Nitrilase immobilization

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. 

In the same group as nitrilase enzymes are the amidases. This includes amino acid amidase (EC 3.5.1.4), used to prepare amino acids, usually through resolution, and also penicillin G acylase (penicillin G amidohydrolase) (EC 3.5.1.11), used in the manufacture of semisynthetic penicillins [102,103]. Immobilized penicillin G acylase has most recently been used to catalyze the formation of A-a-phenylacetyl amino acids, which can then be used in peptide coupling reactions (see Section 13.2.3.2) [104]. 

SP 361 SP 409 Immobilized enzyme mixture from Rhodococcus sp. containing nitrilase, nitril hydratase,esterase, epoxide hydrolase and amidase activity discontinued... 

Kfen, V., and Martmkova, L. (2006) Immobilization of fungal nitrilase and bacterial amidase - two enzymes working in accord. Biocatal. Biotransform., 24, 414-418. 

The immobilized enzyme preparation SP409", possessing both nitrilase and amidase activity, has been used for the hydrolysis of glycosyl cyanides. The a-nitrile 20 underwent hydrolysis to the amide 21, whilst the corre nding P-anomer was converted, at a faster rate, into the analogous carboxylic acid. ... 

Rey, R, Rossi, J.-C., Taillades, J., Gros, G., and Nore, O. (2004). Hydrolysis of nitriles using an immobilized nitrilase Applications to the synthesis of methionine hydroxy analogue derivatives. Journal of Agricultural and Food Chemistry, 52,8155-8162. 

Liu, Z.-Q., Zhou, M., Zhang, X.H., Xu, J.M., Xue, Y.P, and Zheng, Y.G. (2012). Biosynthesis of iminodiacetic acid from iminodiacetonitrile by immobilized recombinant Escherichia coZz harboring nitrilase. Journal of Molecular Microbiology and Biotechnology, 22,35-47. 

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. 

Liu ZQ, Zhou M, Zhang XH, Xu JM, Xue YP, Zheng YG. Biosynthesis of iminodiacetic acid from iminodiacetonitrile by immobilized recombinant Escherichia coli harboring nitrilase [J]. 7Mol Microbiol Biotechnol 2012, 22 (1) 35 7. 

General procedure Dynamic kinetic resolution for the synthesis of (R)-mandelic acid 14 In 10 mL of Tris-HCl buffer (0.1 M, pH 7), immobilized or free cells of Pseudomonas putida MTCC 5110 (harboring a stereoselective nitrilase) at 50mg/mL and racemic mandelonitrile 2 (15 mM) were introduced. The solution was stirred for 4 hours at 35 and then stopped by addition of 1-N HCl (0.3 mL). The enzyme was filtered off and the filtrate neutralized to pH 8.5 with 2-N NaOH, washed with diethylether, readjusted to pH 1.5 with 6-N HCl, extracted with diethylether, and finally concentrated. Yield (free) %%, (immobilized cells) 93% ee 98.8%. 

Hydrolysis of the nitriles 36a and Shh" by an immobilized enzyme system (nitrilase/ amidase activity) prepared from Rhodococcus sp. SP409 provided the corresponding acids 37 in 40-50% yields. However, the more bulky substrate 36c was unreactive under these conditions (Figure 12.18). 

Stereospecific nitrilases were used for the conversion of a-arninonitiiles to optically active L-amino acids (Fig. 4). In an early investigation, L-alanine was formed by an l-specific nitrilase from alginate-immobilized cells of Acinetobacter sp. APN [25]. A decrease of the enantioselectivity with the time was supposed to be caused by a racemase forming d- from L-alanine. The stereoinversion of racemic a-aminopropionitrile led to a conversion yield above 50%. Similar L-a-amino acid preparations showed no stereoinversion and additionally accumulated the D-amide due to the presence of a nitrile hydratase/ amidase system [26,27]. Additionally, a number of L-a-amino acids were synthesized by a 45-kDa monomeric nitrilase from R. rhodochrous PA-34 [28]. Remarkable in this case was the preferential hydrolysis of a-aminopropionitrile to D-alanine in contrast to the l-alanine formation by the Acinetobacter nitrilase (Fig. 4). 

The application of the immobilized nitrilase SP 409 of Rhodococcus sp. from Novo Industri (Denmark), which covers a wide substrate spectrum of aliphatic, aromatic, het-erocylic, and carbohydrate nitriles, proved to be synthetically viable for the mild and stereoselective transformation of base-sensitive carbohydrate nitriles [39]. Preferentially the P anomer of a diastereomeric glycosyl cyanide was hydrolyzed to the corresponding acid (Fig. 8). Using a C-7 alkoxylated glycosyl cyanide, amide intermediates were also detected, indicating the additional presence of a nitrile hydratase. 

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