Biochemical and Immunological Comparison of Aliphatic Amidases Produced by Pseudomonas Species

Abstract

S U M M A R YStrains of Pseudomonas aeruginosa, P. putida, P. acidovorans and P. cepacia were tested for growth on minimal salt media containing aliphatic amides. All strains of P. aeruginosa, P. acidoi7orans, P. cepacia and some strains of P.putida biotype A grew on acetamide; some strains of P.putida and P. acidovorans also grew on butyramide. The inducible aliphatic amidases had both hydrolase and transferase activities and were similar in substrate specificities. All species hydrolysed propionamide more rapidly than acetamide while acetamide was the best substrate for the transferase reaction. The rate of butyramide hydrolysis was 2 to 3 % of the rate of acetamide hydrolysis for all species including those strains able to grow on butyramide. The amidases of P. putida ~8 7and P. acidovorans 14 were inducible by butyramide. Cell-free extracts of P. aeruginosa strains induced with acetamide cross-reacted completely with antiserum prepared against purified P. aeruginosa amidase A. Partial cross-reactions occurred with P. putida and P. acidovorans amidases and a very weak cross-reaction with P. cepacia amidase. I N T R O D U C T I O NKelly & Clarke (1960) showed that a strain of Pseudomonas aeruginosa growing on acetamide as the sole source of carbon and nitrogen possessed an inducible aliphatic amidase. This enzyme had very limited inducer and substrate specificities and acetamide and propionamide were the only aliphatic amides which could support growth. Buhlman, Vischer & Bruhin (1961) found that among the fluorescent pseudomonads only those strains which could be classed as P. aeruginosa were able to produce an alkaline reaction in glucose+ acetamide medium and this reaction was presumably due to the activity of similar aliphatic amidases. Jakoby & Fredericks (1964) isolated a fluorescent pseudomonad which grew in a minimal salts medium with acetamide as the carbon source. This strain produced an amidase with properties similar to those reported for the P. aeruginosa enzyme (Kelly & Clarke, 1962;Brammar & Clarke, 1964;Kelly & Kornberg, 1964). In their survey of the pseudomonads, Stanier, Palleroni & DoudoroE(1966) reported that all the strains of P. aeruginosa which they examined were able to grow on acetamide. In addition, some strains of P. putida biotype A, P. acidovorans and P. cepacia (P. multivorans) were also able to grow on ace t amide.There is considerable homology among some of the enzymes of Pseudomonas species. Queener & Gunsalus (1970) showed that anthranilate synthase from extracts of Pseudomonas aeruginosa, P. putida, P. acidovorans, P. testosteroni, P. stutzeri and P. cepacia could be separated into two subunits and that these subunits could be associated to form hybrid enzymes. The hybrid enzymes made from P. aeruginosa and P. putida (group a-p) subunits, or from P. acidovorans and P. testosteroni (group c-t) subunits, were fully active, whereas hybrid enzymes made with one subunit from each group had lower activities than the native enzymes.Stanier (1968) compared the pathways employed f...

Lipase and esterase-catalyzed acylation of hetero-substituted nitrogen nucleophiles in water and organic solvents

“…We note that the amidase-catalyzed hydroxylaminolysis of carboxylic amides has been known for a long time (Clarke, 1972;Jacobi and Fredericks, 1964); its kinetics have also been reported (Fournand et al, 1998a,b;Thiéry et al, 1986). The lipase-mediated synthesis of acyl hydrazides has briefly been described (Astorga et al, 1991(Astorga et al, , 1993Gotor et al, 1990); the reaction is also catalyzed by amidases (Fournand et al, 1998a;Kobayashi et al, 1999).…”

A Comparative Study of Acquired Amidase Activity in Pseudomonas Species

“…putida biotype A strains has been described as being similar to the enzyme found in P . aeruginosa (Clarke, 1972). The latter is a hexameric protein of subunits with molecular mass 40 kDa.…”

The Utilization of Nitriles and Amides by a Rhodococcus Species

“…If acetamidase activity was not detectable, acetonitrile hydrolysis was assayed indirectly by measuring the rate of ammonia production in the presence of Pseudomonus putidu PPEl (A87). This organism was grown in an acetamide-containing medium (Kelly & Clarke, 1962) to induce aliphatic amidase activity (Clarke, 1972) but no nitrilase activity was detected. The reaction mixture, containing 2 ml amidase-negative Rhodococcus cells, 1 ml P. putidu cells (these contained about 40 nkat amidase activity) and 1 mlO.2 M-acetonitrile in phosphate buffer, was incubated at 25 OC for 30 min.…”

“…Bacterial amidases have been most studied in species of Pseudomonas (Clarke, 1970(Clarke, , 1972) though these organisms are not known to utilize nitriles. Pseudomonas aeruginosa (PAC 1 wild-type) can only utilize acetamide and propionamide, whereas some strains of P. putida and P. acidovorans can also grow on n-butyramide (Clarke & Richmond, 1975).…”