Biocatalytic potetial of degrading enzyme systems belonging to the genus rhodococcus rhodochrous, new approaches towards green chemistry

Abstract

In an attempt to find more efficient and "greener" catalytic processes towards the synthesis of advanced pharmaceutical intermediates, three bacterial strains, namely Rhodococcus rhodochrous ATCC BAA-870, R. rhodochrous A29 and Pimelobacter simplex A99, were evaluated as nitrile hydrolysing biocatalysts. R. rhodochrous ATCC BAA-870 showed constitutive expression of a low-molecular weight cobalt containing nitrile hydratase and a highly enantioselective amidase enzyme that were involved in the kinetic resolution of 3-hydroxy-3-aryloxybutanenitrile and 3-hydroxy-3-arylpropanenitrile. The (R)-3- hydroxy-3-aryloxybutanamide and (S)-3-hydroxy-3-arylpropanamide intermediates were both accumulated with excellent enantioselectivity (>99% ee). Interestingly, the same cells demonstrated enantioselective resolution of β-substituted aminonitriles to amides through a moderately selective nitrile hydratase enzyme when adjusted to a higher pH. With the substrates 3-amino-3-(4-methoxyphenyl)propanenitrile, and 3-amino-3-p-tolylpropanenitrile an enantiomeric purity of the residual nitrile (85% ee and 21% ee respectively) was achieved; while the resultant amide was obtained with 62% ee (3-amino-3-(4-methoxyphenyl)propanamide) and 48% ee (3-amino-3-p-tolylpropanenitrile) respectively. Induction studies demonstrated that both nitrile hydratase and amidase expression in strains A29 and A99 were inducible, with transient expression observed when cultured in the presence of benzonitrile. Furthermore, in a noteworthy discovery, studies on strains ATCC BAA-870, A29 and A99 revealed that the incorporation of 0.5% (v/v) dimethylformamide during cultivation induced nitrilase gene expression. This was evident by the appearance of a new, dominant 40 kDa protein band observed by SDS-PAGE analysis, and confirmed by LCMS-MS sequencing (showing high similarity to Uniprot Q03217). The induced nitrilase in strains A29 and A99 was inhibited by the nitrilase inhibitors benzylamine (61.37% and 79.49%) and benzaldehyde (88.45% and 87.49%) respectively. ATCC BAA-870 cells were however unaffected due to the presence of the alternative nitrile hydratase and amidase system facilitating nitrile hydrolysis. The induced nitrilases converted 3-cyanopyridine to nicotinic acid, and showed excellent enantioselectivity towards 3-amino-3-phenylpropanenitrile (>99% ee), and moderate enantioselectivity towards 3-amino-3-(4-methoxyphenyl)propanenitrile (40% ee). Therefore, Rhodococcal species are capable of expressing various nitrile hydrolysing enzymes that are enantioselective towards classes of beta-substituted nitrile compounds representative of diverse pharmaceuticals such as beta blockers, statins and peptidomimetics. The activity profile of these versatile microbial biocatalyst can be tailored through induction to yield single enantiomer nitriles, carboxamides or carboxyacids.