Nitrilase expression

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. 

The biocatalytic differentiation of enantiotopic nitrile groups in prochiral or meso substrates has been studied by several research groups. For instance, the nitrilase-catalyzed desymmetrization of 3-hydroxyglutaronitrile [92,93] followed by an esterification provided ethyl-(Jl)-4-cyano-3-hydroxybutyrate, a useful intermediate in the synthesis of cholesterol-lowering dmg statins (Figure 6.32) [94,95]. The hydrolysis of prochiral a,a-disubstituted malononitriles by a Rhodococcus strain expressing nitrile hydratase/amidase activity resulted in the formation of (R)-a,a-disubstituted malo-namic acids (Figure 6.33) [96]. 

The regioselectivity of a Rhodococcus rhodochrous nitrilase has been demonstrated for the conversion of 5-fluoro-l,3-dicyanobenzene to 5-fluoro-3-cyano-benzoic acid [62]. The nitrilase was expressed in an Escherichia coli transformant, and a cell-free extract was employed as catalyst (0.14wt% cell-free extract) in 0.1m sodium phosphate buffer (pH 7.2) at 25 °C containing 0.18 m 5-fluoro-l,3-dicyanobenzene. After 72 h, the conversion was >98% and the reaction was stopped by addition of phosphoric acid (pH 2.4) to yield 5-fluoro-3-cyano-benzoic acid as a crystalline product (97% isolated yield). 

The recombinantly expressed nitrilase from Pseudomonas fluorescens EBC 191 (PFNLase) was applied in a study aimed at understanding the selectivity for amide versus acid formation from a series of substituted 2-phenylacetonitriles, including a-methyl, a-chloro, a-hydroxy and a-acetoxy derivatives. Amide formation increased when the a-substituent was electron deficient and was also affected by chirality of the a- stereogenic center for example, 2-chloro-2-phenylacetonitrile afforded 89% amide while mandelonitrile afforded 11% amide from the (R)-enantiomer but 55% amide was formed from the (5)-enantiomer. Relative amounts of amide and carboxylic acid was also subject to pH and temperature effects [87,88]. 

A summary of the industrial-scale process development for the nitrilase-catalyzed [93] route to ethyl (/ )-4-cyano-3-hydroxy-butyrate, an intermediate in the synthesis of Atorvastatin (Pfizer Lipitor) from epichlorohydrin via 3-hydroxyglutaronitrile (3-HGN) was recently reported (Figure 8.15) [94], The reaction conditions were further optimized to operate at 3 m (330 gL ) substrate, pH 7.5 and 27 °C. Under these conditions, 100% conversion and product ee of 99% was obtained in 16 h reaction time with a crude enzyme loading of 6% (based on total protein, 0.1 U mg-1). It is noted that at pH < 6.0 the reaction stalled at <50% conversion and at alkaline pH a slowing in reaction rate was observed. Since the starting material is of low cost and the nitrilase can be effectively expressed in the Pfenex (Pseudomonas) expression system at low cost, introduction of the critical stereogenic center... 

Mueller, P, Egorova, K., Vorgias, C.E. et al. (2006) Cloning, overexpression, and characterization of a thermoactive nitrilase from the hyperthermophilic archaeon Pyrococcus abyssi. Protein Expression and Purification, 47, 672-668. 

R. D. Fallon, J. E. Gavagan, R. DiCosimo, and M. S. Payne, Purification, cloning, sequendng and over-expression in Escherichia coli of a regioselective aliphatic nitrilase from Acidovorax facilis 72W, Appl. Microbiol. Biotechnol. 2003, 61, 118-122. 

Heinemann, U., Engels, D., Burger, S., et al. 2003. Cloning of a nitrilase gene from the cyanobacterium Synechocystis sp. Strain PCC6803 and heterologous expression and characterization of the encoded protein. Applied and Fnvironmental Microbiology, 69 4539-66. 

Kiziak, C., Conradt, D., Stolz, A., et al. 2005. Nitrilase from Pseudomonas fluorescens EBC191 Cloning and heterologous expression of the gene and biochemical characterization of the recombinant enzyme. Microbiology, 151 3639 8. 

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]. 

A method of efficient induction of fungal nitrilases in wild strains was established and seems to be of wide application throughout this enzyme group. However, heterologous expression of fungal nitrilases remains to be solved in order to facilitate the use of these enzymes in industrial biotechnology. To what extent the desirable properties (broad substrate specificity, thermostability, chemoselectivity and enantioselectivity) can be improved by mutagenesis of these enzymes should also be examined. 

It has been noted already that reports can be found in the literature, as far back as the 1960s, of modest amounts of amides being formed in the presence of nitrilases [20-22]. This side activity (see Figure 16.7) could not be accounted for by the commonly accepted nitrilase mechanism [21] and was largely neglected until quite recently. It was then shown, for example, that nitrile hydration in the presence of recombrnantly expressed and purified nitrilases from Arahidopsis thali-ana was a major pathway with some nitriles, in particular electron-deficient ones [23-25]. 

Table 6. Determination of the different recombinant strains expressing the nitrilase and their control strains on ammonium 5-cyanovalerate and adiponitrile... 

Interestingly, if organisms contain non-Bi2 cobalt enzymes such as nitrilases (Koba-yashi and Shimizu 1998), an additional slow, chemisosmotically driven uptake system (NiCoT protein family) is co-expressed with the enzyme (Komeda et al. 1997). ATP-hydrolyzing uptake systems for cobalt (e.g., ABC-transport systems) are not known. This indicates that cobalt for B12-enzymes may indeed be imported as cobalamin, Co(II) for other enzymes by NiCoT transport systems and that Co(II)-import by other systems may not be important in the natural environment of the cells. Co(II) is of medium toxicity and is detoxified by efflux systems (CDF protein family, RND-driven CBA-export systems) in bacteria and yeasts (Nies 2003). 

Sosedov, O., Matzer, K., Burger, S., Kiziak, C., Baum, S., Altenbuchner, J., Chmura, A., van Rantwijk, F., and Stolz, A. (2009) Construction of recombinant Escherichia coli catalysts which simultaneously express an (S)-oxynitrilase and different nitrilase variants for the synthesis of (S)-mandelic acid and (S)-mandelic amide from benzaldehyde and cyanide. Adv. Synth. Catal., 351, 1531-1538. 

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. 

A nitrilase sequenced in the archaeon P. abyssi GE5 was expressed in E. coli to give a highly thermostable enzyme (with an enzyme half-life of 25, 9, and 6h at 70, 80, and 90 °C, respectively [14]). This enzyme is the only experimentally confirmed nitrilase to have been crystallized [13]. The low identity of this enzyme to other characterized nitrilases makes its usefulness for homology modeling low. This enzyme also differs from most other characterized nitrilases in its substrate specificity, its preferential substrates being fumaronitrile and malononitrile [14]. 

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