Acrylamide nitrile hydratases

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. 

Nitrile hydratase (NHase) catalyzes the hydration of nitriles to amides (Figure 1.11) and has been used for production of acrylamide and nicotinamide at large scale. NHases are roughly... 

Nagasawa, T., Shimizu, H. and Yamada, H. (1993) The superiority of the third-generation catalyst, Rhodococcus rhodochrous J1 nitrile hydratase, for industrial production of acrylamide. AppliedMircobiology and Biotechnology, 40, 189-195. 

The significance of the application of immobilized-cell technology in the production of industrially important chemicals is exemplified by the production of acrylamide by immobilized Escherichia coli cells containing nitrile hydratase. The immobilized Escherichia coli cells convert acrylonitrile to acrylamide, yielding 6000 tons of acrylamide per year by this process [28]. 

Yamada, H. and Kobayashi, M. (1996) Nitrile hydratase andits application to industrial production of acrylamide. Bioscience, Biotechnology, and Biochemistry, 60, 1391-1400. 

The microorganism used has a high endogenous nitrile hydratase ratio when urea was used as an inducer in the presence of cobalt ions. (The nitrilase is undesirable as it converts the acrylamide further into acrylic acid). 

Pseudomonas chloraphis cells were used first, and more recently Rhodococcus rhodochrus Jl. Cells are immobilised in polyacrylamide particles and used in column reactors operated at below 10°C. The acrylamide is produced in 100% yield, and is so pure that polymerisation inhibitors have to be added to prevent spontaneous polymerisation. Both acrylonitrile and acrylamide inhibit the nitrile hydratase the nitrile hydratase is extremely stable. Therefore acrylonitrile is fed to maintain a level of 6% resulting in the accumulation of acrylamide of 66% (w/v), after which is it simply decolourised and concentrated (Yamada and Kobayashi, 1996). 

In basic chemicals, nitrile hydratase and nitrilases have been most successful. Acrylamide from acrylonitrile is now a 30 000 tpy process. In a product tree starting from the addition of HCN to butadiene, nicotinamide (from 3-cyanopyridine, for animal feed), 5-cyanovaleramide (from adiponitrile, for herbicide precursor), and 4-cyanopentanoic acid (from 2-methylglutaronitrile, for l,5-dimethyl-2-piperidone solvent) have been developed. Both the enantioselective addition of HCN to aldehydes with oxynitrilase and the dihydroxylation of substituted benzenes with toluene (or naphthalene) dioxygenase, which are far superior to chemical routes, open up pathways to amino and hydroxy acids, amino alcohols, and diamines in the first case and alkaloids, prostaglandins, and carbohydrate derivatives in the second case. 

Nitrile Hydratase Acrylamide from Acrylonitrile, Nicotinamide from 3-Cyanopyridine, and 5-Cyanovaleramide from Adiponitrile... 

Acrylamide from Acrylonitrile with Nitrile Hydratase... 

One of the best examples for discussing biotransformations in neat solvents is the enzymatic hydrolysis of acrylonitrile, a solvent, to acrylamide, covered in Chapter 7, Section 7.1.1.1. For several applications of acrylamide, such as polymerization to polyacrylamide, very pure monomer is required, essentially free from anions and metals, which is difficult to obtain through conventional routes. In Hideaki Yamada s group (Kyoto University, Kyoto, Japan), an enzymatic process based on a nitrile hydratase was developed which is currently run on a commercial scale at around 30 000-40 000 tpy with resting cells of third-generation biocatalyst from Rhodococcus rhodochrous J1 (Chapter 7, Figure 7.1). 

Although biocatalysis is the new kid on the block, more and more companies are using enzymes for chemical manufacture. One reason for this is that biocatalysts give sustainable alternatives to chemical manufacture, and not just for making chiral products. The synthesis of acrylamide via an enzyme-catalyzed water addition to acrylonitrile (2-propenenitrile) is a classic example (Figure 1.15). It uses the Rhodo-coccus enzyme nitrile hydratase. Commercialized in 1985 by Nitto Chemicals in... 

Figure 5.24 Unlike the chemical route, the biocatalytic hydrolysis of acrylonitrile to acrylamide is highly selective, owing to the specific function of the nitrile hydratase enzyme.

Nitto Chemical (now Dia-Nitrix) introduced a biosynthetic route from ACRN to acrylamide in Japan in 1985. This process uses an immobilized nitrile hydratase biocatalyst that converts the ACRN solution to acrylamide with a yield of 99.5%. This high yield allows a concentrated acrylamide solution to be made without the need for ACRN recycle or solution concentration. This process therefore has lower energy costs282. The initial plant capacity was 4,000 tonnes per year it was expanded to 20,000 tonnes per year in 1991 and that is its capacity as of 2002. 

Yamada H, Kobayashi M. Nitrile hydratase and its application to industrial production of acrylamide. Biosci Biotechnol Biochem 1996 60 1391 100. 

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... 

Another example in which a biocatalytic transformation has replaced a chemo-catalytic one, in a very simple reaction, is the Mitsubishi Rayon process for the production of acrylamide by hydration of acrylonitrile (Fig. 1.42). Whole cells of Rhodococcus rhodocrous, containing a nitrile hydratase, produced acrylamide in >99.9% purity at >99.9% conversion, and in high volumetric and space time yields [121]. The process (Fig. 1.42) currently accounts for more than 100000 tons annual production of acrylamide and replaced an existing process which employed a copper catalyst. A major advantage of the biocatalytic process is the high product purity, which is important for the main application of acrylamide as a specialty monomer. 

Scheme 6.37 Nitrile hydratase catalyses the selective formation of acrylamide.

Padmakumar, R. and Oriel, P. 1999. Bioconversion of acrylonitrile to acrylamide using a thermostable nitrile hydratase. Applied Biochemistry and Biotechnology, 79 671-9. 

---