Isolation and primary characterization of an amidase from Rhodococcus rhodochrous

Amidase (EC 3.5.1.4) was purified to homogeneity from Rhodococcus rhodochro us M8 using isopropanol fractionation and exchange chromatography on Mono Q . The isolated amidase consists of four identical subunits with molecular w eight 42 +/- 2 kD. The activity of the enzyme is maximal at 55-60 degrees C and within the pH range 5-8, The amidase from R. rhodochrous M8 is highly sensitive to such sulfhydryl reagents as Hg2+ and Cu2+. Chelators (EDTA and o-phenanthroline) and serine proteinase inhibitors (PMSF and DIFP) did not inhibit the activity of the enzyme. The; enzyme exhibits hydrolytic and ac yl transferase activity and does not possess urease activity. Aliphatic ami des (acetamide and propionamide) were the best substrates for the amidase f rom R. rhodochrous M8, whereas bulky aromatic amides were poor substrates o f this enzyme. The properties of the isolated enzyme are similar to those f ound in the corresponding amidase from Arthrobacter sp. J-1 and an amidase with wide substrate specificity from Brevibacterium sp. R312.