Process for immobilizing proteins on a support containing amino,

Details Process for immobilizing proteins on a support containing amino, Process for immobilizing proteins on a support containing amino,

1990-12-31

1993-09-21

Naff, David M.

Chemistry: molecular biology and microbiology

Carrier-bound or immobilized enzyme or microbial cell;...

435176, 435181, 436524, 436525, 436532, 530811, 530816, C12N 1100, C12N 1114, G01N 33551, G01N 33553

Patent

active

052468461

DESCRIPTION:

BRIEF SUMMARY
The invention relates to a process for immobilizing proteins, peptides, ligands, coenzymes, redox mediators or the like on a support, which exhibits amino, mercapto or hydroxy groups. Such immobilizates, i.a., are known as enzyme immobilizates, which can be used in bioreactors or in the area of biosensory function. Enzyme immobilizates of the initially mentioned type contain, for example, beta-glucosidases, lipases, esterases, phosphatases, oxireductases or naringinases and are used for processing of food, purification of waste water, biocatalyzed organic syntheses, bioanalytics and medical techniques. With now known and used immobilization methods the number and spatial distribution of the reactive groups suitable for coupling can be controlled only in rare cases and only inadequately. Also in most cases it is not possible to activate support materials with amino, mercapto or hydroxy groups with a single process. Also the storage of the activated support material is not possible in most processes.
Immobilizates of the initially mentioned type are produced mostly with the use of glutardialdehyde, and then depending on the type of support, the support has to be derivatized if it exhibits primary amino groups, which later are reacted with glutardialdehyde and still later with the proteins, peptides, coenzymes or the like to be immobilized. Derivatized supports, which are reacted with glutardialdehyde, cannot be dried without loss of activity.
The object of the invention is to provide a process of the initially mentioned type, in which a derivatized support, with amino but also mercapto and hydroxy groups, after a coupling, can be directly reacted with the proteins, peptides, ligands, coenzymes or the like to be immobilized, can be dried without loss of activity, and with which even thin layers of proteins, peptides, coenzymes or the like, especially monolayers, can be immobilized at the same time with high mechanical stressability. To achieve this object, the process according to the invention essentially consists in the fact that the support is reacted with a compound of general formula (I) or (IA) ##STR2## in which X means halogen and R.sub.1, R.sub.2 are the same or different and mean X, R, COR, COOR, in which R means C.sub.1 -C.sub.8 alkyl, COOH, CNS, N.sub.3 or CN, and optionally is dried, after which the peptides, proteins, coenzymes or the like to be immobilized are reacted with the free halogen--in ortho or para position to the C.dbd.O group--of the compound of general formula (I) or (IA). In this case, the compound of general formula (I) or (IA)) first forms by substituent X a covalent bond with the amino, mercapto or hydroxy groups of the support by which an especially stable coupling is achieved. Now the second substituent X, activated by the first coupling, can be reacted with the amino, hydroxy or mercapto groups of the reactant to be immobilized, and an especially stable covalent coupling is achieved. The coupling reaction between the compound of general formula (I) or (IA) and the support or the compound to be immobilized to a high degree is very dependent on the reactivity of the respective substituents. Thus with the compounds of general formula (I) or (IA) in which X=R.sub.1 =R.sub.2 the reactivity according to the following series is decreasing of formula (I) or (IA) therefore are fluorine, chlorine and bromine. Therefore the coupling of o-chloranil or o-bromanil with an amino support material such as, for example, aminosilylated metal or glass surfaces can be considered as model reaction. Also the reactivity of the support is dependent on the respective substituents and decreases corresponding to the following series
The reactivity of the support material thus activated is clearly greater for the coupling of amino and mercapto groups than that for the coupling of hydroxy groups. Therefore most preferred is the reaction of a support which carries a primary amino group with a compound of formula I, in which the substituents X, R.sub.1 and R.sub.2 are the same and each mean fluorine, chlorin

REFERENCES:
patent: 4229537 (1980-10-01), Hodgins et al.
Brandt, et al., Biochimica et Biophysica Acta, vol. 386, 1975, pp. 196-202.
"Immobilized Enzymes," Methods in Enzymology, vol. XLIV, Klaus Mosbach, pp. 134-149, 1976, Academic Press, N.Y.

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