Chemistry: molecular biology and microbiology – Carrier-bound or immobilized enzyme or microbial cell;... – Enzyme or microbial cell is immobilized on or in an...
Reexamination Certificate
1999-01-29
2001-01-30
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Carrier-bound or immobilized enzyme or microbial cell;...
Enzyme or microbial cell is immobilized on or in an...
C435S182000, C436S527000, C530S402000, C530S811000
Reexamination Certificate
active
06180378
ABSTRACT:
BACKGROUND
Lipoxygenase (LOX) enzymes are useful in the oxidation of fatty acids for a variety of commercial purposes. Peroxidases (POD) are oxidoreductases with many applications in medical, environmental and industrial processes including removing aromatic amines or phenols from water by peroxidase-catalyzed transformation (Klibanov, A. M. and Morris, E. D. [1981
] Enzyme Microb Technol.
3:119; Klibanov, A. M. et al. [1983
] Science
221:259). Immobilized peroxidases have been used in biosensors to detect pesticide residues (Sandberg, R. G. et al. [1992
] Biosensor design and application
, Mathewson, P. R. and Finley, J. W. eds., ACS, Washington D.C., pp. 81-88).
Labile proteins such as lipoxygenases, peroxidases and lipases lose most of their activity in aqueous solutions quickly. Immobilizing the enzyme would enable a continuous process that gives high substrate conversions, good product recovery, and minimal loss of enzyme activity. Conventional methods of enzyme immobilization include covalently binding or adsorbing the enzymes onto a solid support. LOX has been immobilized by adsorption to glutenin, gliadin, glass wool, talc, polymer beads, and ion-exchange supports (Cuperus, F. P. et al. [1995
] Catalysis Today
25:441-445; Battu, S. et al. [1994
] J. Agric. Food Chem.
42:2115-2119). The matrices used in covalent binding of LOX include oxirane acrylic beads, CNBr-activated sepharose and agarose, and carbonyidi-imidazole-activated polymer (Parra-Diaz, D. et al. [1993
] Biotech. Appl. Biochem.
18:359-367). Although improving the stability of the enzyme, covalent and ionic bonds formed by these methods can cause a decrease in enzyme activity. For example, the adsorption of
S. tuberosum
lipoxygenase on talc retained only 53% of its activity in immobilized form (Battu et al., supra). Immobilization of enzymes by entrapment has been achieved by encapsulating enzymes through sol-gel processes (Avnir, D. et al. [1994
] Chem. Mater.
6:1605-1614). The entrapped enzymes retained much of their activity and had better stability in the sol-gel matrices. Extension of this technique, however, was limited by two shortcomings of sol-gel materials: their brittleness and narrow pore network (Heichal-Segal et al. [1995
] Biotechnology
13:798-800). Efforts were made to improve the activity of immobilized enzymes by introducing matrix-relaxing additives, such as algenate or polymers (Heichal-Segal et al., supra; Shtelzer, S. et al. [1992
] Biotech. Appl. Biochem.
15:227-235) into sol-gel matrices, or mixing alkyl-substituted silanes in a specific ratio (Reetz, M. T. et al. [1996
] Biotechnol. Bioengineering
49:527-534). Despite these improvements, however, efficient alternative methods are still needed for enzyme immobilization to provide high activity and increased storage stability. Most methods for immobilizing LOX provide materials that are not stable longer than about a month at room temperature. The best immobilization methods in the literature, based on the covalent binding or adsorption of lipoxygenases, typically immobilize lipoxygenases to 70% of protein content with about 50% retainment of enzyme activity.
Clay minerals are naturally occurring phyllosilicates (i.e., layered silicates) with good intercalative properties. Because their layered structures can be broken down into nanoscale building blocks, phyllosilicates can serve as a framework for intercalation. Metal hydroxyl polymeric cations, alkylammonium ions, polymers, and their combinations have been intercalated into phyllosilicates to form a broad spectrum of materials ranging from pillared clays and organoclay, to polymer-clay nanocomposites. The intercalated phyllosilicates exhibit good mechanical and thermal stability, controlled pore size (0.2-1 &mgr;m) and ion mobility, and high adsorption capacity. (Monnier, A. et al. [1993
] Science
261:1299-1303; Pinnavia, T. J. [1983
] Science
220:365-371; Vaia, R. A. et al. [1994
] Chem. Mater.
6:1017-1022; Yan, Y. and Bien, T. [1993
] Chem. Mater.
5:905-907; and Burnside, S. D. and Giannelis, E. P. [1995
] Chem. Mater.
7:1597-1600.)
Compositions providing highly active immobilized bioactive proteins which are storage-stable are needed, as are efficient methods for producing such compositions.
All publications referred to herein are incorporated by reference to the extent not inconsistent herewith.
SUMMARY
This invention provides compositions comprising active, immobilized bioactive proteins, said compositions comprising a phyllosilicate, a bioactive protein intercalated into the galleries of the phyllosilicate, and a crosslinking compound crosslinking said phyllosilicate and said bioactive protein. Activities of up to 170% of free protein are achieved using the immobilized bioactive protein compositions of this invention. The compositions also provide excellent storage stability, retaining up to 98% original activity after being stored at room temperature for two weeks.
Immobilized bioactive proteins, including enzymes such as lipoxygenase, have many commercial uses. For example, lipoxygenases can be used to catalyze the oxidation of polyunsaturated fatty acids containing a Z-1, Z-4 pentadiene structure to give a Z-1, E-3 conjugated diene-5-monohydroperoxy derivative. Reduced derivatives of these hydroperoxy compounds can serve as replacements for ricinoleic acid and hence are useful in a number of industrial applications as lubricants, grease thickeners and drying oils. Currently ricinoleic acid is obtained from caster oil, a commodity that is imported into the United States at a level of thirty thousand metric tons per year. Hydroperoxy fatty add derivatives can be exploited as chemical synthons in pharmaceutical and chemical applications. For example, the perhydroxy derivatives of arachidonic acid serve as precursors in the synthesis of prostaglandins and leukotrienes. The hydroperoxy derivates of linoleic and linolenic acid are useful as fungicides in agricultural applications.
Phyllosilicates are layered silicates and include many naturally-occurring clay minerals such as montmorillonite, vermiculite, illite, mica and kaolinite, and synthetic phyllosilicates such as talc.
Sodium ions or other cations can be used to saturate the phyllosilicate in order to delaminate the phyllosilicate. Any cation not causing significant collapse of the phyllosilicate structure can be used, but sodium is preferred. Alkylammonium ions can be used to replace the sodium ions to make the phyllosilicate more hydrophobic. Hydrophobicity aids in immobilization of bioactive proteins such as lipoxygenase (LOX), Lipase PS30, Lipase SP523 and HPOD lyase, and other such proteins. However, for proteins such as peroxidase (POD) hydrophobicity is undesirable, and the sodium-ion-substituted phyllosilicates work best. Optimization of treatment of the phyllosilicate with alkylammonium after sodium ion delamination can be routinely done depending on the bioactive protein used.
The alkylammonium ion used may have any alkyl group known to the art as a substituent. Preferred alkylammonium ions include trimethylammonium (TMA) and cetyltrimethylammonium (HDTMA). Larger alkyl groups will make the composition more hydrophobic. Hydrophobicity of the composition can be adjusted by selecting alkylammonium ions to optimize the material for specific enzymes by those skilled in the art without undue experimentation.
The bioactive protein (also referred to herein as a biologically active protein) is any protein capable of reacting with another molecule (referred to herein as the “substrate”) in a desired reaction. Enzymes are a preferred class of bioactive protein in this invention. Preferred enzymes include lipoxygenase, peroxidase, trypsin, acid phosphatase, &bgr;-glucosidase, lipase, alkaline phosphatase, hydroxylase, reductase and superoxide dismutase. Other suitable bioactive proteins include albumin and cell-bounded enzymes on living cells, antibodies and b
Foglia Thomas A.
Hsu An-Fei
Shen Siyuan
Tu Shu-I
Fado John D.
Graeter Janelle S.
Naff David M.
Silverstein M. Howard
The United States of America as represented by the Secretary of
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