Nitriles nitrile-degrading enzyme

Aldoximes are prepared from aldehydes and hydroxylamine by condensation reaction, and the dehydration reaction of aldoxime is one of the most important methods of nitrile synthesis in organic chemistry." We speculated that it would become one of the most important examples in Green Chemistry if the dehydration reaction could be realized by an enzymatic method, and started studies on a new enzyme, aldoxime dehydratase, and its use in enzymatic nitrile synthesis. Furthermore, we clarified the relationship between aldoxime dehydratase and nitrile-degrading enzymes in the genome of the microorganisms and the physiological role of the enzyme. 

We also found that the occurrence of aldoxime dehydratase is as wide as that for nitrile-degrading enzymes such as nitrile hydratase, amidase and/or nitri-lase. All of the nitrile degraders hitherto isolated contained aldoxime dehydratase activities. The author would like to propose that the pathway in which aldoximes are successively degraded via nitrile could be named as the aldoxime-nitrile pathway (Fig. 1). 

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. 

Banerjee, A., Sharma, R. and Banerjee, U.C. (2002) The nitrile-degrading enzymes current status and future prospects. Applied Microbiology and Biotechnology, 60, 33 14. 

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 . 

DISTRIBUTION AND PHYSIOLOGICAL ROLE OF NITRILE-DEGRADING ENZYME SYSTEMS... 

The role of ferrous ions in modifying the enzymatic degradation of glucolimnanthin is not completely understood. It is known that species of Brassica produce the so-called nitrile-forming enzymes, such as the epispecifier protein, that require ferrous ions at micromolar concentrations [18]. When we incubated... 

Nitrile hydratase was initially discovered by Yamada and co-workers of our laboratory [46]. The enzyme that catalyzes the conversion of nitriles into amides is clearly distinguishable from nitrilase in the mode of nitrile-degradation. Thus, the enzyme was termed nitrile hydratase (NHase) . This enzyme was purified and characterized from various micro-organisms. NHases are roughly classified into two groups according to the metal involved Fe-type and Co-type [45],... 

The NHase responsible for aldoxime metabolism from the i -pyridine-3-aldoxime-degrading bacterium, Rhodococcus sp. strain YH3-3, was purified and characterized. Addition of cobalt ion was necessary for the formation of enzyme. The native enzyme had a Mr of 130000 and consisted of two subunits (a-subunit, 27 100 (3-subunit, 34500). The enzyme contained approximately 2 mol cobalt per mol enzyme. The enzyme had a wide substrate specificity it acted on aliphatic saturated and unsaturated as well as aromatic nitriles. The N-terminus of the (3-subunit showed good sequence similarities with those of other NHases. Thus, this NHase is part of the metabolic pathway for aldoximes in microorganisms. 

As described above, the procedure and some of our examples of successful isolation of microorganisms having new enzymes were briefly described. Furthermore, historic changes of the way and methods of the research on the degradation of nitrile compounds and the exploitation of some industrially important enzymes including NHase, etc., were introduced. 

The discovery of a Bacillus sp. strain capable of degrading aldoximes via their conversion to nitriles prompted the isolation and purification of an enzyme capable of producing the syn geometrical isomer of phenylacetaldoxime from A-hydroxy-L-phenylalanine, suggesting that amino acid-derived aldoximes are biosynthesized and metabolized in microorganisms like in plants ". 

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