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Characterisation of the substrate specificity of the nitrile hydrolyzing system of the acidotolerant black yeast Exophiala oligosperma R1
The `black yeast' Exophiala oligosperma R1 can utilise various
organic nitriles under acidic conditions as nitrogen sources. The induction of
a phenylacetonitrile converting activity was optimised by growing the strain
in the presence of different nitriles and /or complex or inorganic nitrogen
sources. The highest nitrile hydrolysing activity was observed with cells
grown with 2-cyanopyridine and NaNO3. The cells metabolised the
inducer and grew with 2-cyanopyridine as sole source of nitrogen. Cell
extracts converted various (substituted) benzonitriles and
phenylacetonitriles. They usually converted the isomers carrying a substituent
in the meta-position with higher relative activities than the
corresponding para- or ortho-substituted isomers. Aliphatic
substrates such as acrylonitrile and 2-hydroxy-3-butenenitrile were also
hydrolysed. The highest specific activity was detected with 4-cyanopyridine.
Most nitriles were almost exclusively converted to the corresponding acids and
no or only low amounts of the corresponding amides were formed. The cells
hydrolysed amides only with extremely low activities. It was therefore
concluded that the cells harboured a nitrilase activity. The specific
activities of whole cells and cell extracts were compared for different
nitriles and evidence obtained for limitation in the substrate-uptake by whole
cells. The conversion of 2-hydroxy-3-butenenitrile to 2-hydroxy-3-butenoic
acid at pH 4 demonstrated the unique ability of cells of E.
oligosperma R1 to hydrolyse aliphatic α-hydroxynitriles under
acidic conditions. The organism could grow with phenylacetonitrile as sole
source of carbon, energy and nitrogen. The degradation of phenylacetonitrile
presumably proceeds via phenylacetic acid, 2-hydroxyphenylacetic acid,
2,5-dihydroxyphenylacetic acid (homogentisate), maleylacetoacetate and
fumarylacetoacetate