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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.

Fungal β-N-acetylhexosaminidases are inducible extracellular enzymes with many biotechnological applications. The enzyme from Penicillium oxalicum has unique enzymatic properties despite its close evolutionary relationship with other fungal hexosaminidases. It has high GalNAcase activity, tolerates substrates with the modified N-acyl group better and has some other unusual catalytic properties. In order to understand these features, we performed isolation, biochemical and enzymological characterization, molecular cloning and molecular modelling. The native enzyme is composed of two catalytic units (65 kDa each) and two propeptides (15 kDa each), yielding a molecular weight of 160 kDa. Enzyme deglycosylated by endoglycosidase H had comparable activity, but reduced stability. We have cloned and sequenced the gene coding for the entire hexosaminidase from P. oxalicum. Sufficient sequence identity of this hexosaminidase with the structurally solved enzymes from bacteria and humans with complete conservation of all catalytic residues allowed us to construct a molecular model of the enzyme. Results from molecular dynamics simulations and substrate docking supported the experimental kinetic and substrate specificity data and provided a molecular explanation for why the hexosaminidase from P. oxalicum is unique among the family of fungal hexosaminidases., (© 2011 The Authors Journal compilation © 2011 FEBS.)

Purpose of Work: our aim is to describe new fungal nitrilases whose sequences were published but whose catalytic properties were unknown. We adapted for expression in E. coli three of the genes and confirmed that the enzymes acted on organic nitriles. The genome mining approach was used to search for nitrilases in filamentous fungi. Synthetic genes encoding nitrilases in Aspergillus niger, Gibberella moniliformis and Neurospora crassa were expressed in Escherichia coli. This is the first heterologous expression of fungal enzymes of this type. The recombinant enzyme derived from G. moniliformis was an aromatic nitrilase with an activity of 390 U l(-1) culture with benzonitrile as substrate. This was much less than the activities of the recombinant enzymes derived from A. niger and N. crassa that had activities of 2500 and 2700 U l(-1) culture, respectively, with phenylacetonitrile as substrate.