Nitrilase stability

A novel nitrilase was purified from Aspergillus niger K10 cultivated on 2-cyanopyridine. It was found to be homologous to a putative nitrilase from Aspergillus fumigatus Af293. The nitrilase exhibited maximum activity at 45 °C and pH 8.0 with much less activity observed at slightly acid pH. Its substrate preference was for 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene, thio-phen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile. ( )-2-Phenylpropionitrile was only poorly converted by this enzyme and with minimal enantioselectivity. The enzyme was shown to be multimeric (>650 kDa) and be stabilized in the presence of sorbitol and xylitol [57]. 

Table 14.4 Specific activity, reaction optima, and stability of purified nitrilases from filamentous fungi comparison with prokaryotic nitrilases, cyanide hydratase, and cyanide dihydratase. 

Fungal nitrilases immobilized on these columns were applicable to continuous biotransformation of heteroaromatic nitriles like 3- and 4-cyanopyridine, the products of which, nicotinic and isonicotinic acid, respectively, are of commercial interest. The enzyme from F. solani exhibited a higher stability than that from A. niger at 35 °C. The conversion of 3-cyanopyridine by the former enzyme was nearly quantitative within 24h [50], while it decreased by 30% within 15h in the case of the latter [49]. Similar differences in operational stabiUties were observed during conversion of 4-cyanopyridine. The stabiUty of the enzymes depended on the substrate used, both nitrilases being more stable during the conversion of 4-cyanopyridine in comparison with 3-cyanopyridine. 

However, production of 2,6-difluorobenzamide (Scheme 12.1-18) was effected in 99.5% n-heptane using the nitrile hydratase from Rhodococcus sp. NCIMB 12 21 81841. The enzymatic reaction was found to be activated by light (see 12.1.3.4). More recently, Layh and Willetts have studied nitrile transformations in various organic solvents and biphasic mixtures using a nitrilase from Pseudomonas sp. DSM 11387 and a nitrile hydratase from Rhodococcus sp. DSM 113971 51. The enzymes exhibited good stabilities in biphasic mixtures with hydrophobic solvents when dispersed in... 

Nitrilase, nitrile hydratase Nitrile hydrolysis Chemo- and regioselectivity Enantioselectivity, enzyme stability State of the art, stable (commercial) nitrilases ... 

Nitrilase is a useful alternative to the NHase/amidase cascade, the benefits of nitrilase often being its higher enantioselectivity or stability. However, a drawback of nitrilase is its tendency to form mixtures of carboxylic acids and amides, this phenomenon being caused by an untypical cleavage of the reaction intermediate leading to the amide as the side product [55]. 

Although the combination of HnLs and nitrilases appears at first glance to be a straightforward process, several problems had to be solved for the intended enzyme cascade. For example, synthetic HnL reactions are usually performed at pH < 5 in order to suppress the uncatalyzed (and therefore nonenantioselective) hydro-cyanation reaction. Furthermore, synthetic HnL-mediated reactions are usually performed in an aqueous-organic two-phase system in order to further suppress the uncatalyzed reaction [59]. Unfortunately, nitrilases generally show only a low activity and stability under acidic conditions and are rapidly inactivated in the presence of organic solvents [1]. 

A different method consisting of directed evolution was used with the nitrilases from P. fluorescens [26] and Alcaligenes faecalis [27]. The amide production by the former and the operational stability of the latter at a low pH were improved in this... 

Li et al. [39] recently reported fusing the A. faecalis nitrilase expressed in E. coli with an amphipathic peptides. Their studies found that -90% of nitrilase exists in the form of active inclusion bodies (aggregates) in the host cells. The thermal stability was significantly improved by 6.8- and 4.3-fold at 45 °C and 50 °C, respectively. The nitrilase aggregates were purified and immobilized with sodium alginate. The stability was further increased by 1.5-fold. Immobilized enzymes demonstrated an improvement of substrate tolerance from 30 mM to 200mM with mandelonitrile as the substrate. 

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