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
1994-09-19
1996-05-07
Wityshyn, Michael G.
Chemistry: molecular biology and microbiology
Carrier-bound or immobilized enzyme or microbial cell;...
Enzyme or microbial cell is immobilized on or in an organic...
435212, 435213, 435214, 435215, 435216, 435217, 435218, C12N 946, C12N 968, C12N 948, C12N 970, C12N 976, C12N 966, C12N 974, C12N 972
Patent
active
055145728
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method to avoid binding of polyethylene glycol (PEG) at the active site and its surroundings in proteolytic enzymes.
The method, based on the modification reaction of an enzyme-macromolecularized inhibitor complex in the heterogeneous state, allows to obtain enzyme-PEG adducts in which the proteolytic activity toward macromolecular substrates is preserved.
BACKGROUND OF THE INVENTION
The modification of enzymes with polyethylene glycol (PEG) is a technology that has markedly been developed in recent years to obtain adducts having valuable properties for the use both in the biomedical field and as novel biocatalysts, due to the presence of polyethylene glycol chains linked at the surface.
In fact, the enzyme-PEG adducts lose the major part of the typical properties of naturally occurring enzymes, such as immunogenicity and antigenicity, rapid clearance from circulation, easy degradability by proteases and instability in diluted solutions A. Abuchowski et al., J. B. C., 252 3582, 1977!, that often prevent their use in therapy.
In the use of enzymes in biocatalysis, the PEG-enzyme adduct acquires a quite different characteristic, i.e. the solubility in organic solvents, thus allowing a better use of the enzymes in converting liposoluble substrates Y. Inada et al., Thrends Biotech., 4 190, 1986!.
The properties of such novel biotechnologic products are due to the fact that PEG binds to the enzyme surface, thus protruding with its hydraration cloud toward the outer protein solvent, preventing the access of large molecules, such as proteolytic enzymes, as well as the recognition by the immune system. On the other hand, as PEG also has amphyphilic properties, the PEG-enzyme adduct can acquire solubility in organic solvents.
However, the polymeric cloud surrounding the PEG-enzyme adduct also limits the general use of said derivatives: in fact, the enzymatic activity is maintained toward small substrates, that can have access to the active site diffusing among the PEG polymer chains, but it is prevented toward large substrates, that cannot reach the active site due to steric hindrance.
There fore, convenient PEG-enzyme adducts are obtained with enzymes such as superoxide dismutase, catalase, asparaginase, arginase, urease, adenosine deaminase, phenylalanine ammonium liase etc., which are nowadays under pharmacological and clinical tests, but not with enzymes acting on large substrates such as proteins, nucleic acids and polysaccharides. In fact, substantial activity losses are de scribed following a PEG-modification of -trypsin, chymotrypsin, urokinase, ribonuclease, lysozyme and the like.
A proposed solution consists in preparing adducts having only a few polymer chains linked to the enzyme, thus decreasing the loss in enzymatic activity. However, this result, which can be attained carrying out the reaction in a PEG molar defect, suffers from drawbacks such as attainment of very heterogeneous products and poor reproducibility.
SUMMARY OF THE INVENTION
To allow access of macromolecular substrates, large polypeptides or proteins, in case of proteolytic enzymes, to the active site, the PEG binding to the enzyme is carried out in heterogeneous phase, in which the enzyme is linked to an inhibitor thereof that is, in its turn, immobilized on a highly hydrated insoluble polysaccharide (Sepharose). In such a way, the PEG polymer will bind to enzyme areas far form the active site and its proximity, thus allowing the approach of the substrate macromolecules.
The method was investigated with two serine-dependent proteolytic enzymes, trypsin and urokinase, the first being used in medicine, for instance in the removal of necrotic tissues, in digestive disorders or in ophthalmology in the elimination of protein deposits from contact lenses; the latter, i.e. urokinase, being of specific therapeutical interest as a plasminogen activator.
Benzamidine, an inhibitor of serine enzymes, was used as a linker to keep Sepharose in the surrounding of the active site (example 2). The method could al
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Trends in Biotechnology, vol. 6, No. 6, Jun. 1988, Cambridge GB, pp. 131-134, "Application of PEG-Enzyme and Magnetite-PEG-Enzyme Conjugates for Biotechnological Processes" by Y. Inada, et al. (see p. 132).
Journal of Bioactive and Compatible Polymers, vol. 8, Jan. 1993, pp. 41-50, "Active Site Protection of Proteolytic Enzymes, by Poly(ethylene glycol) Surface Modification" by P. Caliceti, et al. (see whole document).
Inada et al. (1986) Trends in Biotechnol, 4, 190-194.
Caliceti Paolo
Luciana Sartore
Schiavon Oddone
Veronese Francesco
Consiglio Nazionale Delle Ricerche
Weber Jon P.
Wityshyn Michael G.
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