Nitrilase activity carboxylic acids

Nitrilases catalyze the synthetically important hydrolysis of nitriles with formation of the corresponding carboxylic acids [4]. Scientists at Diversa expanded the collection of nitrilases by metagenome panning [56]. Nevertheless, in numerous cases the usual limitations of enzyme catalysis become visible, including poor or only moderate enantioselectivity, limited activity (substrate acceptance), and/or product inhibition. Diversa also reported the first example of the directed evolution of an enantioselective nitrilase [20]. An additional limitation had to be overcome, which is sometimes ignored, when enzymes are used as catalysts in synthetic organic chemistry product inhibition and/or decreased enantioselectivity at high substrate concentrations [20]. 

There are two pathways for the degradation of nitriles (a) direct formation of carboxylic acids by the activity of a nitrilase, for example, in Bacillus sp. strain OxB-1 and P. syringae B728a (b) hydration to amides followed by hydrolysis, for example, in P. chlororaphis (Oinuma et al. 2003). The monomer acrylonitrile occurs in wastewater from the production of polyacrylonitrile (PAN), and is hydrolyzed by bacteria to acrylate by the combined activity of a nitrilase (hydratase) and an amidase. Acrylate is then degraded by hydration to either lactate or P-hydroxypropionate. The nitrilase or amidase is also capable of hydrolyzing the nitrile group in a number of other nitriles (Robertson et al. 2004) including PAN (Tauber et al. 2000). 

Nitrilases convert nitriles to the corresponding carboxylic acids and NH3 through a cysteine residue in the active site [50]. Because of their high enantio- and regio-selectivity, nitrilases are attractive as green catalysts for the synthesis of a variety of carboxylic acids and derivatives (Figure 1.10) [51,52]. Recently, a number of recombinant nitrilases have been cloned and characterized heterologously for synthetic applications [50,53,54]. 

The two enzyme classes nitrile hydratases (RCN + H20 — RCONH2) and nitrilases (RCN + 2H20 —y RCOOH + NH3) actually belong to two distant groups in the EC system, with the hydratases being classified as lyases (EC 4.2.1.84) and nitrilases as hydrolases (EC 3.5.5.1). Microorganisms that produce a nitrile hydratase also seem to produce amidases, which enable them to convert nitriles into carboxylic acids in a two-step reaction. Actually, amidase side-activity can be a problem with commercial nitrile hydratase preparations (if the target structure is the amide). Nitrilases, however, hydrolyze the nitrile without the formation of a free amide intermediate. 

Recently, the potential of bacterial enzymes for the synthesis of aromatic, optically active amides, and carboxylic acids firom racemic nitriles was evaluated. An enantiomer-selective amidase, active on several 2-aryl and 2-aryloxy propionamides, was identifided and purified from Brevibacterium sp. strain R312 [145]. A nitrilase, found in Acinetobacter sp. strain AK226 and able to hydrolyze efihciently both aromatic and aliphatic nitriles, was reported to hydrolyze racemic nitriles to optically active 2-aryl propionic acids [146]. Enzyme system of Rhodococcus butanica could be successfully adapted for the kinetic resolution of a-arylpropionitriles resulting in the formation of (R)-... 

In industrial biotransformations, hydrolytic reactions occupy a prominent position for the production of optically active amines, alcohols, and carboxylic acids. Compared with other reactions, hydrolytic reactions are feasible to scale up because they are cofactor-free, relatively simple, and chemically tunable systems. In addition to home-made whole-cell biocatalysts, which are considered to be more cost-effective for specific syntheses, some commercially available hydrolases, including lipases/esterases, epoxide hydrolases, nitrilases, and glycosidases, are also employed for the enantioselective production of chiral chemicals. 

Nitrilases are classified into branch 1 of the nitrilase superfamily, which is comprised of enzymes acting on various nonpeptide CN bonds [15]. All the proteins of this superfamily are characterized by a conserved catalytic triade (glu, lys, cys) and an additional conserved glu residue that seems to participate in the reaction mechanism [2]. Members of class 1 transform the CN bonds in nitriles and cyanides. The enzymes in which these activities were confirmed share in some cases levels of aa sequence identity as low as about 20%. This sequence diversity is reflected in different substrate specificities and different reaction products (carboxylic acids, amides) in various subtypes of these enzymes (aromatic nitrilases, aliphatic nitrilases, arylacetonitrilases, cyanide hydratases, cyanide dihydratases). 

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. ... 

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