H J Knackmuss

Enrichment strategies for nitrile-hydrolysing bacteria

Abstract A series of enrichments with different nitriles as sole source of nitrogen was performed in order to obtain a relationship between the selective nitrogen source and (i) the enzyme systems that are synthesized by the isolates and (ii) the enzyme specificities for the utilization of the nitriles. Bacteria were enriched with 2-phenylpropionitrile, 2-(2-methoxyphenyl)propionitrile, 2-phenylbutyronitrile, ibuprofen nitrile, naproxen nitrile, ketoprofen nitrile, ketoprofen amide, benzonitrile, or naphthalenecarbonitrile as sole nitrogen source and succinate as sole source of carbon and energy. 2-Phenylpropionitrile as nitrogen source resulted predominantly in the enrichment of gram-negative bacteria, which harboured nitrilase and in some cases also amidase activity. In contrast, with the other nitriles used, a substantial majority of gram-positive strains, mainly of the genus Rhodococcus, were isolated. These strains contained predominantly a nitrile hydratase/amidase system. The nitrilases and nitrile hydratases showed R or S selectivity with generally poor optical yields. In contrast, the amidases were almost exclusively S-selective, often forming the optically pure acids with an enantiomeric excess above 99%. The conversion of different nitriles by the isolates was compared. The nitrile-hydrolysing systems of the new isolates usually showed high activity against those nitriles that were used for the enrichment of the bacteria.

Enantioselective hydration of 2-arylpropionitriles by a nitrile hydratase from Agrobacterium tumefaciens strain d3

Abstract The enantioselective nitrile hydratase from the bacterium Agrobacterium tumefaciens d3 was purified and completely separated from the amidase activity that is also present in cell extracts prepared from this strain. The nitrile hydratase had an activity optimum at pH 7.0 and a temperature optimum of 40 °C. The holoenzyme had a molecular mass of 69 kDa, the subunits a molecular mass of 27 kDa. The enzyme hydrated various 2-arylpropionitriles and other aromatic and heterocyclic nitriles. With racemic 2-phenylpropionitrile, 2-phenylbutyronitrile, 2-(4-chlorophenyl)propionitrile, 2-(4-methoxy)propionitrile or ketoprofen nitrile the corresponding (S)-amides were formed enantioselectively. The highest enantiomeric excesses (ee >90% until about 30% of the respective substrates were converted) were found for the amides formed from 2-phenylpropionitrile, 2-phenylbutyronitrile and ketoprofen nitrile. For the reaction of the purified nitrile hydratase, higher ee values were found than when whole cells were used in the presence of an inhibitor of the amidase activity. The enantioselectivity of the whole-cell reaction was enhanced by increasing the reaction temperature.