3A-C are graphic representations for perfusive immobilized enzyme reactions performed under various reaction conditions.
• the matrix of the enzyme reactors of this invention is characterized by a geometry which is bi- modal or multi-modal with respect to its porosity and which has interactive surface regions capable of immobilizing enzymes.
• the matrix defines a set of pores of larger diameter, such as are defined by the interstices among a bed of particles, which determine pressure gradient and fluid flow velocity through the bed, and a set of pores of smaller diameter (e.g., anisotropic throughpores). The smaller pores permeate the individual particles. At fluid flow velocities above a threshold level, these pores serve to deliver by perfusion a solution of an enzyme or enzyme
• FIGURE 1 shows schematically a matrix particle 10 suitable for use with the present invention. As illustrated, in addition to extra-particle pores or channels 12, which have a relatively large mean
• a matrix utilizing the particle 10 also comprises a second set of throughpores 14, here embodied as pores defined by the body of the particle 10. Also defined by the particle 10 is a set of diffusive transport pores 16.
• the mean diameter of the pores 12 is larger than the throughpores 14.
• the ratio of the mean diameters of pores 12 and 14 is such that, when the particles are close-packed in a bed, there exists a threshold of fluid velocity which can
• the bed is said to be operating in the perfusive domain where contact between enzyme and substrate is no longer bound by diffusive transport. Precisely where this threshold of perfusion occurs depends on many factors, but primarily it depends upon the ratio of the mean diameters of the first and second pore sets, here, pores 12 and 14 respectively. The smaller that ratio, the lower the threshold velocity. In preferred embodiments, the ratio of the mean
• particle diameter to mean intraparticle throughpore diameter is less than 70, most preferably less than 50.
• the particle 10 includes a large surface region 18 within the particles onto which enzymes can be immobilized. Enzymes may be covalently attached to the surfaces 18 at high concentration using any one of a number of techniques well known to those skilled in the art.
• the matrix first may be derivatized to create the desired functional group for a given
• a pendant chain can be attached to the surface having a terminal functional group distal to the surface to which the enzyme can be coupled resulting in an immobilized enzyme which is attached by a "leash" at a distance away from the surface.
• Enzymes also may be covalently bound to the matrix surface by means of difunctional crosslinkers. Further details on methods for covalent immobilization of matrices can be found in a number of references in the art. Included among these are Falb, R.D. pp. 67-76, in Enzyme Engineering Vol 2, Pye, E. at al. eds., (1973) Plenum Press, NY, and White et al., (1980) Enzyme Microb. Technol. 2:82-90.
• enzymes may be noncovalently
• Noncovalent attachment provides a number of advantages over covalent
• a given enzyme can be bound to the matrix using any of a number of well-known noncovalent interactions. Thereafter the enzymatic conversion is run, and the enzyme
• the matrix then can be reloaded with the same or a different enzyme, thereby
• conformational distortion of the enzyme needed to achieve binding It will be understood by those skilled in the art that substrate conversions performed using noncovalently immobilized enzymes will need to be run under conditions that do not induce elution of the enzyme. This may be achieved by manipulation of various reaction parameters, such as buffer pH and/or salt conditions.
• Particularly useful matrices for noncovalent enzyme immobilization include ion exchangers, and matrices that bind by hydrophobic interactions (e.g., "reverse phase" matrices.)
• a primary design criterion is to maximize enzyme activity of the resulting enzyme reactor. Even though the reactor may be highly stable and permit repeated use of the enzyme, a significant loss of enzymatic activity in the
• immobilization should be compatible with the enzyme.
• a reactor should have an enzyme activity of at least 100 u/g of matrix material, where a unit of enzyme activity (U) is defined as one micromole of substrate converted per minute.
• Increases in surface area also can be achieved by reducing particle size, but the column flow properties of the reactor are usually unsatisfactory.
• perfusive systems allow enzymes to be loaded rapidly onto a perfusive reactor system (e.g., in seconds or minutes) in a fraction of the time currently required without loss of binding capacity.
• a perfusive reactor system e.g., in seconds or minutes
• the rapid throughput of the system and its small size allow one to manipulate reaction parameters rapidly and easily when developing a reaction protocol.
• any enzyme can be utilized in the enzyme reactor of this invention for an equally large variety of applications.
• multiple different enzymes may be immobilized on the matrix to catalize reactions with multiple, .different subtrates.
• Useful enzymes include oxido-reductases, transferases,
• hydrolases, lyases and isoraerases Useful applications include a wide range of industrial applications, including those in the chemical, food and fragrance industries; in bioremediation, including the treatment of waste water by immobilization of pesticide-detoxification enzymes; and in pharmaceutics,
• the IMERs of this invention are useful for the biochemical analysis of particular enzymatic and metabolic reactions.
• relatively impure and/or heterogeneous enzyme preparations such as those derived from cell extracts, cell lysates, partially purified enzyme isolates and whole cells can also be used, albeit at some reduction in the enzymatic activity.
• enzyme as used herein is meant to broadly include catalytic enzymes in all of these forms.
• enzymes normally associated as a complex to perform sequential enzymatic reactions in nature may be immobilized in series in a perfusive matrix in the following manner.
• a solution containing a concentration of the first enzyme in the series insufficient to saturate all available binding sites on the matrix is provided to the perfusive matrix, e.g., to packed particles in a column.
• the solution further is provided at a fluid flow velocity sufficient to produce perfusion.
• a second solution containing a second enzyme also at a concentration insufficient to saturate all available binding sites, then is added.
• the enzymes in the second solution also provided to the matrix at a perfusive flow rate, will flow past the zone of
• FIGURE 2 A solution of enzyme substrate 20, is pumped by a pump 22 to an enzyme reactor 24.
• the enzyme reactor 24 is a solution of enzyme substrate 20, is pumped by a pump 22 to an enzyme reactor 24.
• the enzyme reactor 24 is a solution of enzyme substrate 20, is pumped by a pump 22 to an enzyme reactor 24.
• the enzyme reactor 24 is a solution of enzyme substrate 20, is pumped by a pump 22 to an enzyme reactor 24.
• the enzyme reactor 24 is pumped by a pump 22 to an enzyme reactor 24.
• particles 10 is preferred, other matrix forms may be used. It is necessary only that the matrix be rigid, so that it can withstand substantial pressure drops, and that it define throughpore sets capable of
• the solution of enzyme substrate 20 is passed through the reactor 24, thereby inducing an enzymatic reaction resulting in a product stream 26 exiting from the enzyme reactor 24.
• the apparatus also may be part of an automated system.
• the apparatus preferably further comprises a multi-port sampling valve 28 which may provide the enzyme(s) to be loaded, as well as all necessary solvents or buffers, including washing solvents, eluting solvents, and recycling or "stripping" solvents.
• a valve 30 at the exit of the reactor 24 may direct the product stream 26 to a detector 32, to waste collectors 34, or to product collectors 36.
• valve positions may be under computer control.
• the rate at which a solution (e.g., 20) is passed through the enzyme reactor 24 and the dimensions of the throughpores 14 and the enzyme interactive surfaces 18 are important to the practice of the invention. That is, flow rates sufficient to induce convective flow through both the interstitial pores 12 and the throughpores 14 should be achievable without requiring excessive pressure drops. Moreover, for true perfusive operation, the convective flow rate within the throughpores 14 should be greater than the rate of diffusion of the solution 20 within the throughpores 14.
• perfusive particles and their throughpores are of a mean diameter sufficient to permit substrate conversion to be performed in perfusion mode.
• a characteristic Peclet number Pe
• VL/D the convective velocity through.the pore
• L the length
• D the diffusivity of the solute through the pore.
• the Peclet number which describes the ratio of convective to diffusive transport within the pores of a material is always much less than one.
• the Peclet number in the second set of pores e.g., the throughpores
• the threshold flow rate is about 300 cm/hr. Above this threshold, it will be found that increased pressure drop and velocity permit increased throughput per unit volume of matrix above levels heretofore achievable. Extraordinary productivities are achieved at flow rates of within the range of 1000-4000 cm/hr.
• Particle sizes commonly used in IMERs are substantially larger than conventional HPLC matrix particles, often on the order of 100-1000 ⁇ m in
• Perfusive matrices comprising particles in this size range can have intraparticle throughpores in the range of aobut 2-20 ⁇ m.
• the threshold flow rate need only be about 0.3 cm/hr to achieve convection within the pores (or, assuming only 1% bed to pore velocity, 30 cm/hr).
• perfusion can be achieved at very low pressure drops and at sufficiently slow flow rates that enzyme conversion reactions are not compromised, even for reactions requiring flow rates at fractions of a ml/min.
• perfusive matrices comprising smaller particles and requiring concomitantly higher threshold flow rates still will be useful, even when enzyme reactions are run below the perfusion mode. There are several reasons for this. First, the enzymes themselves may be loaded in the perfusion mode, allowing the column to be loaded in seconds, a fraction of the time required using
• intraparticle throughpore size substantially reduces pore effects identified with conventional porous matrices.
• the reduced ratio of interparticle and intraparticle pores, and the substantially reduced intraparticle diffusional pathlengths that characterize these matrices virtually eliminate substrate or enzyme inhibition due to solute build up in a stagnant mobile phase.
• the invention may be further understood from the following, nonliraiting example.
• Calf intestinal alkaline phosphatase (CI-AP) is noncovalently attached to a perfusive reverse phase column (POROSTM RM, PerSeptive Biosystems, Inc.,
• CI-AP reconstituted to 1 mg/ml with 10 mM tris(hydroxymethyl)aminoethane, pH 8.0, is diluted (1:100) in 10 mM phosphate buffer (PB), pH 7.1, and ng quantities loaded onto the column at a given flow rate, generally between 0.1 ml/min to 0.5 ml/min. Typically, the column is loaded in about 10 seconds.
• the enzyme substrate p-nitrophenylphosphate
• PNPP Pierce Co., Rockford, IL
• 10 mM diethylanolamine 0.5 mM MgCl 2 , pH 9.5
• the substrate reaction is run as a continuous flow system at a given flow rate and concentration of enzyme.
• Product is identified by absorbance at 405 nm.
• the column may be stripped by washing with PB, followed by a stripping solvent comprising 80%
• PNPP can be passed over the column before a second enzyme is loaded, and the effluent tested for residual enzyme activity.
• Fig. 3A and 3B represent plots of product formation versus enzyme concentration for 10 different flow rates and two different substrate concentrations (0.1 mg/ml and 1.0 rag/ml in 3A and 3B, respectively).
• a 10-fold increase in substrate concentration appears to have no effect on the system.
• slower flow rates increase sensitivity of the assay.
• preferred substrate reaction flow rates e.g., less than about 0.2 ml/min are below the threshold
• perfusive particles for use in IMERs have a diameter on the order of about 100-1000 ⁇ m, substantially reducing the threshold flow rate for intraparticle convective flow.
• immobilizing enzyme is placed in tandem with the first, and the effluent from the first column passed over it to test for leaching enzyme. No significant substrate conversion can be identified in this second column when substrate is subsequently passed through this second column, indicating that substantially no enzyme has been leached from the first column..

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• 1992
  • 1992-04-09 EP EP92911278A patent/EP0580761A4/en not_active Withdrawn
  • 1992-04-09 WO PCT/US1992/002887 patent/WO1992018636A1/en not_active Ceased
  • 1992-04-09 CA CA002102237A patent/CA2102237C/en not_active Expired - Fee Related
  • 1992-04-09 AU AU19172/92A patent/AU1917292A/en not_active Abandoned
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