Rhodococcus rhodochrous nitrile hydratase

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. 

A potential versatile route into a-amino acids and their derivatives is via a combination of (i) nitrile hydratase/amidase-mediated conversion of substituted malo-nonitriles to the corresponding amide/acid followed by (ii) stereospecific Hofmann rearrangement of the amide group to the corresponding amine. Using a series of a,a-disubstituted malononitriles 14, cyanocarboxamides 15 and bis-carboxamides 16, the substrate specificity of the nitrile hydratase and amidase from Rhodococcus rhodochrous IF015564 was initially examined (Scheme 2.7). The amidase hydrolyzed the diamide 16 to produce (R)-17 with 95% conversion and 98%e.e. Amide 17 was then chemically converted to a precursor of (S)-a-methyldopa. It was found... 

Tauber MM, Cavaco-Paulo A, Robra K-H et al (2000) Nitrile hydratase and amidase from Rhodococcus rhodochrous hydrolyse acrylic fibers and granulates. Appl Environ Microbiol 66 1634-1638... 

Nagasawa, T., Takeuchi, K., Nardi-Dei, V., Mihara, Y. and Yamada, H. (1991) Optimum culture conditions for the production of cobalt-containing nitrile hydratase by Rhodococcus rhodochrous l.Appl. Microbiol. Biotechnol., 34, 783-788. 

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). 

Utilising an overexpressed nitrile hydratase from Rhodococcus rhodochrous in immobilised cells 30000 tons per year are produced by Mitsubishi Rayon Co. Ltd. The selectivity of the nitrile hydratase is 99.99% for the amide, virtually no acrylic acid is detected and since all starting material is converted yields are excellent (>99%). The time space yield is 2 kg product per liter per day (Scheme 6.37). This clearly demonstrates the power of biocatalysis. 

As mentioned in Chapter 1 the same Rhodococcus rhodochrous catalyses the last step in Lonza s >3500 tonnes/year nicotinamide synthesis [94, 111, 112]. Lonza has further developed this technology and currently synthesises a number of relevant fine chemical building blocks with nitrile hydratases [94, 113]. 

Rhodococcus rhodochrous was used for the hydrolysis of both granular PAN and acrylic fibers by nitrile hydratase and amidase (Tauber et al., 2000). Similarly, Agrobacterium tumefaciens (BST05) was found to convert polyacylonitrile into polyacrylic acid by nitrile hydratase and amidase (Fischer-Colbrie et al., 2006). Nitrilase was also used for the surface hydrolysis of PAN from Micrococcus luteus BST20 (Fischer-Colbrie et al., 2007). However, polyamidase from Nocardia farci-nica leads to an increase of polar groups on the surface of PA, which was measured by tensiometry (Heumann et al., 2009). 

Nagasawa, T., Mathew, C.D., Manger, 1., et al. 1988. Nitrile hydratase-catalysed prodnction of nicotinamide from 3-cyanopyridine in Rhodococcus rhodochrous 11. Applied and Environmental Microbiology, 54 1766-9. 

Wieser, M., Takeuchi, K., Wada, Y. et al. 1998. Low-molecular-mass nitrile hydratase from Rhodococcus rhodochrous JI Purification, substrate specificity and comparison with the analogous high molecular-mass enzyme. FEMS Microbiology Letters, 169 17-22. 

J. Mauger, T. Nagasawa, and H. Yamada, Synthesis of various aromatic amide derivatives using nitrile hydratase of Rhodococcus rhodochrous Jl, Tetrahedron, 45 1347 (1989). 

A remarkable example of a lyase-catalyzed commercial process, developed by the Japanese company Nitto and operative since 1985, is the biocatalytic production of acrylamide using immobilized whole cells of Rhodococcus rhodochrous [17]. The enzyme responsible for water addition to the double bond of acrylonitrile is nitrile hydratase ... 

Acrylamide (Ch) Nicotinamide (H) Nitrile hydratase Rhodococcus rhodochrous... 

Current acrylamide production at Mitsubishi using bioconversion is around 40 000 tonnes per year. Using a highly improved cobalt-containing nitrile hydratase from Rhodococcus rhodochrous Jl, final product concentrations of around 700 g L"1... 

With both these strains, the enzymatic system is composed of nitrile hydratases and amidases. The nitrile hydratase gene of Brevibacterium R312 is cloned, sequenced (ref. 17) and over expressed in Rhodococcus rhodochrous ATCC12674 (pKRNH2) (ref. 18). The best selectivity which can be hoped for with this nitrile hydratase is 93 % (ref. 4). Moreover, the cyanovaleramide with its poor solubility must not be accumulated and requires a biocatalyst with a superactivated amidase activity. The nitrile hydratase is less stable than amidase and the biocatalyst with these two enzymes would not be sufficiently robust for an industrial application. 

Production of acrylamide (Fig. 13) by hydration of acrylonitrile under the action of the intracelluar nitrile hydratase in Rhodococcus rhodochrous (Nitto Chemical Industry Co., Ltd., fed-batch process). The annual production amounts to >30000 tons (see also Table 6). Acrylamide is one of the most important commodity chemicals and is required in large quantities as the pre-polymer of polyacrylamide that is widely used in polymer and floccu-lent applications. The advantages of this hydratase approach in comparison with the classical chemical nitrile hydration are higher product end concentration, quantitative yields, no formation of acrylic acid, no need for copper catalyst, and only five chemical/technical operations instead of seven [73,112,113,171]. An analogous process for nicotinamide is being commercialized by Lonza (see also section 6). 

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. 

Acrylamide is the first bulk chemical manufactured using an industrial biotransformation. Acrylamide which is produced 200000 t/a is an important industrial chemical that is mainly processed into water-soluble polymers and copolymers, which find applications as flocculants, paper-making aids, thickening agents, surface coatings, and additives for enhanced oil recovery. The chemical manufacture of acrylamide has been established for a long time, it is based on Cu-catalysis. The production of acrylamide using immobilized whole cells of Rhodococcus rhodochrous is a remarkable example of a lyase-catalyzed commercial process. The enzyme responsible for water addition to the double bond of acrylonitrile is nitrile hydratase (Eq. 4-17) ... 

Catalyst A copper salt the enzyme nitrile hydratase in whole cells of Rhodococcus rhodochrous, immobilized on polyfpropenamide) gel... 

The production of amides from nitriles has been studied by several workers, and most of them focused on the accumulation of acetamide from acetonitrile [126,133-136]. The enz3nnatic production of acrylamide from acrylonitrile by nitrile hydratase of P. chlororaphis B23, Rhodococcus sp. N-774, and Klebsiella pneumoniae, respectively has been reported [137-142]. These microorganisms exhibited a high nitrile hydratase activity and a low amidase activity, allowing the accumulation of the corresponding amide. Nagasawa et al. optimized the reaction conditions for the production of nicotinamide by a nitrile hydratase, found in Rhodococcus rhodochrous Jl. The enzyme contains cobalt, and shows high activity towards 3-cyanopyridine [143,144]. 

Company in Japan now uses a nitrile hydratase from Rhodococcus rhodochrous to hydrolyse acrylonitrile to acrylamide. The established... 

Desymmetrization of Prochiral Dinitriles. Prochiral a,a-disubstituted malono-nitriles can be hydrolyzed in an asymmetric manner by the aid of Rhodococcus rhodochrous [678] (Scheme 2.106). In accordance with the above-mentioned trend, the dinitrile was nonselectively hydrolyzed by the nitrile hydratase in the cells to give the dicarboxamide. In a second consecutive step, the latter was subsequently transformed by the amidase with high selectivity for the pro-(/ ) amide group to yield the (/ )-amide-acid in 96% e.e. and 92% yield. This pathway was confirmed by the fact that identical results were obtained when the dicarboxamide was used as substrate. The nonracemic amide-acid product thus obtained serves as a starting material for the synthesis of nonnatural a-methyl-a-amino acids [679]. 

---