Enzyme catalysed hydrolysis of nitriles and amides.
Described in this thesis is the isolation of two microorganisms containing a nitrile
hydratase and amidase to effect the enantioselective hydrolysis of a-substituted
nitriles to their corresponding amides and acids. Isolate NP3854 was identified as an
atypical Rhodococcus sp. The nitrile hydratase proved to be non-selective for all the
substrates tested. However, carboxylic acids with excellent enantiomeric excess were
obtained from a large number of amides.
nitr-il-e--h-y-d-r.a~tase ~X amidas.e RH CONH2
Optically active acids with an enantiomeric excess of, generally, >98 %, were
obtained when X = NH2, Me and Cl, but proved to be racemic for OH and Br.
R could be a variety of aromatic, cyclic and acyclic alkyl residues without adversely
affecting the enantioselectivity.
The pH-activity profile was determined for the amidase of NP3854 using
propionamide as the substrate. From this data, coupled with inhibition studies, it may
,.tentatively be suggested that the amidase has a histidine residue in the active site,
which may act as a general base for a serine amino acid. The pH-activity profile was
determined for 2-amino-2-phenylacetamide 2b, and this suggested that the
unprotonated form of the amine acted as the substrate. Within a pH range of 3 - 9 the
enantiomeric excess remains high (>98 %) and experimentally invariant.
The amidase was found to have a temperature optimum of 60°C and could tolerate 20
% THF with a loss of only 15 % activity.
Attempts made to hydrolyse 4,5,6-amino nitriles and amides to the corresponding
amino acids and isolate any reaction intermediates failed. This was presumed to be
due to the large fraction of the unprotonated amine due to the higher pKa (- 9 - 10).