Abstrakt
Nitrilases from Aspergillus niger CBS 513.88, A. niger K10, Gibberella moniliformis, Neurospora crassa OR74A, and Penicillium marneffei ATCC 18224 were expressed in Escherichia coli BL21-Gold (DE3) after IPTG induction. N. crassa nitrilase exhibited the highest yield of 69,000 ULMINUS SIGN 1 culture.
Co-expression of chaperones (GroEL/ES in G. moniliformis and P. marneffei; GroEL/ ES and trigger factor in N. crassa and A. niger CBS 513.88) enhanced the enzyme solubility. Specific activities of strains expressing the former two enzymes increased approximately fourfold upon co-expression of GroEL/ES.
The enzyme from G. moniliformis (co-purified with GroEL) preferred benzonitrile as substrate (Km of 0.41 mM, Vmax of 9.7 μmol minMINUS SIGN 1 mgMINUS SIGN 1 protein). The P. marneffei enzyme (unstable in its purified state) exhibited the highest Vmax of 7.3 μmol minMINUS SIGN 1 mgMINUS SIGN 1 protein in cellfree extract, but also a high Km of 15.4 mM, for 4- cyanopyridine.
The purified nitrilases from A. niger CBS 513.88 and N. crassa acted preferentially on phenylacetonitrile (Km of 3.4 and 2.0 mM, respectively; Vmax of 10.6 and 17.5 μmol minMINUS SIGN 1 mgMINUS SIGN 1 protein, respectively), and hydrolyzed also (R,S)-mandelonitrile with higher Km values. Significant amounts of amides were only formed by the G.
Moniliformis nitrilase from phenylacetonitrile and 4-cyanopyridine.