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United States Patent (19) (11) 4,343,901 Defilippi 45) Aug United States Patent (19) (11) 4,343,901 DeFilippi 45) Aug. 10, 1982 54 MAGNETICSUPPORT MATRIX FOR ENZYME IMMOBILIZATION OTHER PUBLICATIONS o b Halling et al., Magnetic Supports for Immobilized En 75)75 InventorI tor: HousL J. DeFilippi,erupp, Mount PFrospec t, zymes and Bioaffinity Adsorbents, Enzyme Microb.w (73).73 Assignee: UOP Inc.,nc., Des Plaines,Pl IIll. Technol., vol. 2, 1980, (pp.- 2-11). Primary Examiner-David M. Naff 21 Appl. No.: 199,161. Attorney, Agent, or Firm-James R. Hoatson, Jr.; 22 Filed: Oct. 22, 1980 Eugene I. Snyder; William H. Page, II 51 Int. Cl............................................... C12N 11/14 (57) ABSTRACT 52 U.S. C. .................................... 435/176; 252/430; A magnetic support matrix for enzyme immobilization 435/177,435/180 is prepared which comprises a porous, refractory inor 58) Field of Search ............... 435/174, 176, 177, 180, ganic oxide containing ferromagnetic particles dis 435/182; 252/430, 448, 466; 424/1 persed throughout its interior and a polyamine cross 56) References Cited linked with an excess of a bifunctional reagent impreg nated therein so as to furnish pendant functional groups. U.S. PATENT DOCUMENTS Such a magnetic support matrix does not otherwise 2,620,314 12/1952 Hoekstra ............................. 252/448 substantially decrease loading of subsequently immobi 3: 3, 3. g 4.E. lized enzyme, nor in any other way substantially alter 4,152,210at is 5/1979 RobinsonCVS Cal. et . .al. 435/176 the properties R the immobilized- - - enzyme system when 4, 177,253 12/1979 Davies et al. ........................... 42/1 compared to that prepared on a non-magnetic support. 4,199,614 4/1980 Ziolo ................................... 430/111 4,289,655 9/1981 Bailey .............................. 252/466 B 17 Claims, No Drawings 4,343,901 1. 2 thus presenting substantial limitations on the amount of MAGNETC SUPPORT MATRIX FOR ENZYME enzyme which can be immobilized. Additionally, be MMOBILIZATION cause a surface coating of ferromagnetic material neces sitates a method of preparation which is reasonably BACKGROUND OF THE INVENTION 5 elaborate, complex, and demanding, such supports can Enzyme-catalyzed reactions have the advantages of be expected to be reasonably expensive and commer proceeding with great chemical specificity under rela cially unfeasible. Magnetic supports for immobilized tively mild conditions, and often accomplish what man enzymes and bioaffinity adsorbents has been reviewed finds difficult, if not impossible, to duplicate in the labo by Halling and Dunnill, Enzyme Microb. Technol, 2, ratory. For such reasons there is increasing emphasis on 10 2-10 (1980). the use of enzymatic processes on a commercial scale. It is highly desirable to have a magnetic support ma One example, of many which could be cited, is the trix whose only difference from a conventional one is its conversion of glucose to fructose using glucose isomer magnetic properties, and which can be readily prepared aSC. by simple procedures. In part this dictates a magnetic Enzymes often are water soluble, and if they are 15 support whose ferromagnetic materials are not bound as merely used in aqueous solutions recovery of enzymes a surface coating and which may be prepared by a vari for reuse is difficult and expensive. Using the enzyme ation in the basic method of preparation. only once affords a process which is relatively expen sive. Consequently, many techniques have been devel SUMMARY OF THE INVENTION oped for immobilizing the enzyme in such a way that 20 An object of this invention is to provide a magnetic, substantial enzymatic activity is displayed while the porous support matrix. An embodiment comprises a enzyme itself remains rigidly attached to some water porous refractory inorganic oxide through the interior insoluble support, thereby permitting reuse of the en of which are dispersed particles of ferromagnetic mate zyme over substantial periods of time and for substantial amounts of feedstock. One illustration of a method for 25 rials, said oxide being impregnated with a polyamine immobilizing an enzyme is found in U.S. Pat. No. cross-linked with an excess of a bifunctional reagent so 4,141,857, where a polyamine is absorbed on a metal as to furnish pendant functional groups. In a more spe oxide such as alumina, treated with an excess of a bi cific embodiment the particles are at least 0.05 microns functional reagent, such as glutaraldehyde, so as to in size and selected from the group consisting of iron, cross-link the amine, and then contacting the mass with 30 cobalt, and nickel. In a still more specific embodiment, enzyme to form covalent bonds between the pendant the polyamine is polyethyleneimine and the bifunctional aldehyde groups and an amino group on the enzyme. reagent is glutaraldehyde. The support matrix prepared according to the afore DESCRIPTION OF THE INVENTION mentioned invention has great utility in immobilizing reactive chemical entities, enzymes being but one class 35 The support matrices of U.S. Pat. No. 4,141,857 are of such reactive chemical entities. broadly effective and have enjoyed wide success for An immobilized enzyme system is the material which diverse enzymes. This application describes support results from immobilization of an enzyme onto a sup matrices incorporating advantages accruing to a mag port matrix. Magnetic support matrices and immobi netic support matrix while utilizing the benefits of the lized enzyme systems offer several advantages when support matrix described in the cited patent. Briefly compared to non-magnetic systems. For example, sepa described, the support matrix of this invention com ration of such materials from other non-magnetic solids prises a porous, refractory inorganic oxide through the by use of a magnetic field can permit separations other interior of which are dispersed particles of ferromag wise difficult or impossible to perform. Such a situation netic materials, said oxide being impregnated with a is represented by the case where two enzymes, with 45 polyamine cross-linked with an excess of a bifunctional different deactivation times, are used concurrently on a reagent so as to furnish pendant functional groups. substrate, with one immobilized enzyme system mag Among the porous refractory inorganic oxides of this netic and the other non-magnetic. In this case, after one invention are included alumina, thoria, magnesia, silica, enzyme is exhausted, the other may be readily recov and combinations thereof, with alumina being pre ered and reused by magnetic separation of the two 50 ferred. Such oxides may be in the form of granules or immobilized enzyme systems, which will be recognized powder with a size as small as about 100 mesh, as as offering great benefits in economy. Another advan spheres, as pellets, as extrudate, and so forth. tage of such magnetic materials is their use in a magneti A point of novelty of this invention is that there is cally stabilized fluid bed, thereby presenting further dispersed throughout the interior of such oxides parti options in continuous reactor systems. 55 cles of magnetic or ferromagnetic materials. The disper U.S. Pat. No. 4,152,210 describes a support matrix sion of ferromagnetic particles occurs more or less uni comprised of particulates of ferromagnetic materials. formly throughout the body of the oxide. Because of Enzymes are bound thereto by reagents which react such uniform dispersion, some ferromagnetic particles with a film of metal oxide on the surface of the particu will be on the surface of the oxide. However, it is to be lates, or to polymeric material attached to the support. recognized and emphasized that the presence of some A serious disadvantage of such a support matrix is its ferromagnetic materials on the surface is incidental to high density. This disadvantage is overcome in U.S. this invention and does not form any part of it. It must Pat. No. 4, 177,253 which describes a ferromagnetic also be recognized that only a relatively small fraction composite comprising a low density core whose surface of the ferromagnetic particles will be on the surface, this is coated with magnetic materials. Because more than 65 fraction being less than, and generally substantially less 50% of the surface must be so coated, the latter support than, about 10% of the total. matrix presents the disadvantage of reducing the num The particle size of the ferromagnetic materials gen ber of sites available for subsequent enzyme bonding, erally will be greater than about 0.05 microns. A lower 4,343,901 3 4. limit is dictated by the necessity of preventing a substan dispersed particles of ferromagnetic materials, resulting tial portion of the ferromagnetic material from being in the latter becoming more or less uniformly dispersed oxidized to a non-ferromagnetic state. Where the prod throughout the oxide. The sol is then calcined, for ex uct of the ferromagnetic material is itself ferromagnetic, ample, at about 650 C. for about two hours, under this lower limit in particle size may not be applicable. conditions where the ferromagnetic material is not oxi An upper limit is dictated by the desirability that the dized to a nonferromagnetic state. For example, where diameter of the formed oxide particles be at least about particles of nickel greater than about two microns are ten times the diameter of ferromagnetic materials dis used, calcination may be performed in air. However, persed through its interior. Thus, particle size may be as when magnetite of the same size is used, calcination great as about 0.5 mm, with the range from about 0.1 O must be performed in an inert or non-oxidizing atmo micron to about 0.1 mm being preferred, and sizes from sphere, such as nitrogen, hydrogen, argon, and so forth. about 0.5 micron to about 50 microns being still more After preparing the oxide with ferromagnetic parti preferred.
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