Purification and Characterization of an Enantioselective Amidase from Pseudomonas chlororaphis B23

Abstract

An amidase produced by Pseudomonas chlororaphis B23 was purified and characterized. The purification procedure used included ammonium sulfate precipitation and hydrophobic, anion-exchange, gel filtration, and ceramic hydroxyapatite chromatography steps. This amidase has a native molecular mass of about 105 kDa and is a homodimer whose subunits have a molecular mass of 54 kDa. The enzyme exhibited maximal activity at 50؇C and at pH values ranging from 7.0 to 8.6. We found no evidence that metal ions were required, and the enzyme was inhibited by several thiol reagents. This amidase exhibited activity against a broad range of aliphatic and aromatic amides and exhibited enantioselectivity for several aromatic amides, including 2-phenylpropionamide (enantiomeric excess [ee] ‫؍‬ 100%), phenylalaninamide (ee ‫؍‬ 55%), and 2-(4-chlorophenyl)-3-methylbutyramide (ee ‫؍‬ 96%), but not 2-(6-methoxy-2-naphthyl)propionamide (the amide form of naproxen) (ee ‫؍‬ 0%). The characteristics of the P. chlororaphis B23 amidase are the same as the characteristics of enantioselective amidases described by Mayaux et al.

Improved efficiency of asymmetric hydrolysis of 3-substituted glutaric acid diamides with an engineered amidase

“…We determined partial amino acid sequences of the purified enzyme, which allowed us to clone and sequence the gene encoding the enzyme. The deduced amino acid sequence of CoAM shared the highest identity (87%) with an amidase from P. chlororaphis B23, a catalyst for the industrial production of acrylamide (Nagasawa et al 1989;Ciskanik et al 1995). Thus, the amidase from P. chlororaphis B23 and its homologues might be active towards 3-substituted glutaric acid diamides.…”

Acyl Transfer Activity of an Amidase from Rhodococcus sp. Strain R312: Formation of a Wide Range of Hydroxamic Acids

“…This finding differs markedly from the data obtained with the enantioselective amidase from P. chlororaphis B23, which, despite a high level of sequence homology (level of strict identity, 46%) with the enantioselective amidase from Rhodococcus sp. strain R312, exhibited the highest levels of hydrolysis activity with isobutyramide, nicotinamide, and DLphenylalaninamide (5).…”

“…Amidases from P. chlororaphis B23, R. erythropolis MP50, Rhodococcus sp. strain R312, Rhodococcus sp., and R. rhodochrous J1 were shown to exhibit an (S)-enantioselective hydrolysis activity towards several 2-arylpropionamides (5,12,14,20,21). The amidase from Rhodococcus sp.…”

Isolation and characterization of an acrylamide‐degrading yeast Rhodotorula sp. strain MBH23 KCTC 11960BP

“…Several microbes capable of utilizing acrylamide as a source of carbon and nitrogen have been isolated, for example: Klebsiella pneumonia [15], Helicobacter pylori [16], Ralstonia eutropha [17,18], Rhodococcus rhodochrous [19], Pseudomonas stutzeri [20], Bacillus cereus [21], Pseudomonas sp. [22], Pseudomonas chlororaphis [23], Pseudonocardia thermophila [24] and the fungi Aspergillus oryzae [25]. The introduction of microorganisms capable of degrading acrylamide into polluted soils and aquatic bodies is a potential bioremediation tool [8, 17 -25] and bioremediation works on partially hydrolyzed polyacrylamide pollutant has shown promising results [26].…”