Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1992-02-28
1994-01-18
Naff, David M.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435180, 435182, C12P 1924, C12N 1108, C12N 1104
Patent
active
052799484
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to a particulate immobilized enzyme preparation, to use of the immobilized enzyme preparation in an enzyme-catalyzed process and to a process for immobilizing enzymatically active biological material.
BACKGROUND ART
It is known that enzymes can be immobilized without using a carrier by cross-linking with polyethylene imine (PEI) and a cross-linking agent such as glutaraldehyde or polyazetidine (e.g. U.S. Pat. No. 4,288,552, U.S. Pat. No. 4,355,105, EP 297,912). Such methods can be used to produce immobilized enzymes in particle form with good activity and physical strength, suitable for continuous use in a fixed-bed column. However, PEI is an expensive material, so it is desirable to find alternatives to this. Also, immobilization of some enzymes with PEI some times leads to poor flocculation, resulting in difficult dewatering.
It is the purpose of the invention to provide a method with improved flocculation and dewatering and without the need for PEI for producing an immobilized enzyme preparation with satisfactory properties for fixed-bed column use: activity, stability (half life) and physical strength (pressure drop).
SUMMARY OF THE INVENTION
It has surprisingly been found that the object can be obtained by replacing polyethyleneimine with a certain polymer that has never been described for use in immobilization.
Accordingly, the invention provides a particulate immobilized enzyme preparation obtainable by a process comprising the sequential steps of: material N-vinyl formamide,
The invention also provides use of the immobilized enzyme preparation in an enzyme-catalyzed process. Finally, the invention provides a process for immobilizing enzymatically active biological material, characterized by comprising the above sequential steps.
DETAILED DESCRIPTION OF THE INVENTION
The biological material to be immobilized according to the invention is enzymatically active. It may comprise enzymatically active microbial cells in the form of a culture broth containing intact cells or cell paste consisting of partly or fully disrupted cells. The biological material may also consist of or comprise a cell-free enzyme solution or purified enzyme.
The biological material may also comprise inactive protein, preferably 0-50% by weight. Thus, if highly purified enzyme is to be immobilized, it may be preferable to add inert protein such as albumin or gelatin.
The quantity of water present in the reaction mixture is not critical. Excess water will be removed during dewatering without any serious loss of active material. Thus, water may be added to obtain a convenient consistency. Conveniently, the biological material is added in the form of an aqueous dispersion or solution typically with 1-25% (w/w) of dry substance, particularly 1-10% in the case of culture broth or 10-25% in the case of a purified enzyme.
The polymer to be used in the invention may be a homopolymer of 1-aminoethylene or a copolymer of this monomer and N-vinyl formamide. It preferably contains 10-100 mole % (most preferably 25-50%) of --CH.sub.2 --CH(NH.sub.2)-- units and 0-90% mole % (most preferably 50-75%) of --CH.sub.2 --CH(NH--CHO)-- units. It preferably has a molecular weight in the range 50,000-500,000. Such polymers may be produced by hydrolysis of N-vinylformamide homopolymer, e.g. according to U.S. Pat. No. 4,421,602 or EP 71,050. The molecular weight of the polymer is described in this application by the Fikentscher K value of the non-hydrolysed N-vinylformamide homopolymer as described in U.S. Pat. No. 4,421,602. This K value can vary between 10 and 200. The amount of polymer is preferably 2-30% by weight of the dry matter in the biological material and most preferably 2-15%.
Chitosan may be used in addition to the above-mentioned polymer. Preferably, 1-15% of chitosen and 1-15% of the polymer are used (% by weight of the dry matter in the biological material). The chitosan should be introduced before the addition of cross-linking in step c); it may be added together with the polymer.
The cross-l
REFERENCES:
patent: 4288552 (1981-09-01), Gestrelius
patent: 4421602 (1983-12-01), Brunnmueller et al.
Barta et al., Chemical Abstracts, vol. 107, p. 347 abstract No. 92712n (1987).
Yutaka Moroishi, abstract of JP 59-14789 (1984).
Jorgensen Ole B.
Pedersen Sven
Lambiris Elias J.
Naff David M.
Novo Nordisk A S
Zelson Steve T.
LandOfFree
Immobilization of enzymes with a cross-linking agent and a polym does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Immobilization of enzymes with a cross-linking agent and a polym, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Immobilization of enzymes with a cross-linking agent and a polym will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1135778