Chemistry: molecular biology and microbiology – Carrier-bound or immobilized enzyme or microbial cell;... – Enzyme or microbial cell is immobilized on or in an organic...
Patent
1983-11-08
1987-03-03
Naff, David M.
Chemistry: molecular biology and microbiology
Carrier-bound or immobilized enzyme or microbial cell;...
Enzyme or microbial cell is immobilized on or in an organic...
435178, 435182, 424 93, 424DIG7, 424493, C12N 1102, A01N 2500, A01N 6300
Patent
active
046475362
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method of immobilizing bio material such as animal cells, plant cells, bacteria, algae, fungi, viruses or proteins by encapsulation in bead polymers.
During recent years there has been a considerable interest in immobilizing bio material. The most usual polymers used for encapsulating bio material are alginate, polyacrylamide, carrageenan, agar or agarose. Of these alginate and carrageenan are the only ones which can be manufactured simply in spherical form with encapsulated material. This is done by ionotropic gelling, i.e. the alginate is dropped down into calcium solution and the carrageenan in a potassium solution. However, relatively large beads (2-3 mm diameter) are usually obtained in this way, and beads that are stable only in the presence of ions (calcium and potassium ions, respectively). In the use of agar, agarose, collagen, polyacrylamide, gelatine or fibrinogen, the biomaterial is usually mixed with polymer or polymer solution which is then caused to gel. In order to obtain a suitable size, this gel is fragmented and possibly further cross-linked to attain higher stability. In the preparations produced in this way there is leakage of the encapsulated material in the fragmentation, and due to their heterogeneity they are found to have poor flow in column processes.
Spherical polyacrylamide particles with encapsulated enzymes can be produced by a bead polymerization process, where the monomer solution together with enzyme and catalysts are dispersed in a hydrophobic phase (see Swedish Pat. No. 7204481-1). The hydrophobic phase comprises an organic solvent (exemplified by toluene plus chloroform) and an emulsifier. Since both these solvents and emulsifiers have a denaturing effect, this method is suitable only for relatively insensitive material.
There has therefore been a large need for a more gentle dispersion medium for the production of spherical polymer particles. The present invention discloses that sensitive bio material can be encapsulated with full viability and with retained growth ability if the organic solvent is exchanged for either of the following dispersion media: saturated or unsaturated animal or vegetable fats or oils, tri-n-butylphosphate, liquid silicone, paraffin oil or phthalic acid dibutyl ester. Certain of these dispersion media have been used before in the preparation of spherical polymer particles. In the U.S. Pat. No. 4 169 804 there is described a method of producing magnetic microspheres by dispering an albumin solution in vegetable oil with subsequent heating thereof to 140.degree. C. or cross-linking the albumin polymer with aldehydes. This technique cannot be used for encapsulating sensitive bio material, since either the high temperature or the cross-linking aldehyde then result in inactivation.
A method is described in "Biotechnology Letters" vol. 3, pages 65-70, 1981, of producing spherical polyurethane particles by dispersing the monomers in paraffin oil. By including bacteria in the monomer mixture there is obtained a catalytically active preparation (although whether the bacteria retain their propagating ability is doubtful, furthermore there is great risk that they are linked covalently to the carrier). On the other hand, by combining these gentle dispersion media with a suitable immobilization-method carrier there may be obtained, as with the preparations described here, 100% viability and retained growth ability. The denaturing action of different dispersion media on agar-encapsulated plant cells is compared in table 1. It will be seen from the table that these dispersion media give a retained viability compared with the usual standard method for the production of spherical polymer particles (Swedish patent 7204481-1). In table 2, this relationship is also shown to apply to yeast cells encapsulated in polyacrylamide.
The polymer which is combined with the dispersion medium is selected on the basis of the bio material sensitivity. Insensitive materials such as certain proteins can be linked covalently to the polymer with
REFERENCES:
patent: 4169804 (1979-10-01), Yapel et al.
patent: 4352883 (1982-10-01), Lim
patent: 4353888 (1982-10-01), Sefton
patent: 4389330 (1983-06-01), Tice et al.
patent: 4518693 (1985-05-01), Kuu
Derwent Abs. 64776 B/36 (1979).
Mosbach Klaus
Nilsson Kjell
Foley Shawn P.
Naff David M.
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