Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound having a 1-thia-4-aza-bicyclo
Patent
1996-05-30
1998-07-14
Naff, David M.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound having a 1-thia-4-aza-bicyclo
435 44, 435 45, 435 47, 435 49, 435 50, 435 51, 435175, 435176, 435177, C12P 3700, C12P 3500, C12N 1118, C12N 1114
Patent
active
057802602
DESCRIPTION:
BRIEF SUMMARY
The application is a 371 of PCT/EP94/04132, filed Dec. 13, 1994.
The present invention concerns enzymes immobilized on carriers selected from the group comprising penicillin-G amidase, glutaryl-7-ACA acylase and D-amino acid oxidase, the use of these enzymes in an enzymatic synthesis reaction as well as a process for improving the volume activity and stability of these enzymes immobilized on carriers.
The immobilization of biologically active substances, e.g. enzymes, on solid carrier materials can be carried out by numerous methods and is described in many monographs and publications (see e.g. Characterization of Immobilized Biocatalysts, Buchholz, K., (publisher), DECHEMA Monographs No. 1724-1731, vol. 84 (1979), Protein Immobilization - Fundamentals and Applications, Taylor R. F. (publisher), Marcel Dekker, Inc. (1991)). Apart from non-covalent immobilization techniques such as adsorption, entrapment, microencapsulation, chelation and aggregation, covalent immobilization methods have gained prominence for industrial applications.
Organic as well as inorganic materials of a synthetic or natural basis are known as carrier materials for immobilizing biologically active substances. The covalent immobilization of proteins on carrier materials is generally carried out by coupling the proteins via the reactive side chains of the amino acids to the carrier. A chemical activation of the carrier or/and the protein is usually necessary for this. The coupling chemistry is determined in each case by the type of protein as well as by the carrier matrix used. Due to the quite considerable differences in the secondary, tertiary and quarternary structures of proteins it is fundamentally impossible to predict the suitability of specific carrier materials for specific enzymes.
Organic carrier materials into which numerous reactive groups can be introduced have prevailed for most commercial applications for the immobilization of biologically active substances. Examples of these are natural polymers such as polysaccharide derivatives and structural proteins as well as synthetic macromolecular substances based on polystyrene or polyacrylate. These organic carrier materials can be activated by conventional chemical methods or already have reactive groups which enable linkages to be made with a protein to be immobilized. However, these organic carrier materials still have some disadvantages which limit their usability. Thus natural polysaccharide derivatives are often sensitive towards microbial degradation (cellulose), they have unfavourable particle properties (cellulose, fibres of different length) and poor mechanical or/and swelling properties.
Synthetic materials are usually insensitive towards microbial attack, but they have other disadvantages. Carrier materials based on polyacrylamide which are usually used in protein-monomer coimmobilizations are composed of potentially cancerogenic monomers (acrylamide) and exhibit a strong swelling in aqueous systems. Polyacrylates, polymethacrylates, hydroxyalkyl-methacrylates and polymethacrylamides can be prepared by polymerization of the appropriate monomers using suitable cross-linking agents and some are commercially available. Carriers that are already activated (e.g. Eupergit.RTM., Biosynth.RTM.) can be produced from these materials, in particular by using glycidyl derivatives in copolymerizations or by subsequent modifications. These carrier materials exhibit a relatively strong swelling and often only low volume activities are achieved in the immobilization of enzymes which is why the use of these carrier materials is less suitable in enzymatic synthesis reactions.
Inorganic carriers exhibit much more favourable mechanical properties, thermal stability and resistance towards organic solvents and microbial attack than organic carriers. Examples of inorganic carriers are minerals such as bentonite, attapulgite and diatomaceous earth. They often exhibit a broad distribution of pore sizes. Non-porous materials such as metals and metal oxides usually only have small binding sur
REFERENCES:
Danzig, et. al., Indian Journal of Chemistry, vol. 32 B, Jan. 1993, pp. 40-43.
Brink, et. al., Enzyme Microb. Technol., vol. 10, Dec. 1988, pp. 736-743.
Daser Adelheid
Tischer Wilhelm
Wedekind Frank
Boehringer Mannheim GmbH
Naff David M.
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