Immobilization of proteins on inorganic support materials
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IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C07G-007/02
출원번호
US-0688469
(1976-05-20)
발명자
/ 주소
Messing
Ralph A.
Yaverbaum
Sidney
출원인 / 주소
Corning Glass Works
대리인 / 주소
Giblin, James A.Janes, Jr., Clinton S.Patty, Jr., Clarence R.
인용정보
피인용 횟수 :
49인용 특허 :
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초록▼
Biologically active proteins such as enzymes and antibodies can be chemically bonded to a variety of high surface area, inorganic supports by reacting the support surface with polymeric isocyanates and then reacting the coated surface with a dispersion of the proteins.BACKGROUND OF THE INVENTION1. F
Biologically active proteins such as enzymes and antibodies can be chemically bonded to a variety of high surface area, inorganic supports by reacting the support surface with polymeric isocyanates and then reacting the coated surface with a dispersion of the proteins.BACKGROUND OF THE INVENTION1. FieldThe present invention is concerned generally with the immobilization of biologically active proteins and specifically with the chemical attachment of such proteins to high surface area inorganic supports.2. Prior ArtA variety of techniques have been devised to immobilize various biologically active proteins such as enzymes and antibodies onto and within high surface area inorganic supports. See, for example, U.S. Pat. No. 3,556,945 (enzymes adsorbed to porous glass); U.S. Pat. No. 3,804,719 (enzymes crosslinked within the pores of porous glass); U.S. Pat. No. 3,519,538 (enzymes coupled via intermediate silanes to various inorganics); U.S. Pat. No. 3,652,761 (antibodies coupled via silanes to various inorganics); U.S. Pat. No. 3,850,751 (enzymes adsorbed within the pores of various ceramics); U.S. Pat. No. 3,839,175 (enzymes immobilized via electrodeposition on various porous ceramics); U.S. Pat. No. 3,930,951 (enzymes coupled via BMBD to inorganics); U.S. Pat. No. 3,912,593 ("chelation" of various biologically active materials to oxides of certain metals); U.S. Pat. No. 3,933,589 (enzymes coupled via a mixture of dialkylamines and alkane dihalides to inoganics); and U.S. Pat. No. 3,705,084, enzymes bonded to the surfaces of various macroporous reactors. Methods of covalently bonding proteins to various organic polymers using cyanates are disclosed in U.S. Pat. No. 3,788,948. Although the above list is not intended to be complete, it is clear that a wide variety of methods have been discovered for attaching biologically active proteins to inorganic materials. Most of the above discoveries are relatively recent, thus indicating the present need for exploring alternative methods for immobilizing proteins in an acceptably active state on the surfaces of inorganic supports. Although the presently known bonding techniques have various advantages and disadvantages relative to each other, and to similar techniques used with organic carriers, we have now found a novel method of bonding proteins to inorganic surfaces using a relatively inexpensive and simple class of polymers previously not used. Details of our immobilization method are described in detail herein.SUMMARY OF THE INVENTIONOur method of bonding biologically active proteins to the surfaces of high surface area inorganic support materials comprises the steps of reacting an organic solution of polymerized isocyanate compounds having a molecular weight of at least about 250 with a high surface area inorganic material having surface hydroxyl or oxide groups to form a polymeric surface having reactive isocyanate groups, and then reacting the treated support with a dispersion of the proteins under conditions sufficient to assure bonding of the protein in a biologically active state to the surface of the support via the isocyanate groups. In preferred embodiments the inorganic support is titania, alumina, silica or porous glass, and the bonded proteins are enzymes or antibodies.SPECIFIC EMBODIMENTSThe polymeric isocyanates useful for bonding the proteins may be represented as follows: ##STR1## where n is an interger having a value of at least 2. Such polymeric isocyanates are available commercially (e.g. sold under tradename PAPI, Polymer Chem. Div., Upjohn Company).We have found that a very effective polymeric isocyanate for use with porous titania, silica, alumina is that sold under the tradename PAPI-901.All of these polymeric isocyanates are insoluble in water and they must be applied from an organic solvent. We have found the most effective solvent is acetone. Toluene is also a good solvent for this purpose; however, since toluene is not readily soluble in water, and acetone is, we found that we can accomplish the attachment of the proteins much more rapidly when the solvent is acetone. Alcohols are not very useful as a solvent since they will react with the coupling agent to form urethanes. Solvents having either hydroxyl or amine groups should not be utilized for this coupling procedure since they react to form either urethanes or substituted ureas.The reaction of the polymeric isocyanate coupling agent with the carrier (e.g. titania) results in the formation of a metal carbamate. This may be represented as follows: ##STR2## The isocyanate groups that have not reacted with the carrier remain available for coupling to the enzyme in the following manner: Reaction 1 (under alkaline conditions) -- to yield substituted ureas. ##STR3## Reaction 2 (under mildly acid conditions) -- to yield urethanes. ##STR4##From the above, it can be seen that the polymeric isocyanates of this invention can be used under a variety of pH conditions, both alkaline and acidic.For efficient and practical protein loading, the inorganic supports should have a high surface area (e.g. >0.2 m.sup.2 /g) and may be porous. A preferred average pore size range is about 100 to 2500A. Although any essentially water insoluble inorganic having surface hydroxyl or oxide groups capable of reacting with the polymer may be used, a very preferred support or carrier consists of porous titania particles, 30-80 mesh, having an average pore size in the range of about 320 to about 1500A.
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