Purification and characterization of heterologously expressed nitrilases from filamentous fungi
作者： Alena Petříčková , Alicja Barbara Veselá , Ondřej Kaplan , David Kubáč , Bronislava Uhnáková
DOI: 10.1007/S00253-011-3525-7
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摘要: Nitrilases from Aspergillus niger CBS 513.88, A. K10, Gibberella moniliformis, Neurospora crassa OR74A, and Penicillium marneffei ATCC 18224 were expressed in Escherichia coli BL21-Gold (DE3) after IPTG induction. N. nitrilase exhibited the highest yield of 69,000 U L−1 culture. Co-expression chaperones (GroEL/ES G. moniliformis P. marneffei; GroEL/ES trigger factor 513.88) enhanced enzyme solubility. Specific activities strains expressing former two enzymes increased approximately fourfold upon co-expression GroEL/ES. The (co-purified with GroEL) preferred benzonitrile as substrate (Km 0.41 mM, Vmax 9.7 μmol min−1 mg−1 protein). (unstable its purified state) 7.3 protein cell-free extract, but also a high Km 15.4 for 4-cyanopyridine. nitrilases 513.88 acted preferentially on phenylacetonitrile 3.4 2.0 respectively; 10.6 17.5 protein, respectively), hydrolyzed (R,S)-mandelonitrile higher values. Significant amounts amides only formed by
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