WO1990012873A1

WO1990012873A1 - Micro-particules for immobilising macromolecules

Info

Publication number: WO1990012873A1
Application number: PCT/GB1990/000572
Authority: WO
Inventors: Richard Andrew Williams; Colin Webb; Bernard R. Pieters
Original assignee: Davy Research And Development Limited
Priority date: 1989-04-17
Filing date: 1990-04-17
Publication date: 1990-11-01
Also published as: GB8908662D0; AU5422790A

Abstract

A micro-particle is described as a support for an immobilised biocatalyst, such as an enzyme (e.g. glucoamylase), which comprises a cross-linked natural polymer, such as a carrageenan, the surface of which micro-particle has been treated with a modifier compound having a first functional group capable of reacting with said natural polymer to effect hardening thereof and a further functional group capable of permitting immobilisation of a biocatalyst on said micro-particle either by reaction with said biocatalyst or with a surface modifying agent which can itself react with said biocatalyst.

Description

MICRO-PARTICLES FOR IMMOBILISING MACROMOLECULES

The present invention relates to micro-particles on which a maσromoleσule or other form of biocatalyst, such as a bacterium or yeast, may be immobilised, and more particularly (although not exclusively) to such particles which also possess magnetic properties.

Magnetic micro-particles on which a macromolecule (e.g. an enzyme) is immobilised are particularly suited for use in biotechnological processes, e.g. processing involving controlled mass transport or recovery of enzymes.

Magnetism has been used in a number of different areas encompassing biotechnology. For example, cell recovery, enzyme and cell immobilisation, affinity chromatography, immunoassays, drug delivery, capture of carcinogens, and waste -water -treatπtent. The common feature of all these systems is that they require a suitable device containing a magnetic field (separator or reactor) and a mechanism to magnetically label the species of interest (e.g. a magnetic particle which fixes the biocatalyst by physical or chemical coupling to the surface, or by direct entrapment within the structure) .

The key advantage of magnetic tagging and subsequent separation is that selectivity, simplicity, speed and high efficiency can usually be guaranteed, even in viscous and colloidal media. Hence such techniques are well developed in medical applications which use sophisticated and expensive magnetic supports. However, the potential range of application in the present application is directed towards, but not limited to, "low value high-throughput" processes, which require inexpensive magnetic supports for which many separation applications can be achieved without recourse to expensive high field gradient superconducting magnetic systems.

Magnetic properties need not be used only for separations but may be exploited to control transport of magnetic material in a process (e.g. residence time, stability of fluidised beds, transport along pipes. , mixing within reactors, enhanced magnetic flocculation etc. ) .

For biocatalysts the magnetic carrier particles must possess certain characteristics. For example, if the beads are to be used in a magnetically stabilised fluidised bed reactor (MSFBR) such properties would include:

(i) a low apparent density, to minimise sedimentation under the influence of gravitation forces;

(ii) high rigidity and high abrasion resistance; (iii) high magnetic susceptibility; (iv) uniform shape and size; (v) small size (<200ιm) to minimise the flαidis tion -velocity;

(vi) beads or materials contained within them should not affect the material to be bound on their surface (e.g. to cause deactivation, in the case of an enzyme) ;

(vii) beads must be manufactured from low-cost materials to yield a high concentration of reactive sites for biocatalysts attachment, using a simple method allowing scale-up for large scale production, whilst retaining close control on the quality of the bead being produced (e.g. size, shape, density) ;

(viii) the beads and their bound material must be chemically stable with time for conditions (pH, temperature etc) prevalent in the environment where the process is to be applied; (ix) beads should not aggregate during the processing operation.

According to the present invention there is provided a micro-particle having a maximum cross-sectional dimension of 2mm characterised in that it comprises a cross-linked natural polymer and that its surface has been treated with a modifier compound having a first functional group capable of reacting with said natural polymer to effect hardening thereof and a further functional group capable of permitting immobilisation of a biocatalyst on said micro-particle either by reaction with said biocatalyst or with a surface modifying agent which can itself react with said biocatalyst. In a preferred aspect there are provided micro-particles (as herein defined) of a cross- linked natural polymer, such as a carrageenan, whereof the surface or surfaces of the particles have been treated with a modifier compound having an amino group which has reacted with the natural polymer (e.g. carrageenan) to effect hardening thereof and having a further functional group which is adapted to allow the immobilisation of a biocatalyst, typically a macromolecule, either by reaction with the biocatalyst or with a surface modifying agent which will itself react with the biocatalyst.

The term micro-particles as used herein refers to particles with a maximum cross-sectional dimension of 2mm.

As examples of natural polymers there can be mentioned alginates and polymers extracted from seaweed, such as carrageenan.

The carrageenans are naturally occurring polymers which may be extracted from seaweed, typically Chondras crispus or Eucheuma cottonii Type III. The carrageenans comprise alternating galactose and anhydrogalactose units. There are three principal carrageenans (referred to as k, i and -carrageenan) , and it is the former polyanionic gel (i.e. k - carrageenan) which is particularly suitable for use in the invention since it is comparatively cheap and readily gelled and hardened by bridging the sulphate groups with cations (e.g. potassium ions) and/or by cross-linking with diamines. Preferably the micro-particles comprise a magnetic material embedded in a matrix of the cross-linked carrageenan. Preferably this is a colloidal magnetic material (e.g. magnetite) incorporated in the micro- particles during their production.

The modifier compound may be, for example, an amino compound. In this case the amino compound can serve the dual function of providing one amino group which reacts with, and thereby hardens, the carrageenan via a salt-type interaction and one functional group (preferably a further amino group) for reaction (e.g. via a Schiff's base reaction) with a macromolecule or other biocatalyst or, more preferably, with a surface modifying agent which will itself react with the macromolecule or other biocatalyst. The amino compound in this case can also act as a "spacer" be ween the surface of the particle and the macromolecule or other biocatalyst to avoid steric/conformational hindrance which could adversely affect the biocatalyst activity of the macromolecule or other form of biocatalyst.

Preferably also the modifier compound containing the amino group is a di-, tri- or higher functionality amine, most preferably an aliphatic amine with a chain of 4 to 8, preferably 6, carbon atoms separating two primary amino groups. The preferred compound is 1,6-hexamethylene diamine.

The biocatalyst may be, for example, a bacterium or a yeast cell, in which case it is immobilised on the micro- particle by reaction with a surface component thereof, such as a surface protein. The bacterium or yeast cell will be selected to express a desired chemical product when grown on a suitable nutrient medium. The bacterium or yeast cell may have been .subjected to genetic modification by known genetic engineering techniques so as to enable or to enhance production of the desired chemical product. Production of ethanol by fermentation using a yeast such as Saccharomyces cerevisiae or production of an antibiotic such as a penicillin by use of a bacterium are examples of processes in which the immobilised biocatalysts of the invention can be used.

The invention is also applicable to the immobilisation of enzymes as the biocatalyst. Examples of such enzymes include aminoacylase for the production of L- amino acids from acetyl-DL-amino acids, penicillin amidase for the production of 6-aminopenicillamic acid, glucose isomerase for the production of high fructose syrup, and glucoamylase for the production of glucose from dextrin. Also worthy of mention are lactases, such as β- galactosidase. In the case of glucoamylase the amino compound is preferably 1,6-hexamethylene diamine. Preferably also a di- or higher functionality aldehyde e.g. a trialdehyde or a polyaldehyde, is used as a -surface modifying agent. Preferably the aldehyde is an aliphatic aldehyde, e.g. one with a 2 or 4 carbon chain separating the aldehyde groups. A preferred aldehyde is glutaraldehyde. Similar techniques can be used for other enzymes.

The present invention thus provides a relatively simple and cheap method by which to manufacture a derivatized magnetic micro-particle covered with aldehyde groups, that is suitable for immobilisation of macromolecules (such as enzymes) and other biocatalysts such as bacteria and yeasts and fulfils the criteria listed above.

The cross-linked micro-particles may be produced by known techniques in which the carrageenan is rapidly agitated to form an emulsion under conditions which effect polymerisation/cross-linking of the carrageenan molecules. Typically the micro-particles will be prepared by emulsion polymerisation of k - carrageenan by mixing this polymer at 70-80°C (e.g. 75°C) with water (and optionally with colloidal magnetic material, e.g. magnetite, if magnetic micro- particles are to be produced) and adding the resultant mixture to a heated vegetable oil (particularly corn oil) which acts as polymerisation initiator for the carrageenan. During the polymerisation, the mixture is agitated at a fast rate, the actual rate determining the size of the final particles.

Polymerisation is terminated by rapidly cooling the mixture, e.g. by pouring it into a flask cooled in an ice bath.

The resultant beads may then be washed in potassium chloride which provides a degree of hardening for the micro-particles. The micro-particles are highly spherical and are sufficiently coherent to be screened if necessary.

The micro-particles may be further hardened with potassium chloride, e.g 0.05 mol dm^"3 KC1 in a 0.3 mol d ■buffer (pH * 7). This treatment may be e ected simultaneously with the hardening of the cross-linked - caxxageenan hy, for example, an aliphatic diamine (e.g. 1,6- hexamethylene diamine - HMDA) . For this purpose, this KC1 solution may additionally contain HMDA to a concentration of about 0.2 mol. The treatment may be effected at 50°C for about 10 minutes.

The free amino groups of the particles thus obtained may be derivatized with glutaraldehyde, for example using a 5% solution of the glutaraldehyde in a 0.005 mol phosphate buffer (pH = 7) at 50°C for about 30 minutes.

The biocatalyst, such as glucoamylase, may be immobilised on the particles thus obtained by reaction with the free aldehyde group of the glutaraldehyde. This procedure may be effected by washing the particles (e.g. with sodium acetate buffer) , contacting the particles with a glucoamylase solution and then washing successively with solutions of sodium acetate buffer, sodium chloride and sodium acetate buffer, urea, and sodium acetate buffer.

The invention will now be further described, by way of example only, with reference to the accompanying drawings in which:

Figure 1 schematically illustrates micro-particles (beads) in accordance with the invention on which the enzyme glucoamylase has been immobilised; and

Figure 2 illustrates a magnetically stabilised fluidised bed reactor in which the beads may be used.

As shown in Figure 1, a micro-particle 1 comprises a matrix of cross-linked carrageenan incorporating colloidal Fe^ ^. The sur ace of the particle 1 has been treated with 1,6-hexamethylene diamine (HMDA). Only one molecule of HMDA is shown, but it will be appreciated that many such molecules are bound to the surface. The HMDA acts as a hardening agent and also as a spacer between the surface and the next layer of absorbed macromolecules. Additionally, the HMDA guards against deactivation from the solution species originating from the magnetic material within the particle.

After treatment with the HMDA, the particle has been further treated with glutaraldehyde which acts as a surface modifier so that the enzyme glucoamylase may be immobilised more efficiently on to the surface of the particle.

The particles illustrated in Figure 1 have distinct advantages over previous varieties of beads which have been used to immobilise glucoamylase. For example, the method based on silanization of magnetite (Sambamurthy, 1987) results in magnetic aggregation of the carrier beads; the use of transition metal titanium (IV)-activated coatings on magnetic particles produces a toxic denser bead and direct coupling to the enzyme did not produce a stable coupling (Cabral & Kennedy, 1983, Cabral, 1986) unless the surface is chelated with hexanediamine and cross-linked with glutaraldehyde (Tosa, 1979), and the drying step in the production process is quite difficult to perform without affecting the quality of the beads; the production of beads of calcium alginate containing magnetite followed by surface treatment (Burns, 1985) is not easily achieved on a large scale, and the resultant beads may display superparamagnetic behaviour; the use of aerosol jets to produce such beads often yields non-spherical particles and aerosol blockage problems (Lockmuller, 1987); the use and physical properties of amine-cured k-carrageenan gels has been studied by Chao (1986).

One specific example of the use of the micro- particles (or beads) is in the starch industry for the production of fructose syrups, where biomagnetic processes offer several advantages over conventional routes. In the production of fructose syrup starch is treated with solubilised -κ-amylase to produce dextrin. This is in turn converted by treatatent with glucoaaπylase to produce glucose, which upon treatment with glucose isbmerase yields ethanol and fructose syrup. The beads produced here overcome previous problems in their use of enzyme immobilisation onto high-cost support particles which proved to be unstable, and to cause severe downstream recovery problems. For example, glucoamylase immobilised on beads or about 600/ιm can be used in conjunction with a magnetically stabilised fluidised bed reactor (Figure 2) or stirred tank reactor to produce glucose by maltose hydrolysis.

A second example is in the dairy industry for the conversion of lactose to glucose and galactose using β- galactosidase immobilised on micro-particles according to the invention. In this way lactose can be produced from milk and/or by treatment of whey used in cheese manufacture. M.A. Burns (1985), Biotech. Bioeng. 27., 137-145 J.M.S. Cabral and J.F. Kennedy (1983), Che . Eng. J., 27. 1349-1355

J.M.S. Cabral (1986), J. Chem. Tech. Biotech. 36. (8), 247- 254 K.C. Chao, (1986), Biotech. Bioeng. 28._/ 1289-1293

CH. Lock uller et al (1987), J. Chem. Tech. Biotech., 40,

33-40

K. Sambamurthy et al (1987), J. Microb. Biotech., 2. (1),

15-21

T. Tosa (1979), Biotech. Bioeng., 2_1, 1697-1709.

The invention is further illustrated in the following Example. Example

1.2g of k-carrageenan was melted by magnetic agitation in 40ml of distilled water at about 90°C. 4g of magnetite was then added and the mixture temperature was maintained at 75°C.

The mixture was poured into 400ml of corn oil at 65°C contained in a 800ml round bottomed flask equipped with a overhead stirrer and a thermometer and set in a large basin. The mixture was agitated using a fast stirrer speed (8 blade impeller 500 rpm) to obtain very small beads.

After 5 minutes, ice-water was poured in the basin to cool the bead suspension to about 10°C. After allowing the beads to settle, the oil was removed and replaced afterwards by a solution of 0.3M KCl to harden the k- carrageenan beads.

Several washings with 0.1M KCl were realised to remove the remaining oil. Beads were then sieved to obtain a narrow size range. The main bead fraction ranged from 500um to 85Oμm and was subsequently separated with sieves of 600um and 710jιm. Beads were then soaked in a 0.3M KCl solution and put in a refrigerator. lg of beads were then subjected to hardening and derivatisation by removing the KCl from the beaker containing the beads, followed by adding 5ml of a solution containing 0.05M KCl, 0.3M phosphate buffer (pH 7) and 0.09M 1,6-hexamethylene diamine (HMDA) cooled at 5°C. The beads and solution were then stirred for 10 minutes, following which the HMDA solution was removed. Next there was added 5ml of 5% w/v glutaraldehyde solution in 0.05M phosphate buffer (pH 7), and the mixture was stirred for 30 minutes at 5°C. Next the glutaraldehyde solution was removed and the beads washed with 3 x 10ml of distilled water and 3 x 10ml of 0.02M sodium acetate buffer (pH 4.5). Following removal of the buffer, there was then added 5ml of glucoamylase solution (500Uml^_J- diluted in 0.02M sodium acetate buffer (pH 4.5) and the mixture was stirred for 2 hours at 5°C. The resulting immobilised enzyme was washed in turn with 10ml of each of the following solutions:

0.02M sodium acetate buffer, pH 4.5 (10 minutes stirring) ,

1M sodium chloride in 0.02M sodium acetate buffer (10 minutes stirring),

€M urea {30 minutes stirring),

0.02M sodium acetate buffer, pH 4.5 (10 minutes st rring) .

The resulting beads having glucoamylase immobilised thereon can then be used successfully in a magnetically stabilised fluidised bed reactor as illustrated in Figure 2 for the production of glucose from dextrin.

Claims

1. A micro-particle having a maximum cross-sectional dimension of 2mm characterised in that it comprises a cross-linked natural polymer and that its surface has been treated with a modifier compound having a first functional group capable of reacting with said natural polymer to effect hardening thereof and a further functional group capable of permitting immobilisation of a biocatalyst on said micro-particle either by reaction with said biocatalyst or with a surface modifying agent which can itself react with said biocatalyst.

2. A micro-particle according to claim 1, characterised in that said natural polymer is a polymer extracted from seaweed.

3. A micro-particle according to claim 1 or claim 2, characterised in that said natural polymer is a carrageenan.

4. A micro-particle according to claim 3, characterised in that said natural polymer comprises k- carrageenan.

5. A micro-particle according to any one of claims 1 to 4, characterised in that the natural polymer comprises a polyanionic gel containing sulphate groups cross-linked by a bridging component selected from cations and diamines.

6. A micro-particle according to any one of claims 1 to 5, characterised in that it further comprises a magnetic material.

7. A micro-particle according to claim 6, characterised in that it comprises particles of magnetite embedded therein.

8. A micro-particle according to any one of claims 1 to 7, characterised in that said modifier compound is an amino compound containing two or more amino groups.

9. A micro-particle according to claim 8, characterised in that said amino groups are primary amino groups.

10. A micro-particle according to claim 8 or claim 9, characterised in that said modifier compound is 1,6- hexamethylene diamine.

11. A micro-particle according to any one of claims 8 to 10, characterised in that the surface thereof has been treated with said modifier compound and with a surface modifying agent which is selected from dialdehydes, trialdehydes and polyaldehydes.

12. A micro-particle according to claim 11, characterised in that said surface modifying agent is glutaraldehyde.

13. A micro-particle according to any one of claims 1 to 12, characterised in that it has immobilised thereon a biocatalyst selected from a bacterium, a yeast cell or an enzyme.

14. A micro-particle according to claim 13, characterised in that the biocatalyst is a glucoamylase or lactase.

15. A process for the production of a desired chemical product by contacting a feed solution with an immobilised biocatalyst in a treatment zone and recovering a reaction solution containing the desired chemical product from the treatment zone characterised in that the immobilised biocatalyst comprises a micro-particle according to any one of claims 1 to 14.

16. A process according to claim 15, characterised in that the micro-particle incorporates one or more magnetic particles and said treatment zone comprises a magnetically stabilised fluidised bed reactor.

17. A process according to claim 15 or claim 16, characterised in that the biocatalyst is glucoamylase, the feed solution is a solution or suspension containing maltose, and said desired product is fructose.