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
  • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier

Definitions

• the present invention relates to a carrier for immobilising biologically active organic material.
• biologically active organic material are meant, in the context of this invention, substances having some type of biological activity, such as enzymes and microorganisms, including bacteria, fungi, algae, cells and the like.
• the carrier is a solid material which is substantially inert to the active material and its reaction products.
• the carrier usually is in the form of relatively large grains readily separable- from a fluid.
• the advantages of anchoring biologically active material are well known and obvious and imply, above all, that one can better economise on the expen ⁇ sive active material in that it can be more readily separated and recovered from a fluid.
• the known carriers comprise a multiplicity of different materials, such as pumice, silica, ceramics, polymeric materials etc.
• glass is a known carrier material, primarily in the form of microporous glass spheres.
• Filter bodies of glass fibres are also known within the art, but these are not sintered glass fibre matrices, and the filter body is held together by a polymeric binder, such as an acrylate resin.
• a polymeric binder such as an acrylate resin.
• This structure is unsuitable for the use which is contem ⁇ plated in the present invention because the polymeric binder may affect the biologically active material and also, in the production of dense compressed filter bodies, may flow out and block the pores so that a homogeneous and uniform porosity is not obtained.
• DE 2,443,502 which relates to a carrier of the type filter candle for enzyme immobilisation.
• the carrier can be built up either of helically wound material or of porous par- ticulate material.
• the porous particulate material may either be sintered or held together by curing by means of a polymeric binder.
• the inner pores of the particles presumably are fused together and become inaccessible, and similarly the use of a cured polymeric binder will cause the surface of the particles to be covered by the polymeric binder, such that the pore openings are covered and the pores also in this instance become inaccessible.
• the present invention aims at providing a novel carrier for immobilising biologically active material, said carrier being solid, inert, uniform and entirely open, and having excellent dimensional stability, a high inner total surface, homogeneous and controllable porosity, and a structure which is characterised by even and smoothly rounded inner surfaces.
• the carrier accord ⁇ ing to the invention is a porous body consisting of joined-together particles of a porous sintered glass fibre matrix.
• the particles are preferably held to ⁇ gether at their points of contact with one another by means of a bond obtained in conneccion with the carbonisation of an organic binder.
• a channel system is formed which permits liquid flow and, thus, good liquid contact with the individual particles.
• the inner pore walls of the particles are formed by solid glass fibre "rods" which have a well-defined diameter and are sintered together at the points of contact.
• Such sintering gives a glass fibre matrix having a three-dimensional lattice or network structure with even, homogeneous and smoothly rounded inner surfaces, which structure provides high strength and rigidity and is highly useful for the transport of substrates and products.
• the glass fibre matrix is characterised in that it can be formed with extremely fine pores and still retain its open pore structure, which is not possible with other methods of making porous glass.
• the pores are given the form of spherical voids interconnected by constricted apertures.
• the pores form comparatively closed channels which are obtained by selective acid dissolu- tion of a phase in a multiphase gaseous mixture.
• conventional powder sintering technique a low volume proportion of pores is obtained.
• fine pore dimen ⁇ sions a considerable proportion of the pores will, besides, be of the closed type.
• a sintered glass fibre matrix is meant that the original glass fibres have been compressed under pressure at elevated temperature, such that the fibres at their points of contact are fused together to pro ⁇ vide a uniform matrix.
• the sintered glass fibre matrix forms a three-dimensional network with a regular open structure having high permeability to gas and liquid. Blocks consisting of the sintered glass fibre matrix are then comminuted into particles or disks.
• the carrier according to the invention consists of joined-together porous particles, it provides, in a manner of speaking, a double porosity, i.e. both an "outer" porosity and an “inner” porosity.
• outer porosity is here meant the ratio of the void volume formed between the joined-together particles to the total volume of the carrier, while the inner porosity is the ratio of the pore volume of the joined-together porous particles to the total volume thereof.
• the carrier according to the invention exhibits, be- cause of its composition, both an outer porosity and an inner porosity, the inner porosity being utilised primarily for safe and interference-free immobilisation of the biologically active material, such as an enzyme, while the outer porosity contributes to an effective contact between the biologically active material and the fluid upon which it is intended to act, thereby to achieve an optimal reaction and an efficient removal of products formed.
• the biologically active material such as an enzyme
• the inner porosity of the sintered glass fibre matrix particles is determined by the density of the matrix and the diameter of the fibres employed. At a density corresponding to the one of the glass employed, there is obtained an entirely compact and nonporous matrix which lies outside the scope of the present invention.
• a general rule is that the particles in the carrier according to the invention have a density within the range 20-2000 kg/m , preferably 300-2000 kg/m , a density in the range 1500-2000 kg/m being especially preferred.
• the average diameter of the glass fibres utilised in the production of the sintered particles for the carrier according to the invention generally is within the range 0.3-100 um, preferably 0.5-5 am, a fibre diameter of 0.5-3 um being especially preferred.
• a general rule is that the smaller fibre diameters make it possible to obtain particles with fine pores, simul ⁇ taneously as the favourable three-dimensional network structure is maintained. In this respect, a fibre diameter below about 5 ⁇ m is especially advantageous.
• particles having fine pores and an open network structure are preferred because they are especially well suited for the immobi ⁇ lisation of biologically active material of small dimensions, such as enzymes and bacteria, simultaneously as an efficient transport of substrate and products is made possible.
• the pore size of the sintered particles in the carrier according to the invention is determined by the density of the particles and the diameter of the fibres utilised in the production of the particles. By suitable selec ⁇ tion of these parameters, it is thus possible to ob ⁇ tain the desired pore size of the particles.
• the par- tides according to the invention suitably have a pore size of at most about 20 ⁇ m, usually about 1-20 ⁇ m. A pore size within the range 1-15 ⁇ m is preferred, a pore size of about 5-10 ⁇ m being especially preferred.
• the particles in the carrier according to the invention consist of a sintered glass fibre matrix.
• the composition of the glass employed is not critical, although the glass must, of course, be a glass that can be converted into fibres. Thus, it is possible, within the scope of the invention, to vary the glass composition of the particles in many ways.
• the form of the porous sintered glass fibre matrix is not critical and may be varied in innumerable ways, it is preferred to produce it origi ⁇ nally in the form of a planar disk.
• a simple method of making such a disk is to sinter the glass fibre matrix between two press plates at elevated pressure and temperature.
• the finished disk may have optional ' thickness, but preferably the thickness lies within the range 0 A 05-5 cm, most preferably 0.05-2 cm.
• the finished disk is then comminuted into fragments or regular pieces having an average diameter of about 0.005-5 cm, preferably 0.005-0.05 cm.
• average diameter is here meant the diameter of a sphere having the same volume as the fragment or piece concerned.
• the fragments may also be in the form of disks having a thickness of 0.02-0.5 cm, in which case the diameter is the average disk width.
• the glass fibre matrix After sintering into blocks, the glass fibre matrix thus is comminuted into particles, each of which consists of a three-dimensional network struc ⁇ ture by which high mechanical strength is imparted to the particles which thus obtain good tolerance to being stacked in columns or subjected to strain during mechanical stirring.
• the particles Before their use for the object of the present invention, the particles are joined together to form a three-dimensional network structure in connection with carbonisation of an organic binder.
• the structure is ' then impregnated either with a binder which then is caused to solidify, or with a binder monomer which is polyme ⁇ rised, and in this manner a solid self-supporting structure is obtained which is then heated, preferably in air, for "carbonisation” of the organic binder which thus is substantially completely removed from the free particle surfaces of the structure but imparts a bonding effect to the adjacent or adjoining contact surfaces of the particles, such that the initially loosely combined structure forms a firmly held-together unit with an open structure consisting of joined-together particles of a porous sintered glass fibre matrix, the particles being held together at their points of contact by a carbonised organic binder.
• the finished carrier will have the above-mentioned advantageous combination of outer and inner porosity.
• the organic binder employed in the context of this invention preferably is an organic polymeric binder.
• preferred polymeric binders are, for example, acrylate plastics, such as polymethacrylate and polymethyl methacrylate, including monomers to form these polymers; vinyl acetate plastic, such as polyvinyl acetate; and vinyl acetal plastic, such as polyvinyl acetal and polyvinyl butyral , but it should be noted that the invention is not restricted to these polymeric binders.
• Highly complicated shapes are obtainable by an alternative and comparatively simple production method, according to which the par ⁇ ticles are first impregnated with a highly viscous liquid polymer (.such as urethan dimethacrylate or the reaction product of bisphenol-A and glycidyl me- thacrylate (BIS-GMA) ) which cause the particles to stick together.
• a highly viscous liquid polymer such as urethan dimethacrylate or the reaction product of bisphenol-A and glycidyl me- thacrylate (BIS-GMA)
• the particle mass can now be given the desired shape which may be fixed initially by curing at least the superficially located polymer. This can be done for example by heating or irradiation with UV light or, alternatively, visible light.
• the method of curing is decided by the initiators and accelerators that have been added according to prior art technique.
• the body thus shaped and, possibly, fixed is then heated, optionally after embedding it in a refractory material,
• the carbonisation conditions for the polymeric binder are not critical, except that the temperature must not be so high that the softening point of the glass fibre matrix particles is exceeded and the par ⁇ ticles soften and their pores collapse. During carbo- nisation of the binder, the temperature must always be maintained sufficiently low to ensure that the pores of the glass fibre matrix particles will be kept intact. Generally, a suitable temperature range is about 500-700°C, preferably about 550-650°C, about 600 C being the temperature which is especially pre ⁇ ferred at present.
• the carbonisation atmosphere is not critical and preferably is air.
• the carbonisation time must be sufficiently long to effect complete carbonisation of the organic binder, and generally a time of about 15-60 min. is satisfac ⁇ tory. In most cases, a time of about 30 min. is suffi ⁇ cient.
• the porosity of the glass fibre matrix particles i.e. the inner po ⁇ rosity of the carrier, may be controlled within wide limits by controlling the fibre diameter and the sinter ⁇ ing pressure.
• the outer porosity of the carrier may be controlled by suitable selection of the dimensions of the glass fibre matrix particles.
• the sintered porous glass fibre matrix is comminuted into particles having an average diameter of about 0.005-5 cm, preferably about 0.005-0.05 cm.
• the particles have a relatively narrow particle size di ⁇ stribution, i.e. all particles have substantially the same average diameter, thereby to provide a carrier of substantially homogeneous outer porosity.
• the carrier may also be built up by different layers of different particle sizes, such that the outer porosity will be essentially homogeneous in each individual layer but vary from one layer to another.
• the carrier preferably is produced in that the glass fibre matrix particles are loosely joined together in a mould, whereupon the organic binder is added. Using a mould also makes it possible to vary the shape of the carrier in many different ways, such as disk shape, spherical shape, cylindrical shape, cubic shape, or the like.
• the biologically active material concerned for example an enzyme
• the carrier for example by being impregnated with a solu ⁇ tion or dispersion of the active material. If this is a microorganism suspension, it will be sucked up as if by a sponge and can then be developed by the formation of micro-colonies in the network structure.
• the carrier with the immobilised active material is then ready for use, for example for carrying out an enzyme-catalysed chemical reaction.
• the carrier may be in the form of an aggregate or lumps which simply are introduced into the reaction medium which may consist of a substrate solution for the enzyme.
• the carrier When the carrier is brought into contact with the reaction medium, the reaction starts, and to facilitate and accelerate the reaction, the reaction medium suitably is stirred. At the end of the reaction, or when the reaction has advanced to the desired stage, the carrier is separated from the reaction medium by filtration or simply by removing the carrier lumps from the reac ⁇ tion medium.
• the carrier In another type of application of the carrier according to the invention, the carrier is in the form of a disk or plate.
• the thickness of the carrier disk may be varied as desired, but preferably lies within the range 0.05-2 cm.
• the carrier disk may have optional form, such as circular, elliptical or polygonal, for example rectangular or square.
• the biologically active material is first im ⁇ mobilised in the carrier disk, for example by impregnat ⁇ ing the disk with a solution or dispersion of the active material, whereupon the carrier disk is ready for use, for example for analytical purposes.
• the fluid which is to be affected by the immobilised bio ⁇ logically active material is then contacted with the carrier disk, for example by letting the fluid flow through the disk.
• An example of this is a substrate solution flowing through a carrier disk in which an enzyme is immobilised.
• the reaction may be carried out in batches, but also continuously in that the carrier disk is inserted across the cross-sectional area of a tubular reactor so that the reagent fluid (such as a substrate solution) * passes the thickness of the disk.
• the present invention provides a carrier intended for the immobilisation of biologically active material and having highly desirable and advantageous properties, such as great dimensional stability and strength, an open grid-like structure, and having a well-defined and reproducible, uniform porosity and pore size. Furthermore, the carrier is inert to the biologically active materials concerned and their reaction products, while at the same time having affinity to the immobili- sation of biologically active materials.
• the glass surface may be given positive groups by silanisation.
• the glass composition may vary, . and tracer elements may be incorporated which, by ion exchange with the surroundings, can affect the condi ⁇ tions of life of the microorganisms.
• Other advantages of the glass structure are its hydrophilic character (facilitates substrate absorption etc. ) and its excel ⁇ lent mechanical and thermal properties.
• the excellent mechanical properties protect, for example, accommodat ⁇ ed cells during stirring or stacking in a column.
• the carrier is not decomposed under the action of cells which are being divided, or the action of an inner pressure during gas-forming reactions.
• the thermal stability provides for hardiness against high process temperatures and sterilisabilit .
• the method as referred to above for producing porous glass in the .form Of a three-dimensional network structure •has the advantage that it gives an entirely open struc- ture having a high pore volume and large total inner surface, which structure does not contain spherical and wholly or partly closed voids. In this manner, optimal conditions for substrate and product transport through the carrier medium are established.
• the fibres have a diameter of less than 5 ⁇ m, this method also makes it possible to produce extremely fine-pore (fine-mesh) structures with pores having an average diameter of less than 10 ⁇ m, while retaining the above-mentioned advantages. This is not possible with any other method previously disclosed.
• the pore size be about 5 times the cell size, but not greater than 10 times the said cell size.
• fine-pore structures having pore sizes of less than 10 ⁇ m, in particular structures with pores within the range 3-10 ⁇ m, are especially desir ⁇ able as carriers of microorganisms.
• pore sizes of less than 3 ⁇ m are optimal in connection with enzyme processes, while pore sizes greater than 10 ⁇ m are desirable for the cultivation of, for example, mammal cells.

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• Chemical & Material Sciences (AREA)
• Genetics & Genomics (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Wood Science & Technology (AREA)
• Health & Medical Sciences (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Zoology (AREA)
• Biomedical Technology (AREA)
• Biotechnology (AREA)
• Inorganic Chemistry (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Microbiology (AREA)
• Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
• Solid-Sorbent Or Filter-Aiding Compositions (AREA)

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Cited By (10)

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Citations (6)

• 1985
  • 1985-01-09 SE SE8500082A patent/SE455103B/sv not_active IP Right Cessation
  • 1985-12-16 AU AU53147/86A patent/AU5314786A/en not_active Abandoned
  • 1985-12-16 EP EP19860900674 patent/EP0213147A1/en not_active Ceased
  • 1985-12-16 WO PCT/SE1985/000524 patent/WO1986004088A1/en not_active Application Discontinuation
  • 1985-12-16 JP JP50059686A patent/JPS62501678A/ja active Pending
• 1986
  • 1986-09-02 DK DK419686A patent/DK419686A/da not_active Application Discontinuation

Patent Citations (6)

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