We have also acetanilide, 4 chloroacetanilide, 4 nitroacetanilide DNA-PK inhibitor in clinical trials and phenacetin using the same reaction conditions tested above. All substrates were purchased from Sigma Co. The hydrolysis products based diazocoupling described by a modified method based on the reaction was measured by adding 750 l mixture, 50 l of 2% NaNO 2 and held 1 ml of cold water. After standing for 5 min at 0 for diazotization, 100 l Sulfamins Acid 10%, diamine 50 l of a 1% naphthylethylene and 300 liters of water were added and incubated for 20 min at room temperature. The purple color developed in the mixture was determined at 540 nm spectrophotometerically. Phenetidine was determined from the molar extinction coefficient. One unit of activity was t catalyzes than the amount of enzyme that catalyses the hydrolysis of the substrate 1 mol for 1 min at 37. The kinetic analysis was performed in a 96 well microplate with the same reaction mixture by Ver Change in the concentrations of enzyme and substrate. The kinetic parameters were calculated using a Lineweaver Burk classic. Activity tstest For the reverse reaction, the reverse reaction activity to test t, we used 6.2 g protein and 80 mM substrate in 250 l of reaction buffer. The reaction was for 3 h at the 37th The reduced amount of aniline was prepared by a method modified diazocoupling determined as described above. RESULTS Identification and cloning of a new acylamidase aryl amidases are omnipresent Rtige enzymes found in living organisms, and are divided into different subfamilies according to substrate specificity and molecular cox2 inhibitor function t. Among these subfamilies, we focused on the activity t of arylacylamidase based on the hydrolysis of amide bonds in aryl, acyl amides potential for biotechnological applications. The typical response of the AAA is in Figure 1 To clone a gene AAA, as we have a soil bacterium with activity T with a AAA enrichment media with acetaminophenol p as the sole carbon source. We extracted genomic DNA of soil bacteria and a genomic library constructed. The microorganism was originally isolated as a Pseudomonas sp. However accidentally w during the experiment before the 16S rRNA analysis lost and therefore the taxonomic origin was not identified, au he that it is a Gram-negative. Since AAA hydrolyses the amide bond in aryl acyl amides such as p acetaminophenol, we developed a screening method for fast and easy on the basis of the color of the reaction product. Acetaminophenol p is essentially a colorless compound, however, gives his reaction product of the AAA, paminophenol, a purple color. Therefore, a clone of the activity was t the genomic AAA color Change in the colony when it was grown on solid media enriched acetaminophenol p. We have extracted a single colony by Ver Change the color by visual inspection, the plasmids and sequenced the entire insert DNA fragment. The Gr E Nelarabine of the inserts of 2.3 kb, and we identified A 496 residue open reading frame of a Mutma Lichen AAA insertion in the fragment. The Pfam domain analysis of the ORF sequence showed that the ORF of the family go Rte amidase signature. The ORF was also searched against the GenBank using NCBI BLAST. Look up the top of the results was a glutamyltRNA subcontractors amidotransferase