Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1981-08-03
1983-09-20
Naff, David M.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435179, 435181, C12P 1902, C12N 1112, C12N 1106
Patent
active
044057156
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a process for fixing an enzyme on a water-insoluble solid support. It also comprises as its object an enzyme-support complex and an application procedure for such a complex.
PRIOR ART
For several years already work has been done to render insoluble enzymes or other agents such as antibodies, antigens or nuclear acids so as to obtain the capability of purifying, modifying, or insulating certain substances with which these "catalysts" form complexes or else favor a specific reaction.
The endeavors to achieve insoluble enzyme-support combinations are due to the fact that numerous reactions involve enzymes, and not only is there an economic incentive to recover an expensive enzyme, but moreover the reaction products should not be soiled.
Numerous works have been performed on the selection of the supports and the methods of fixing or combining enzymes: the scientific literature is quite abundant and describes several supports such as silica, glass, synthetic polymers, polysaccharides and in particular cellulose.
Most of the publications relating to cellulose refer to a very pure cellulose, whereas as a rule cellulose is associated with hemicelluloses and lignin.
Thus, N. WELIKY and H. H. WEETHALL in 1965 (Immunochemistry Pergamon Press, Vol. 2, pp. 293-322) published an article entitled "The Chemistry and Use of Cellulose Derivatives for the Study of Biological Systems."
The cellulose reactivity is that of a polyalcohol and can be subjected to oxidation, esterification, etherification, halogenation reactions, etc.
In particular, oxycelluloses are coupled to alcohols, amines or proteins, forming thereby esters or amides.
In view of the reactions used to modify cellulose, it is clear why as a rule the attempt has been made to eliminate lignin, a crosslinked polymer of which the numerous reactive groups use up the bodies being employed to modify the cellulose or else inhibit the desired reactions.
However, the U.S. Pat. No. 3,841,969 (A. N. EMERY et al.) describes a method for preparing immobilized enzymes by chelation with a metal complex and with substances comprising hydroxyl functions: microcrystalline cellulose, diethylaminoethylated cellulose, carboxylmethyl cellulose, but also sawdust and wood chips.
In addition to the difficulty of using industrially such metal chlorides as titanium tetrachloride or tin chloride, it is also known that the chelates are not stable over wide pH ranges.
On the other hand, the French Pat. No. 2,247,472 (SNPA) describes a method for fixing enzymes on lignin associated cellulose and indicates in surprising manner that the cellulose-enzyme association reaction is enhanced by lignin present by 5 to 25% by weight and preferably 10 to 20% by weight.
Cereal stalk granules between 0.3 and 10 mm in size first are surface-delignified and following washing are treated with thionyl chloride in pyridine, and lastly are placed in contact with a buffered enzyme solution.
It appears obvious that the lignin exerts no favorable influence at all on the reaction that allows immobilizing the enzyme, because it is mandatory to begin with a stage of surface delignification of the granules.
DESCRIPTION OF THE INVENTION
The present invention concerns a method for obtaining enzyme derivatives wherein the enzyme is fixed by a covalent bond on a water-insoluble support, characterized by the following steps:
(a) a substrate essentially containing cellulose and lignin is washed with water,
(b) controlled oxidation of the oside fraction of the substrate,
(c) condensation of a diamine on the aldehyde groups of the oxidized support,
(d) stabilization by reduction of the amine-substrate bond,
(e) activation by means of a dialdehyde of the aminated groups,
(f) coupling the enzyme on the activated substrate.
Preferably the oxidizer in stage (b) is sodium periodate, the diamine is ethylene diamine, the reducer is sodium borohydride or cyanoborohydride, dialdehyde, glutaraldehyde. The enzyme in particular may be invertase, saccharase levan, lactase or dextran
REFERENCES:
patent: 3669841 (1972-06-01), Miller
patent: 3706633 (1972-12-01), Katchalski et al.
patent: 3741871 (1973-06-01), Weeks et al.
patent: 3841969 (1974-10-01), Emery et al.
patent: 3947352 (1976-03-01), Cuatrecasas et al.
patent: 3960666 (1976-06-01), Bourdeau et al.
patent: 4013514 (1977-03-01), Wildi et al.
patent: 4279998 (1981-07-01), Shahani et al.
Zaborsky, O., Immobilized Enzymes, CRC Press, Cleveland, Ohio, 1974, (pp. 6-7).
Beghin-Say S.A.
Breiner A. W.
Naff David M.
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