Enhancing the catalytic potential of nitrilase from Pseudomonas putida for stereoselective nitrile hydrolysis

Abstract

(R)-mandelic acid was produced from racemic mandelonitrile using free and immobilized cells of Pseudomonas putida MTCC 5110 harbouring a stereoselective nitrilase. In addition to the optimization of culture conditions and medium components, an inducer feeding approach is suggested to achieve enhanced enzyme production and therefore higher degree of conversion of mandelonitrile. The relationship between cell growth periodicity and enzyme accumulation was also studied, and the addition of the inducer was delayed by 6 h to achieve maximum nitrilase activity. The nitrilase expression was also authenticated by the sodium dodecyl phosphate-polyacrylamide gel electrophoresis analysis. P. putida MTCC 5110 cells were further immobilized in calcium alginate, and the immobilized biocatalyst preparation was used for the enantioselective hydrolysis of mandelonitrile. The immobilized system was characterized based on the Thiele modulus (phi). Efficient biocatalyst recycling was achieved as a result of immobilization with immobilized cells exhibiting 88% conversion even after 20 batch recycles. Finally, a fed batch reaction was set up on a preparative scale to produce 1.95 g of (R)-(-)-mandelic acid with an enantiomeric excess of 98.8%.

Cloning and biochemical properties of a highly thermostable and enantioselective nitrilase from Alcaligenes sp. ECU0401 and its potential for (R)-(−)-mandelic acid production

“…The nitrilase was labile at pH 9.0 exhibiting a half life of 4 h. However, the half lives of the Pseudomonas putida 412 NA 7.0 40 [29] nitrilase from Pseudomonas putida were previously reported to be only 110 and 53 min at pH 7.0 and 8.0, respectively [29]. Because the cost of a biocatalytic process is closely related to the biocatalyst generation, increased frequency of catalyst replacement due to its unstable nature is reflected by high cost associated with such process [11]. The better thermal and pH stabilities of this nitrilase would reduce the frequency of catalyst replacement in practical application, thus reducing the cost of an enzymatic process.…”

“…Recently, literatures on the nitrilase-mediated production of (R)-(-)-mandelic acid have been reported [9][10][11][12]. However, the low activity, low substrate/product tolerance, and most importantly, the poor thermostability of these nitrilases hinder their industrial application.…”

Isolation and Characterization of a Nitrile-Hydrolysing Bacterium Isoptericola variabilis RGT01

“…Acetonitrile was finally chosen as the inducer for the cultivation, due to its cheaper price and good effectivity than other inducers. It was found to act as an efficient inducer for nitrilases of Pseudomonas putida [5] and Rhodobacter sphaeroides [21]. MSM supplemented with 50 mM acetonitrile, an incubation temperature of 30°C with 2 % v/v inoculum, at 200 rpm and incubation of 48 h was found to be the optimal condition for maximum production (2.64 ± 0.12 U/mg) of nitrile-hydrolysing activity by I. variabilis RGT01 (Fig.…”

“…Nitriles are toxic in nature, but can be hydrolysed to corresponding less-toxic and valuable carboxylic acids either by nitrilase (EC 3.5.5.1), or by nitrile-hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4). Nitrilases have been reported in a variety of microbes including Alcaligenes [1], Bacillus [2], Burkholderia [3], Labrenzia [4], Pseudomonas [5], Pyrococcus [6] and Rhodococcus [7]. In spite of many nitrilases reported in literature, the quest for their new sources has never ended.…”

Biocatalytic synthesis of (R)-(−)-mandelic acid from racemic mandelonitrile by cetyltrimethylammonium bromide-permeabilized cells of Alcaligenes faecalis ECU0401

“…(R)-(-)-Mandelic acid and its derivatives are a class of chiral synthons for the production of various pharmaceuticals [1,9,16,25], such as semisynthetic penicillins, cephalosporins, antiobesity agents, and antitumor agents, and is also used as a chiral resolving agent [12][13][14]. Production of (R)-(-)-mandelic acid can be achieved by physicochemical methods [13] as well as by different enzymatic routes.…”