C - Chemistry – Metallurgy – 12 – P
Patent
C - Chemistry, Metallurgy
12
P
195/35
C12P 1/00 (2006.01) C12N 11/00 (2006.01) C12N 11/06 (2006.01) C12Q 1/00 (2006.01) C12Q 1/68 (2006.01) G01N 33/535 (2006.01)
Patent
CA 1299123
ABSTRACT A method for amplifying enzyme activity is disclosed. Enzyme amplifica- tion 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-support 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. As a prototype enzyme amplification system the S-peptide and S-protein fragments of ribonuclease S (RNase S) have been immobilized onto separate. Sepharose gels via a polycytidylic acid (poly C) substrate leash. Each of these gels releases its RNase fragment when treated with the complementary enzyme fragment or with RNase A. Also the released fragments recombine to give RNase S activity. Thus this system provides more enzymatic activity from the system than is applied. A 100 pg sample of Nase is amplified by 1.9 x 104 in 20 hours to yield activity equivalent to 1.9 µg of RNase, by applying it to the S-peptide gel followed by combination of the released S-peptide with excess S-protein. Also, application of 1 ng of RNase A to a three-stage amplification system in which each stage consists of a pair of S-peptide and S-protein gels produced cumulative amplifications of 4.9, 52, and 25x after each of the stages, respectively. Only 1-2 mg amounts of each gel are used per stage in these experiments, reflecting the significant production of RNase S activity. -2-
534718
Cecchini Douglas J.
Ehrat Markus
Giese Roger W.
Northeastern University
R. William Wray & Associates
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