Nitrile hydratase reactions catalyzed

The commercial bioconversion process employs the enzyme nitrile hydratase, which catalyzes the same reaction as the chemical process (Figure 31.15). The bioconversion process was introduced using wild-type cells of Rhodococcus or Pseudomonas, which were grown under selective conditions for optimal enzyme induction and repression of unwanted side activities. These biocatalysts are now replaced with recombinant cells expressing nitrile hydratase. The process consists of growing and immobilizing the whole cell biocatalyst and then reacting them with aqueous acrylonitrile, which is fed incrementally. When the reaction is complete the biocatalyst is recovered and the acrylamide solution is used as is. The bioconversion process runs at 10°C compared to 70°C for the copper-catalyzed process, is able to convert 100 percent of the acrylonitrile fed compared to 80 percent and achieves 50 percent concentration... 

UF-Membrane Bioreactors for Kinetics Characterization of Nitrile Hydratase-Amidase-catalyzed Reactions a Short Survey... 

We found a new microbial enzyme named "nitrile hydratase" which catalyzes the hydration reaction of nitrile to amides. it has been proven that acrylonitrile and methacrylonitrile are easily converted to the corresponding amides. When Rhodococcus rhodochrous J1 resting cells were used as the catalyst, more than 600 g of acrylamide was produced in 1 liter of reaction mixture with a yield of nearly 100 % for acrylonitrile. Since 1991, immobilized R. rhodochrous cells have been used for the industrial production of acrylamide (Fig. 1). At present, more than 10,000 tonnes of acrylamide is produced per year by Nitto Chemical Industries Ltd. 

Nitrile hydratases (NHases) catalyze the hydration of organic nitriles to amides under very benign reaction conditions (neutral aqueous environment and room temperature) and therefore offer a chemoselective alternative to classical approaches, where functional group compatibility is often limited due to the harsh acidic or basic solutions used [1], Starting with their application in acrylamide production [2,3], this enzyme class is one of the most prominent in industrial processes with respect to production volume (>3 X 10 kg/a for acrylonitrile hydration) [4]. Hence, Lonza (Switzerland) uses a nitrile hydratase to convert 3-cyanopyridine into nicotinamide (6 X 10 tons/year). Very recently, a one-pot industrial protocol for the synthesis of a chiral intermediate for dlastatin was published that employed a nitrile hydratease/amidase approach [5],... 

The dehydration reaction of aldoxime to form nitriles using the resting cells of Rhodococcus sp. YH3-3 was optimized. We found that the enzyme was induced by aldoxime and catalyzed the stoichiometric synthesis of nitriles from aldoximes at pH 7.0 and 30°C. Phenylacetonitrile once synthesized from phenylacetaldoxime was hydrolyzed to phenylacetic acid, since the strain has nitrile degradation enzymes such as nitrile hydratase and amidase. We have been successful in synthesizing phenylacetonitrile and other nitriles stoichiometrically by a selective inactivation of nitrile hydratase by heating the cells at 40°C for 1 h. Various nitriles were synthesized under optimized conditions from aldoximes in good yields. 

Notably, nitrile-degrading enzymes (e.g. nitrilase that converts the CN group to carboxylic acid, and nitrile hydratase that produces an amide function) have been described, and they co-exist with aldoxime-degrading enzymes in bacteria (Reference 111 and references cited therein). Smdies in this area led to the proposal that the aldoxime-nitrile pathway, which is implemented in synthesis of drugs and fine chemicals, occurs as a natural enzymic pathway. It is of interest that the enzyme responsible for bacterial conversion of Af-hydroxy-L-phenylalanine to phenacetylaldoxime, an oxidative decarboxylation reaction, lacks heme or flavin groups which are found in plant or human enzymes that catalyze the same reaction. Its dependency on pyridoxal phosphate raised the possibility that similar systems may also be present in plants . 

Strategic importance of biocatalyzed synthetic transformations in terms of eco-compatibility and cheaper processes has been widely stressed previously. Among the developed biotransformations catalyzed by nitrilases or nitrile hydratases/ amidases systems, a special interest is focused toward stereoselective reactions able to give access to molecules otherwise impossible to obtain by classical chemical routes. Hereby, selected examples aim to offer an overview of research in this direction. Examples of industrial processes using nitrile hydrolyzing biocatalysts are also illustrated. 

Nitrile biodegradation is performed by a variety of microorganisms and proceeds through two different enzymatic pathways direct transformation to carboxylic acids and ammonia, with some exceptions, catalyzed by a nitrilase (EC 3.5.5.1) [1-3] or a two-step reaction, the first catalyzed by nitrile hydratase (EC 4.2.1.84) that produces an amide intermediate, which is further hydrolyzed to the acid and ammonia by an amidase (EC 3.5.1.4) [4, 5],... 

The operational thermal stability of enzymes can be easily evaluated in experiments carried out in a CSMR fed with a saturating substrate concentration, while varying the temperature but maintaining all the other parameters constant. Each enzyme of the cascade system was tested by feeding the CSMR with the appropriate substrate. The kinetic characterization of amidase-catalyzed reactions in runs fed with a nitrile was hampered by the fact that the intracellular enzyme works in cascade with nitrile hydratase. The concentration of amide, produced in situ in the first step, varied with the time and reaction conditions and did not assure the differential conditions needed for an accurate analysis, the amide being completely converted by amidase in some runs. Hence, amidase activity was characterized independently by feeding the reactor with amide as the substrate [35]. 

During the course of our research on nitrile hydratase- and amidase-catalyzed reactions we had to deal with different aspects of substrate concentration effects on the enzyme kinetics and all were suitably investigated making use of a CSMR, as shown by the following case studies. 

The propionitrile nitrile hydratase-catalyzed reaction presented kinetics inhibited by high substrate concentrations that were clearly evidenced in the CSMR, as revealed in [33]. The kinetic parameters for nitrile hydratase-catalyzed reactions of... 

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]. 

Biotransformations for the synthesis of asymmetric compounds can be divided into two types of reactions those where an achiral precursor is converted into a chiral product (true asymmetric synthesis) and those involving the resolution of a racemic mixture. Both types of reaction are used at Lonza, which is a leading producer of intermediates for the life science industry. Lonza also uses biocatalysis for the synthesis of achiral molecules, for example, an immobilized whole-cell biocatalyst is used for the nitrile hydratase-catalyzed synthesis of thousands of tons per year of nicotinamide from 3-cyanopyridine. 

The addition of water to carbon-carbon double bonds is a reaction that is catalyzed by lyases belonging to the subclass of the hydro-lyases (E.C. 4.2.1.-), which have been grouped under the carbon-oxygen lyases. Not all members of this subgroup are capable of water addition to carbon-carbon double bonds. Nitrile hydratase (E. C. 4.2.1.84, discussed in Section 12.1) for instance, is categorized in this subclass and catalyzes the addition of water to nitriles. The nomenclature of the hydro-lyases subgroup, which contains hydratases and dehydratases, does not preclude any direction of the reaction, but rather reflects the context in which the enzyme was originally discovered. 

Since many enzymes have capacities to catalyze reactions with even unnatural substrates and to produce unnatural compounds, hybrid use of enzymes as biocatalysts with chemical synthesis can realize processes to produce useful substances with higher flexibility than processes with growing cells. Discovery of novel microbial enzymes with required specificity by screening is a key to the establishment of such a hybrid processes. Many successful achievements in Japan are observed in this unique field of biotechnology. Application of nitrile hydratase to production of acrylonitriles has proved that biocatalysts can be applied to production of commodity chemicals beyond the presumed limitation of fine chemicals. Discovery of the enzymatic reactions to produce trehalose from starch is an example that reveals the possibility of microbial screening or what remains undiscovered in the microbial world. The importance of developing new application is also crucial in this field as shown in the case of transglutaminase and alkaline cellulase. 

Fe-(lll) Nitrile Hydratases Nitrile hydratases function as Lewis acids, in contrast to the mode of action of most nonheme iron center enzymes, which catalyze redox type reactions. The nitrile hydratase activity found in Rhodococcus N-771 [54-56] was found to be photoregulated, with light reversing the in vivo inhibitory nitric oxide (NO) binding and thereby activating the nitrile hydratase [56]. 

Nitriles may be converted to amino acids by nitrilases, which catalyze a two-step nitrile hydrolysis reaction, and can provide p -amino acids in high enantiopurity. The same reaction can be catalyzed by a combination of nitrile hydratase and... 

Types of selectivity exhibited by enzymes. Biocatalytic processes can provide chemo-, regio-, and stereoselective conversions. Representative examples can be observed in reactions catalyzed by nitrile hydratases, lipases and dehydrogenases. 

Enzymes of the hydroxynitrilase dass catalyze the addition of HCN to aldehydes, produdng cyanohydrins. Recendy, the reaction has been extended to a few ketones with modified hydroxynitrilase enzymes. In many cases, these are formed with good optical purities and such reactions are the simplest type of enzyme catalyzed carbon-carbon bond formation. By pairing hydroxynitrile lyases with nitrilases or nitrile hydratases, one-pot, multistep conversions become possible, and this also shifts the equilibrium to favor the addition products. Such concerns are particularly important when applying these catalysts to ketones where the equilibrium generally favors the starting carbonyl compound (Figure 1.17). 

Hydrolysis of nitriles Hydrolysis of [ CJnitrile functions represents a well-established route for the synthesis of [ C]carboxylic acids. For cases in which the harsh reaction conditions of chemical hydrolysis (e.g., 2 N NaOH, reflux) are incompatible with sensitive functionalities, application of enzymes might provide an alternative (Figure 12.17). Enzymatic hydrolysis of nitriles results in amides if catalyzed by nitrile hydratases or in the corresponding carboxylic acids if catalyzed by nitrilases". ... 

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