Nitrile hydratases hydrolysis with enzymes

The hydrolysis of nitriles can be carried out with either isolated enzymes or immobilized cells. Eor example, resting cells of P. chlororaphis can accumulate up to 400 g/L of acrylamide in 8 h, provided acrylonitrile is added gradually to avoid nitrile hydratase inhibition (116). The degree of acrylonitrile conversion to acrylamide is 99% without any formation of acryUc acid. Because of its high efficiency the process has been commercialized and currentiy is used by Nitto Chemical Industry Co. on a multithousand ton scale. 

Generally, enzymatic hydrolysis of nitriles to the corresponding acids can either proceed stepwise, which is the case for catalysis by the nitrile hydratase/amidase enzyme system, or in one step in the case of nitrilases. Both systems have been investigated for surface hydrolysis of PAN [10], Complete hydrolysis with either system was monitored by quantification of ammonia and/or polyacrylic acid formed as a consequence of hydrolysis of nitrile groups [70-72], As a result, considerable increases in colour levels (e.g. 156% for commercial nitrilase) were found upon dyeing [72],... 

The nitrile group is a versatile building block, in particular since it can be converted into acids or amides. It undergoes hydrolysis but requires relatively harsh reaction conditions. Nature provides two enzymatic pathways for the hydrolysis of nitriles. The nitrilases convert nitriles directly into acids, while the nitrile hy-dratases release amides. These amides can then be hydrolysed by amidases (see also above). Often nitrile hydratases are combined with amidases in one host and a nitrile hydratase plus amidase activity can therefore be mistaken as the activity of a nitrilase (Scheme 6.32). A large variety of different nitrilases and nitrile hydratases are available [100, 101] and both types of enzyme have been used in industry [34, 38, 94]. 

Glycolic acid can be produced via fermentation process [6] from glycolo-nitrile hydrolysis by mineral acid, such as sulfuric acid [7,8]. Both processes produce a multi-component solution with the acid concentration typically less than 10 wt% for fermentation technology, and less than 40 wt% for glycolo-nitrile hydrolysate. The acid can be produced by the enzymatic conversion (typically the enzyme catalyst used is nitrilase or a combination of a nitrile hydratase and an amidase) of glycolonitrile which results in the production of an aqueous solution of ammonium glycolate [9]. 

In 2003, Griengl s group reported the hydrolysis of cyanohydrins by treatment with bacterial cells of Rhodococcus erythropolis NCIMB 11540, which have a highly active nitrile hydratase/amidase enzyme system. In this manner, (R)-2-chloromandelic acid and (R)-2-hydroxy-4-phenylbutyric acid, two important pharmaceutical intermediates, could be prepared in high optical and chemical yield after short reaction times (3 and 1.5 h, respectively) (Scheme 3.43). 

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". ... 

Evidence for the enantioselectivity of the nitrile hydratase was given by the purified enzyme catalyzing the hydration of the (5)-nitrile at least 50 times faster than the hydrolysis of the (i )-nitrile [51]. The strain was also capable of a two-step hydrolysis of racemic ibuprofen and naproxen nitriles to the corresponding (S)-acids in enantiomeric purities above 90% e.e., however, with the stereoselectivity residing primarily in the amidase. In this case, the analysis of enantioselectivity was complicated due to the product inhibition of (i )-nitrile hydration by enzymatically formed (5)-amide. On the basis of the initial rate of appearance, the nitrile hydratase showed a slight preference for the (R) enantiomers of... 

Amidases catalyze the second step in die two-enzyme step conversion of nitriles to the corresponding acids, the hydrolysis of amides. Among stereoselective nitrile-converting enzymes, the highest enantioselectivities were found in amidases. Amidases have been mostly characterized as thiol-dependent homodimers (aa) sharing amino acid sequence homologies with other amidases. The functional relationship between nitrile hydratases and amidases is structurally documented by the close proximity of their genes [71,72]. In this chapter, stereospecific amidases alone or with nonselective nitrile hydratases are described. 

Besides a highly active nitrile hydratase employed in the industrial production of acrylamide and nicotinamide [7-10] and a nitrilase [14,20-22], R. rhodochrous J1 contains an amidase with a high (5)-specificity in the hydrolysis of 2-phenylpropionamide. The corresponding gene was cloned and overexpressed in E. coli [71,72]. The recombinant enzyme was used for the preparation of (5)-2-phenylpropionic acid with high enantiomeric purity (Fig. 24) but could not recognize the configuration of 2-chloropropionitrile presumably due to the requirement of a bulky moiety for enantioselectivity. 

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