Isolation of an Inducible Amidase from Pseudomonas acidovorans AE1

Abstract

S U M M A R YA bacterial strain, AEI, which hydrolysed acetanilide, was isolated from soil and identified as Pseudomonas acidovorans. Numerous amides, esters and enzyme inhibitors were tested as amidase inducers. Phenacetin was chosen as inducer for the large scale cultivation of these organisms because it was less toxic to the bacteria than acetanilide. The induction increased the enzymic activity 250-fold. In comparison, the type culture strain of P. ucidovovans, ~~~~1 5 6 6 8 , had no amidase activity which could be induced by phenacetin. Optimal growth conditions were established with respect to the concentration of carbon source and inducer so that about 10 % of the extractable bacterial protein consisted of the amidase. The organisms were lysed with lysozyme in the presence of EDTA and the enzyme was isolated mainly by column chromatography procedures. A preparation from 60 g (wet wt) bacteria yielded about IOO mg highly purified amidase with a specific activity of I 37 pmol substrate hydrolysed/min/mg protein. In addition to acetanilide, the purified enzyme hydrolysed several other amides and esters. As standard substrate, p-nitroacetanilide was chosen.

N-Acetylanthranilate Amidase fromArthrobacter nitroguajacolicusRü61a, an α/β-Hydrolase-Fold Protein Active towards Aryl-Acylamides and -Esters, and Properties of Its Cysteine-Deficient Variant

“…Application of o-nitroacetanilide (5 mM in BR buffer, pH 8) to the tributyrin plate after the incubation period resulted in formation of yellow 2-nitroaniline by His 6 Amq, indicating that the enzyme was still functional. As a positive control, 2.5 M of an esterase (accession no.…”

“…The supernatant was applied to an Ni-Sepharose High Performance column (GE Healthcare Europe, Munich, Germany; 10-ml bed volume in Bio-Scale MT10 column from Bio-Rad Laboratories, Munich, Germany), equilibrated in 50 mM sodium phosphate buffer containing 10 mM imidazole, 3 mM dithiothreitol (DTT), and 300 mM NaCl (pH 8.0). After washing with the same buffer, His 6 Amq was eluted with a linear gradient (30 ml) of 20 to 250 mM imidazole in equilibration buffer. Fractions containing His 6 Amq were pooled and washed in 40 mM Britton-Robinson (BR) buffer (58) (pH 7.5; 12.5% [vol/vol] glycerol added) by ultrafiltration (Vivaspin 20; molecular weight cutoff, 10,000; Vivascience, Hannover, Germany).…”

“…In the spectrophotometric standard assay, carried out in BR buffer (40 mM, pH 8) with 100 M EDTA at 25°C, enzyme-catalyzed formation of anthranilate from N-acetylanthranilate (5 mM) was measured at 325 nm (ε 325 ϭ 2.012 mM Ϫ1 cm Ϫ1 ). In preliminary tests for His 6 Amq-catalyzed conversion of other (aromatic) amides and esters, UV/Vis spectra (250 to 600 nm) were taken at appropriate time intervals of an assay mixture that contained 5 mM of the potential substrate and 0.06 M of His 6 Amq. Quantitative spectrophotometric determinations of products generated by enzyme-catalyzed hydrolysis of aryl-acylamides or -esters were based on the following molar extinction coefficients (in assay buffer): 2-nitroaniline, ε 420 ϭ 4.438 mM Ϫ1 cm Ϫ1 ; 2-chloroaniline, ε 290 ϭ 1.670 mM Ϫ1 cm Ϫ1 ; 2-bromo-4-methylaniline, ε 295 ϭ 1.966 mM Ϫ1 cm Ϫ1 ; aniline, ε 285 ϭ 1.152 mM Ϫ1 cm Ϫ1 ; phenol, ε 270 ϭ 1.068 mM Ϫ1 cm Ϫ1 ; p-nitrophenol, ε 400 ϭ 14.847 mM Ϫ1 cm Ϫ1 ; salicylic acid, ε 298 ϭ 3.162 mM Ϫ1 cm Ϫ1 ; 3,4-dichloroaniline, ε 305 ϭ 1.99 mM Ϫ1 cm Ϫ1 .…”

“…The concentrated protein solution was diluted in 50 mM Tris-HCl buffer (pH 8) and loaded onto an UNOsphere Q column (2-ml bed volume; Bio-Rad) equilibrated in the same buffer. After a washing step, His 6 Amq was eluted in a linear gradient (32 ml) from 0 to 300 mM NaCl in 50 mM Tris-HCl buffer (pH 8.0). Fractions containing the enzyme were washed and concentrated in BR buffer (pH 7.5, with 12.5% [vol/vol] glycerol) by ultrafiltration and stored at Ϫ20°C.…”

“…Biochemical data have been published on enzymes from Pseudomonas striata (37), Bacillus sphaericus ATCC 12123 (18), P. acidovorans AE1 (6,30), P. fluorescens ATCC 39004 (26), Rhodococcus erythropolis NCIB 12273 (68), P. pickettii (31), and Nocardia globerula IFO 13510 (70). Recently, a urethane hydrolase was isolated from Rhodococcus equi TB-60, which besides catalyzing cleavage of urethane bonds to release amines effectively hydrolyzes p-nitrophenylacetate and acetanilides (3).…”