Physical, biochemical, and immunological characterization of a thermostable amidase from Klebsiella pneumoniae NCTR 1.
An amidase capable of degrading acrylamide and aliphatic amides was purified to apparent homogeneity from Klebsiella pneumoniae NCTR 1. The enzyme is a monomer with an apparent molecular weight of 62,000. The pH and temperature optima of the enzyme were 7.0 and 65 degrees C, respectively. The purified amidase contained 11 5,5-dithiobis(2-nitrobenzoate) (DTNB)-titratable sulfhydryl (SH) groups. In the native enzyme 1.0 SH group readily reacted with DTNB with no detectable loss of activity. Titration of the next 3.0 SH groups with DTNB resulted in a loss of activity of more than 70%. The remaining seven inaccessible SH groups could be titrated only in the presence of 8 M guanidine hydrochloride. Titration of SH groups was strongly inhibited by carboxymethylation and KMnO4, suggesting the presence of SH groups at the active site(s). Inductively coupled plasma-atomic emission spectrometry analysis indicated that the native amidase contains 0.33 mol of cobalt and 0.33 mol of iron per mol of the native enzyme. Polyclonal antiserum against K. pneumoniae amidase was raised in rabbits, and immunochemical comparisons were made with amidases from Rhodococcus sp., Mycobacterium smegmatis, Pseudomonas chlororaphis B23, and Methylophilus methylotrophus. The antiserum immunoprecipitated and immunoreacted with the amidases of K. pneumoniae and P. chlororaphis B23. The antiserum failed to immunoreact or immunoprecipitate with other amidases.
Nawaz
M S
MS
Division of Microbiology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arkansas 72079, USA.
Khan
A A
AA
Bhattacharayya
D
D
Siitonen
P H
PH
Cerniglia
C E
CE
D020106:Acrylamide; D000178:Acrylamides / Q000378:metabolism; D000577:Amides / Q000378:metabolism; D000581:Amidohydrolases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004795:Enzyme Stability; D006358:Hot Temperature; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D011196:Potassium Permanganate / Q000494:pharmacology; D013045:Species Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D017186:Titrimetry
D000178:Acrylamides; D000577:Amides; D007461:Iodoacetates; D013439:Sulfhydryl Reagents; D011196:Potassium Permanganate; D020106:Acrylamide; D004228:Dithionitrobenzoic Acid; D000581:Amidohydrolases; D019807:Iodoacetic Acid