Enzyme amplification by using free enzyme to release enzyme from an immobilized enzyme material
First Claim
1. A method for amplifying enzymatic activity, comprising:
- providing a support-leash-enzyme conjugate wherein the support is a solid, the leash comprises molecular chain which is cleavable by the enzyme in the free state, the leash is attached to the support and to the enzyme by covalent bonds, and the conjugate has a plurality of -leash-enzyme units connected to the support;
said conjugate being structured such that the bound enzyme of a given -leash-enzyme unit is unable to cleave the leash portion of that unit;
adding to said conjugate a first amount of said enzyme in the free state, to produce a mixture of free enzyme and support-leash-enzyme conjugate;
incubating said mixture a predetermined period of time, to cause enzyme from said conjugate to be released into the free state; and
recovering said enzyme in the free state from said conjugate in a second amount greater than said first amount of enzyme.
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Abstract
A method for amplifying enzyme activity is disclosed. Enzyme amplification is achieved by covalently bonding enzyme to a supporting material via a molecular chain which is a substrate for the enzyme, then introducing a small amount of enzyme in the free state to this system, causing release of a large amount of bound enzyme. In an alternative embodiment, complementary enzymatically inactive fragments of an active enzyme, which fragments can recombine to form active enzyme, are covalently attached to separate support materials by a molecular chain material which is a substrate for the active enzyme, and these two fragment-supported conjugates are connected in series. Upon application of free enzyme or free complementary enzyme to one of these fragment-support conjugates, followed by application of the resulting product mixture to the second fragment-support conjugate, a large amount of free enzyme is ultimately produced. In a second alternative embodiment, two different active enzymes are each attached to separate supporting materials by different leashes, in which the leash for the first enzyme only is cleaved in the system by the second enzyme, and the leash for the second enzyme only is cleaved in the system by the first enzyme. These two materials are connected in series, and upon application of the second enzyme to the first enzyme-support conjugate, followed by application of the released first enzyme to the second enzyme-support conjugate material, ultimately a large amount of released active second enzyme is produced. Also disclosed are molecular conjugates of enzyme material on supports, required for the enzyme amplification.
42 Citations
24 Claims
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1. A method for amplifying enzymatic activity, comprising:
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providing a support-leash-enzyme conjugate wherein the support is a solid, the leash comprises molecular chain which is cleavable by the enzyme in the free state, the leash is attached to the support and to the enzyme by covalent bonds, and the conjugate has a plurality of -leash-enzyme units connected to the support;
said conjugate being structured such that the bound enzyme of a given -leash-enzyme unit is unable to cleave the leash portion of that unit;adding to said conjugate a first amount of said enzyme in the free state, to produce a mixture of free enzyme and support-leash-enzyme conjugate; incubating said mixture a predetermined period of time, to cause enzyme from said conjugate to be released into the free state; and recovering said enzyme in the free state from said conjugate in a second amount greater than said first amount of enzyme. - View Dependent Claims (2, 3, 4)
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- 5. A molecular conjugate composition having the generalized formula
- space="preserve" listing-type="equation">support-leash-enzyme
wherein; the support is a solid support material; the leash comprises molecular chain which is cleavable by the enzyme in the free state; the leash is attached to the support and to the enzyme by covalent bonds; the support-leash-enzyme conjugate has a plurality of -leash-enzyme units connected to the support; and said support-leash-enzyme conjugate is structured such that the bound enzyme of a given -leash-enzyme unit is unable to cleave the leash portion of that unit. - View Dependent Claims (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
- 19. A molecular conjugate composition having the generalized formula
- space="preserve" listing-type="equation">support-leash-enzyme fragment
wherein; the enzyme fragment is one of a plurality of complementary enzymatically inactive fragments of an active enzyme, and is capable of recombining with the remainder of said fragments to regenerate said active enzyme; the support is a solid support material; the leash comprises molecular chain which is cleavable by said active enzyme in the free state; the leash is attached to the support and to the enzyme fragment by covalent bonds; the support-leash-enzyme fragment conjugate has a plurality of -leash-enzyme fragment units connected to the support; and said support-leash-enzyme fragment conjugate is structured such that bound active enzyme produced by recombination of the complementary enzyme fragments is unable to cleave the leash to which it is attached. - View Dependent Claims (20, 21, 22, 23, 24)
Specification