title: Purification and characterization of heterologously expressed nitrilases from filamentous fungi.
abstract: 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 U L(-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 (K(m) of 0.41 mM, V(max) of 9.7 μmol min(-1) mg(-1) protein). The P. marneffei enzyme (unstable in its purified state) exhibited the highest V(max) of 7.3 μmol min(-1) mg(-1) protein in cell-free extract, but also a high K(m) of 15.4 mM, for 4-cyanopyridine. The purified nitrilases from A. niger CBS 513.88 and N. crassa acted preferentially on phenylacetonitrile (K(m) of 3.4 and 2.0 mM, respectively; V(max) of 10.6 and 17.5 μmol min(-1) mg(-1) protein, respectively), and hydrolyzed also (R,S)-mandelonitrile with higher K(m) values. Significant amounts of amides were only formed by the G. moniliformis nitrilase from phenylacetonitrile and 4-cyanopyridine.
authors: Institute of Microbiology, Centre of Biocatalysis and Biotransformation, Prague, Czech Republic.
authors: Alena
authors: A
authors: Petříčková
authors: Alicja Barbara
authors: AB
authors: Veselá
authors: Ondřej
authors: O
authors: Kaplan
authors: David
authors: D
authors: Kubáč
authors: Bronislava
authors: B
authors: Uhnáková
authors: Anna
authors: A
authors: Malandra
authors: Jürgen
authors: J
authors: Felsberg
authors: Anna
authors: A
authors: Rinágelová
authors: Philip
authors: P
authors: Weyrauch
authors: Vladimír
authors: V
authors: Křen
authors: Karel
authors: K
authors: Bezouška
authors: Ludmila
authors: L
authors: Martínková
mesh_terms: D000619:Aminohydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D005658:Fungi / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D000619:Aminohydrolases; C021867:nitrilase