Vojtěch Vejvoda

Fungal nitrilases as biocatalysts: Recent developments.

Of the numerous putative fungal nitrilases available from protein databases only a few enzymes were purified and characterized. The purified nitrilases from Fusarium solani, Fusarium oxysporum f. sp. melonis and Aspergillus niger share a preference for (hetero)aromatic nitriles, temperature optima between 40 and 50 degrees C and pH optima in the slightly alkaline region. On the other hand, they differ in their chemoselectivity, i.e. their tendency to produce amides as by-products. The production of fungal nitrilases is increased by up to three orders of magnitude on the addition of 2-cyanopyridine to the culture media. The whole-cell and subcellular biocatalysts were immobilized by various methods (LentiKats(R); adsorption on hydrophobic or ion exchange resins; cross-linked enzyme aggregates). Operational stability was examined using continuous stirred membrane bioreactors. Fungal nitrilases appear promising for biocatalytic applications and biodegradation of nitrile environmental contaminants.

Hyperinduction of nitrilases in filamentous fungi.

Abstract2-Cyanopyridine proved to act as a powerful nitrilase inducer in Aspergillusniger K10, Fusarium solani O1, Fusarium oxysporum CCF 1414, Fusarium oxysporum CCF 483 and Penicillium multicolor CCF 2244. Valeronitrile also enhanced the nitrilase activity in most of the strains. The highest nitrilase activities were produced by fungi cultivated in a Czapek-Dox medium with both 2-cyanopyridine and valeronitrile. The specific nitrilase activities of these cultures were two to three orders of magnitude higher than those of cultures grown on other nitriles such as 3-cyanopyridine or 4-cyanopyridine.

Immobilization of fungal nitrilase and bacterial amidase – two enzymes working in accord

A nitrilase from Aspergillus niger and an amidase from Rhodococcus erythropolis co-immobilized on a 1-mL Butyl Sepharose column were used for the hydrolysis of 4-cyanopyridine into isonicotinic acid. The former enzyme converted the nitrile into the acid:amide mixture (molar ratio ca. 3:1), while the latter enzyme hydrolyzed the amide by-product. Therefore, the ratio of amide in the total product decreased to about 5%. Sodium sulfate was used as a component of the elution buffer, as the commonly used ammonium sulfate (0.8 M) acted as an amidase inhibitor. The hydrolysis of 4-cyanopyridine by a nitrilase from F. solani gave isonicotinic acid and isonicotinamide at a molar ratio of about 98:2. When using this enzyme and the amidase immobilized on two columns operated in tandem, the percentage of isonicotinamide in total product decreased to <0.2%.

Erratum to: Purification and characterization of a nitrilase from Aspergillus niger K10

Erratumto:ApplMicrobiolBiotechnol(2006)73:567–575DOI 10.1007/s00253-006-0503-6The previous article reported on the biochemical characteriza-tion of a nitrilase purified from Aspergillus niger K10. Theamino acid sequence of this enzyme was recently analyzed bymassspectroscopywhichreveale dthattheN-terminalsequencereported in Fig. 3A (by KB) in the previous article was incor-rect. This N-terminal sequence (XAPVLKKYKAAXVNXE),which was highly homologous to those of a number of hypo-thetical proteins in genus Aspergillus (Aspergillus fumigatusAf29, Aspergillus oryzae, Aspergillus nidulans FGSC A4) didnot belong to the enzyme purified and characterized in theprevious article. Mass spectrum analyses of this enzyme wererecently performed as follows. Briefly, the peptides wereextracted after in gel digestion of the enzyme withtrypsin and analyzed by MALDI-ToF MS using BrukerBiflex IV (Bruker Daltonics, Germany). Alternatively,the peptides were analyzed by using UHPLC DionexUltimate3000 RSLC nano (Dionex, Germany) equippedwith a ESI-Q-ToF Maxis Impact (Bruker Daltonics,Germany) mass spectrometer. Spectra were interpreted us-ing Mascot software (Matrix Science, UK). These analyses(Fig. S1) suggested a 42.5-58.1 % sequence coverage ofthe enzyme with a putative nitrilase from Aspergilluskawachii IFO 4308 (gi|358373570) having N-terminalsequence MSHDGPKTIRVAAVQA (Fig. 1).The N-terminal amino acid sequence reported in theprevious article belonged to another enzyme encoded inthe same strain (gb|ABX75546). This enzyme was laterexpressed in E. coli, purified and characterized, and itssubstrate specificity was found to be different from that ofthe nitrilase purified in A. niger K10 (Kaplan et al. 2011).This was hypothesized to be caused by a misfolding or by aposttranslational modification (Kaplan et al. 2011) but thishypothesis has been corrected according to the new MSanalyses (Kaplan et al., Corrigendum to: Heterologous ex-pression, purification and characterization of nitrilase fromAspergillus niger K10 (BMC Biotechnol (2011) 11:2).BMC Biotechnol, submitted manuscript). The aforemen-tioned enzyme from A. nidulans FGSC A4 was later char-acterized as a cyanide hydratase (Basile et al. 2008). In

Erratum to: Hydrolysis of benzonitrile herbicides by soil actinobacteria and metabolite toxicity

M. Mackova, P. Lovecka and P. Janů wish to correct a typographical error in Table 3 which reports the results of acute toxicity determination implemented by them. The units of EC50 ± SD were not properly given and should have appeared in lM. The correct representation of Table 3 is herewith re-published and should be treated as definitive by the reader. Table 3 Determination of acute toxicity of chloroxynil, bromoxynil, ioxynil, dichlobenil and standards of their biodegradation products using the luminescent bacterium Vibrio fischeri