Purification and characterization of an amidase from an acrylamide-degrading Rhodococcus sp.
A constitutively expressed aliphatic amidase from a Rhodococcus sp. catalyzing acrylamide deamination was purified to electrophoretic homogeneity. The molecular weight of the native enzyme was estimated to be 360,000. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified preparation yielded a homogeneous protein band having an apparent molecular weight of about 44,500. The amidase had pH and temperature optima of 8.5 and 40 degrees C, respectively, and its isoelectric point was pH 4.0. The amidase had apparent K(m) values of 1.2, 2.6, 3.0, 2.7, and 5.0 mM for acrylamide, acetamide, butyramide, propionamide, and isobutyramide, respectively. Inductively coupled plasma-atomic emission spectometry analysis indicated that the enzyme contains 8 mol of iron per mol of the native enzyme. No labile sulfide was detected. The amidase activity was enhanced by, but not dependent on Fe(2+), Ba(2+), and Cr(2+). However, the enzyme activity was partially inhibited by Mg(2+) and totally inhibited in the presence of Ni(2+), Hg(2+), Cu(2+), Co(2+), specific iron chelators, and thiol blocking reagents. The NH2-terminal sequence of the first 18 amino acids displayed 88% homology to the aliphatic amidase of Brevibacterium sp. strain R312.
Nawaz
M S
MS
Division of Microbiology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arkansas 72079.
Khan
A A
AA
Seng
J E
JE
Leakey
J E
JE
Siitonen
P H
PH
Cerniglia
C E
CE
D020106:Acrylamide; D000178:Acrylamides / Q000378:metabolism*; D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001673:Biodegradation, Environmental; D007526:Isoelectric Point; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012240:Rhodococcus / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D000178:Acrylamides; D000596:Amino Acids; D008670:Metals; D020106:Acrylamide; D000581:Amidohydrolases; C019665:amidase