Nitrilase activity

The nitrilase activity of Arthrobacter sp. F-73 retains substantial activity in aqueous solutions containing a significant concentration of organic co-solvent [84]. More than 10% of nitrilase activity remains at acetone concentrations up to 60%, whereas no activity... 

The preparation of malonic acid monoesters has been demonstrated using the microbial nitrilase activity of Corynebacterium nitrilophilus ATCC 21 419, Gordona terrae MA-1, or Rhodococcus rhodochrous ATCC 33 025 to hydrolyze methyl cyanoacetate, ethyl cyanoace-tate, M-propyl cyanoacetate, isopropyl cyanoacetate, M-butyl cyanoacetate, tertbutyl cyanoacetate, 2-ethylhexyl cyanoacetate, allyl cyanoacetate, and benzyl cyanoacetate [96]. By maintaining the concentration of nitrile in a reaction mixture at <5 wt%, significant inactivation of the nitrilase activity was avoided for example, a total of 25 g of M-propyl cyanoacetate was added in sequential 5g portions to a lOOmL suspension of Rhodococcus rhodochrous ATCC 33 025 cells (OD630 = 5.6) in 50 mM phosphate buffer (pH 7.0) over 30h at 25 °C to produce mono-M-propyl malonate in 100% yield (Figure 8.17). 

Figure 8.17 Production of mono-n-propyl malonate from M-propyl cyanoacetate using the microbial nitrilase activity of Rhodococcus rhodochrous ATCC 33025...

Organisms with Characterized Nitrilase Activity and Substrate Classification... 

Nolan, L.M., Harnedy, P.A., Tinner, R, et al. 2003. The cyanide hydratase enzyme of Eusarium lateritium also has nitrilase activity. EEMS Microbiology letters, 221 161-5. 

The expression of nitrilase may be enhanced and, thus, screening for its activity fadhtated by the choice of an appropriate nitrilase inducer. For instance, in five strains belonging to three fungal genera 2-cyanopyridine enhanced the nitrilase activity by two to three orders of magnitude. Therefore, this nitrilase inducer appears to be very efficient and of wide use in filamentous fungi [25]. 

Some filamentous fungi, mainly representatives of the genera Fusarium, Aspergillus, and Penicillium, degrade nitriles preferentially through nitrilase activity [18, 19] (see Chapter 14). 

The degradation of nitriles by nitrilases (EC 3.5.5.1) has been the subject of intense study, especially as it relates to the preparation of the commodity chemical acrylamide. Nitrilases catalyze the hydrolysis of nitriles to the corresponding acid plus ammonia (Figure 1 reaction 5), whereas nitrile hydratases (EC 4.2.1.84) add water to form the amide. Strains such as Rhodococcus rhodo-chrous Jl, Brevibacterium sp., and Pseudomonas chlororaphis have been used to prepare acrylamide from acrylonitrile, which contain the hydratase and not nitrilase activity [12]. A comparison of these strains has been discussed elsewhere [98]. Other uses of nitrilases, however, have primarily been directed at resolution processes to stereoselectively hydrolyze one enantiomer over another or regiose-lectively hydrolyze dinitriles [99-101]. 

The purification steps were mainly carried out in 50 mM TrisHCl buffer, pH 7.5, 1 mM dithioerythrytol. At each step, the nitrilase activity of the fractions was determinated at pH 7 and at 25 °C in 10 mM phosphate buffer in the presence of 10 mM adiponitrile. The protein concentration was determined by the Commassie blue method. The protein pannel was analysed by SDS PAGE (Phasystem pharmacia ). The data from each of the steps are listed in Table 4. 

For the adiponitrile, the nitrilase activity was independant of pH and for ammonium 5-cyanovalerate, the higher its ionic feature the worse the nitrilase activity. Therefore, the chemo-selectivity of the nitrilase between adiponitrile and ammonium 5-cyanovalerate was 64 % at pH 4 and 97 % at pH 7. 

The nitrilase activities were determined for other dinitriles and compared to that of adiponitrile (Fig. 3). For each compound, the chemo-selectivity was identical to that obtained for adiponitrile. 

Subsequently, bienzymatic whole cell catalysts were constracted by coexpressing the (S)-HnL and nitrilase activities simultaneously in the yeast Pichia pastoris and the bacterium Escherichia coli. The recombinant E. coU cells exhibited much higher HnL and nitrilase activities compared to the P. pastoris catalysts and were therefore studied in greater detail [63, 64]. The recombinant E. coli cells were... 

A. (2012) Application of a recombinant Escherichia coli whole-cell catalyst expressing hydroxynitrile lyase and nitrilase activities in ionic liquids for the production of (S)-mandelic acid and (S)-mandeloamide. Adv. Synth. Catal., 354, 113-122. 

Mahadevan and Thimann [43] postulated the first nitrilase reaction mechanism, suggesting that the nitrile carbon present in the substrate displays a partial positive charge that is subjed to nudeophihc attack by one of the two SH groups in the nitrilase active site. The resulting thioimidate is then hydrolyzed to a thioester, with the release of ammonia as a by-product Hydrolysis of the acyl-enzyme then results in the release of the final acid product. 

Recently we determined that two R. rhodochrous strains (A29 and A99) expressed nitrilase activity after induction. These strains were capable of enantioselectively hydrolyzing racemic 3-amino-3-phenylpropanenitrile directly to the corresponding (R)-3-amino-3-phenylpropanoic acid with >95% ee (Table 14.1) in a kinetic resolution. Various inhibitors were used, that indicated the observed hydrolytic activity was due to the presence of a nitrilase rather than a nitrile hydratase and amidase pair [47]. 

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