1. A process for the stereoselective decarboxylation of malonic acid derivatives of the formula (I)
the process comprising:
using a decarboxylase to produce a compound of the formula (II)
wherein X is selected from the group consisting of OH, SH, C2-C10 alkyl, C1-C10 alkoxy, and NH2 and R is selected from the group consisting of aryl, heteroaryl, condensed aryl, and condensed heteroaryl radical, wherein R is substituted or unsubstituted and optionally wherein a substituent is selected from the group consisting of a halogen, alkyl, alkenyl, and alkynyl radical.
2. A decarboxylase comprising an amino acid sequence corresponding to SEQ ID NO:1, which has at least one mutation compared with SEQ ID NO:1, the said mutation being present at the amino acid positions selected from the group consisting of 17, 19, 22, 24, 25, 32, 41, 42, 46, 47, 53, 60, 61, 63, 68, 74, 83, 84, 85, 87, 94, 103, 105, 112, 116, 119, 121, 139, 142, 155, 168, 173, 178, 199, 201, 202, 203, 204, 205, 210, 221, 222, 224, 225, 226, 227, 228, 229, 230, 235, 238, 239, 240, and combinations thereof, wherein the amino acid(s) at said position(s) in SEQ ID NO:1 are replaced by any other amino acid, except that, at position 188, the cysteine residue is not replaced, and not more than 10 amino acids are inserted into andor deleted from the sequence according to SEQ ID NO:1.
3. The decarboxylase according to claim 2, wherein the amino acid sequence has at least one mutation compared with SEQ ID NO:1, and the mutation is present at an amino acid positions selected from the group consisting of 17, 19, 24, 32, 41, 42, 46, 47, 53, 60, 61, 63, 68, 74, 84, 85, 87, 94, 103, 116, 119, 142, 199, 202, 210, 225, 229, 230, 238, and combinations thereof.
4. The decarboxylase according to claim 2 wherein the mutation is present at the positions selected from the group consisting of 19, 24, 32, 42, 46, 47, 60, 61, 63, 68, 84, 85, 103, 199, 202, and combinations thereof, and wherein said mutation is a replacement of the amino acid at said position in SEQ ID NO:1.
5. The decarboxylase according to claim 2 wherein the amino acid sequence has at least one mutation for improving the thermal stability, wherein said mutation is that the amino acid in SEQ ID NO:1 at a positions selected from the group consisting of 15, 32, 74, 75, 128, 163, 165, and combinations thereof is replaced.
6. The decarboxylase according to claim 2 which is able to convert malonic acid derivatives of the formula III
into a compound of the Formula (IV)
wherein X is selected from the group consisting of F, CH3, and H, and R is selected from the group consisting of aryl, heteroaryl, condensed aryl, and condensed heteroaryl radical, wherein R is substituted or unsubstituted, and optionally wherein a substituent is selected from the group consisting of a halogen, alkyl, alkenyl, and alkynyl radical,
wherein an amino acid in SEQ ID NO:1 at the positions selected from the group consisting of 17, 19, 22, 25, 32, 41, 42, 47, 53, 60, 61, 63, 68, 74, 84, 85, 87, 94, 103, 112, 119, 121, 139, 142, 155, 168, 173, 178, 199, 201, 203, 205, 207, 210, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 238, 240, and combinations thereof of the decarboxylase is replaced by an unspecified amino acid.
7. The decarboxylase according to claim 6, wherein the cysteine residue at position 188 of SEQ ID NO:1 is not mutated, and the compounds of the Formula (II) have the R-configuration.
8. The decarboxylase according to claim 6, wherein an amino acid between positions 69 and 81 of SEQ ID NO:1 is replaced by a cysteine and the cysteine residue at position 188 is replaced by an amino acid other than cysteine, and wherein the compound of the Formula (II) has the S-configuration.
9. The decarboxylase according to claims 2 comprising a polyhistidine sequence of amino acids at the C-terminus andor at the N-terminus.
10. A process for the preparation of a decarboxylase according to claims 2, comprising:
expressing the decarboxylase in a suitable host organism optionally purifying said decarboxylase,
wherein said organism contains a vector which contains the polynucleotide sequence ID NO:2 with base exchanges coding for the mutation(s), andor the chromosome of the host organism contains the gene having the mutated polynucleotide sequence ID NO:2 with regulatory units.
11. The process according to claim 10, wherein the host organism is Escherichia coli.
12. A transformed host organism, capable of expressing a decarboxylase according to claims 2.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. A method for testing efficacy of a protease enzyme assay, the method comprising:
providing an enzyme which acts as a surrogate enzyme control for a protease enzyme;
combining the surrogate enzyme control with an assay substrate for the protease enzyme; and
determining a change in the assay substrate resulting from the surrogate enzyme control acting on the assay substrate;
wherein the protease enzyme is selected from the group consisting of metalloproteinases, serine proteases, and cysteine proteases.
2. A method for conducting a protease enzyme assay, the method comprising:
testing efficacy of the protease enzyme assay comprising:
providing an enzyme which acts as a surrogate enzyme control for a protease enzyme;
combining the surrogate enzyme control with an assay substrate for the protease enzyme; and
determining a change in the assay substrate resulting from the surrogate enzyme control acting on the assay substrate; and
determining a level of the protease enzyme in a biological sample by contacting the assay substrate with at least a portion of the biological sample;
wherein the protease enzyme is selected from the group consisting of metalloproteinases, serine proteases, and cysteine proteases.
3. The method of claim 2, wherein the biological sample is selected from the group consisting of animal tissues, wound fluids, body fluids, saliva, tears, urine, blood serum, and blood plasma.
4. The method of claim 1, wherein the protease enzyme is a matrix metalloproteinase.
5. The method of claim 4, wherein the surrogate enzyme control is selected from the group consisting of thermolysin, porcine trypsin, bovine trypsin, and recombinant trypsin.
6. The method of claim 5, wherein the surrogate enzyme control is thermolysin.
7. The method of claim 1, wherein the protease enzyme is a serine protease.
8. The method of claim 7, wherein the surrogate enzyme is subtilisin.
9. The method of claim 1, wherein the protease enzyme is a cysteine protease.
10. The method of claim 1, wherein determining the change in the assay substrate is carried out by measuring a property of the assay substrate.
11. The method of claim 10, wherein when the property is determined to be within a predetermined range, and the efficacy of the assay is verified.
12. The method of claim 10, wherein the property is fluorescence.
13. The method of claim 12, wherein the assay substrate undergoes an increase in fluorescence when acted upon by the protease enzyme and by the surrogate enzyme control.
14. The method of claim 10, wherein the property is ultra violet or visible light absorbance.
15. The method of claim 14, wherein the assay substrate undergoes a change in absorbance when acted upon by the protease enzyme and by the surrogate enzyme control.
16. The method of claim 10, wherein the property is visually observable or optically measurable at a binding zone of a flow device.
17. The method of claim 16, wherein the assay substrate causes the binding zone of a lateral flow device to exhibit a first color or level of color when the assay substrate is acted upon by the protease enzyme and by the surrogate enzyme control, and to exhibit a second color or level of color when the assay substrate is not acted upon by the protease enzyme and by the surrogate enzyme control.
18. A kit including:
an enzyme which acts as a surrogate enzyme control for a protease enzyme in testing efficacy of a protease enzyme assay comprising a predetermined assay substrate for the protease enzyme;
a reconstitution buffer; and
an expected fluorescence range andor an ultra violet or visible light absorbance range of the predetermined assay substrate; wherein the predetermined assay substrate at a specified concentration has a fluorescence or absorbance within the expected fluorescence or absorbance range when acted upon by the surrogate enzyme control at a specified concentration; andor
an expected color range or color level range of a binding zone of a flow device when contacted with the predetermined assay substrate; wherein the predetermined assay substrate causes the binding zone of the flow device to exhibit a color or level of color within the expected color range or color level range when the assay substrate is acted upon by the surrogate enzyme control at a specified concentration.
19. The kit of claim 18, wherein having a fluorescence or absorbance within the expected fluorescence or absorbance range indicates that the predetermined assay substrate is verified for use in assaying the protease enzyme.
20. The kit of claim 18, wherein the binding zone of the flow device exhibiting a color or level of color within the expected color range or color level range indicates that the predetermined assay substrate is verified for use in assaying the protease enzyme.
21. The kit of claim 18, wherein the protease enzyme is selected from the group consisting of metalloproteinases, serine proteases, and cysteine proteases.
22. The kit of claim 21, wherein the protease enzyme is a matrix metalloproteinase.
23. The kit of claim 22, wherein the surrogate enzyme control is selected from the group consisting of thermolysin, porcine trypsin, bovine trypsin, and recombinant trypsin.
24. The kit of claim 23, wherein the surrogate enzyme control is thermolysin.
25. The kit of claim 21, wherein the protease enzyme is a serine protease.
26. The kit of claim 25, wherein the surrogate enzyme is subtilisin.
27. The kit of claim 21, wherein the protease enzyme is a cysteine protease.
28. The kit of claim 18, further comprising the predetermined assay substrate.
29. The kit of claim 18, further comprising a device for assaying the protease enzyme.
30. (canceled)
31. The kit of claim 18, further comprising a sampling tool.
32. The kit of claim 18, wherein the predetermined assay substrate undergoes an increase in fluorescence when acted upon by the surrogate enzyme control.
33. The kit of claim 32, wherein the kit includes the expected fluorescence range of the predetermined assay substrate.
34. The kit of claim 18, wherein the predetermined assay substrate undergoes a change in absorbance when acted upon by the surrogate enzyme control.
35. The kit of claim 34, wherein the kit includes the expected ultra violet or visible light absorbance range of the predetermined assay substrate.
36. The kit of claim 18, wherein the predetermined assay substrate causes a binding zone of a lateral flow device to exhibit a first color or level of color when the assay substrate is acted upon by the surrogate enzyme control, and to exhibit a second color or level of color when the assay substrate is not acted upon by the surrogate enzyme control.
37. The kit of claim 36, wherein the kit includes the expected color range or color level range of the binding zone of the lateral flow device when contacted with the predetermined assay substrate acted upon by the surrogate enzyme control.
38. A method of releasing a kit of claim 18, the method comprising:
determining the expected fluorescence or ultra violet or visible light absorbance range of the predetermined assay substrate at a specified concentration when acted upon by the surrogate enzyme control at a specified concentration; and
providing the expected fluorescence or absorbance range for including in the kit.
39. The method of claim 38, further comprising measuring the fluorescence or absorbance of the predetermined assay substrate at the specified concentration in combination with the protease enzyme at at least one concentration; and determining if the measured fluorescence or absorbance fits a standard plot of fluorescence or absorbance of the predetermined assay substrate versus protease enzyme concentration.
40. A method of releasing a kit of claim 18, the method comprising:
determining the expected color range or color level range of the binding zone of the lateral flow device when contacted with the predetermined assay substrate acted upon by the surrogate enzyme control at a specified concentration; and
providing the expected color range or color level range of the binding zone of the lateral flow device for including in the kit.