Black fungal extremes - CBS

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s u b s t r at e s p e c i f i c i t y o f t h e n i tr i l e h y d r o ly s i n g s y s t e m o f Ex o p h i a l a o l i g o s p e r m aspecific activity was calculated from the known molar extinctioncoefficient of maleylacetoacetate (ε 330nm= 14 mM -1 cm -1 ; Adachi etal. 1966) as 0.11 U mg -1 of protein. The reaction terminated whenthe initial concentration of homogentisate was converted and thecalculated almost stoichiometric amounts of maleylacetoacetatewere formed. The maleylacetoacetate was stable in the cuvettesfor more than an hour. In most organisms maleylacetoacetateis converted by glutathione-dependent maleylacetoacetateisomerases (Fernánandez-Canón & Penalva 1998). Therefore,glutathione (2 mM) was added to the cuvettes and an immediatedecrease in the absorbance at λ max= 317 nm observed. Thissuggested that homogentisate is metabolised by E. oligospermaR1 via maleylacetoacetate and fumarylacetoacetate to fumarateand acetoacetate (Fig. 5). Thus, it appears that homogentisate isan important ring-fission substrate in “black yeasts”, because it hasalready been suggested that E. jeanselmei (nowadays Phialophorasessilis) and E. lecanii-corni degrade compounds such as styreneand ethylbenzene via homogentisate (Cox et al. 1996; Gunsch etal. 2005; Prenafeta-Boldú et al. 2006).DISCUSSIONThe main aim of the present study was to obtain sufficient amounts ofactive biomass in order to allow a comparison of the nitrile convertingsystem of E. oligosperma R1 with other (fungal) nitrile hydrolysingsystems. The initial induction experiments demonstrated that thehighest nitrile hydrolysing activities were achieved after growth of E.oligosperma R1 in the presence of 2-cyanopyridine. This compoundhad already been previously identified as a powerful inducer forthe nitrile hydrolysing systems of different filamentous fungi suchas A. niger K10, Fusarium oxysporum CCF1414, F. oxysporumCCF 483, F. solani O1, and P. multicolor CCF 244 (Kaplan et al.2006b). 2-Cyanopyridine did not only serve as inducer of the nitrileconverting activity of E. oligosperma R1 but was also metabolisedand utilised as sole source of nitrogen as has been previouslyreported for F. solani O1 (Kaplan et al. 2006b).The experiments with whole cells and cell extractsdemonstrated that E. oligosperma R1 converted nitriles primarilyto the corresponding acids and that the organism formed only fromvery few substrates low amounts of the corresponding amides. Inaddition, the cell extracts exhibited only a rudimentary amidaseactivity. Thus, it can be concluded that the nitriles were convertedby a nitrilase. It therefore appears that most fungi convert organicnitriles using nitrilases and that nitrile hydratases are not commonamong fungi.The extracts prepared from cells of E. oligosperma whichhad been grown in the presence of 2-cyanopyridine convertedall available isomers of methyl-, hydroxy-, or chloro-substitutedbenzonitriles and phenylacetonitriles as well as phenylpropionitrile,2-, 3- and 4-cyanopyridine. These cell extracts exhibited for thesesubstrates compared to benzonitrile as substrate significantly higherrelative activities than cell extracts or purified nitrilase fractions fromF. solani O1, P. multicolor CCF 2244, or A. niger K10 (see Table 2).This was especially evident for the meta-substituted benzonitriles,because cell extracts of E. oligosperma R1 in general convertedthese substrates with higher specific activities than benzonitrile. Incontrast, the opposite was reported for cell extracts from F. solaniO1, P. multicolor CCF 2244, and the purified nitrilase of A. niger K10(Kaplan et al. 2006a, c). The enzyme from E. oligosperma R1 alsoconverted different ortho- substituted benzonitriles (e.g. 2-tolunitrile,www.studiesinmycology.org2-hydroxy- and 2-chlorobenzonitrile) which, probably due to stericalhindrances, could not be converted by the other strains (Kaplanet al. 2006a, c). In addition to the (substituted) benzonitrile(s) andphenylacetonitrile(s), the cell extracts from E. oligosperma R1 alsoconverted aliphatic substrates such as acrylonitrile and 2-hydroxy-3-butenenitrile. The turn-over of acrylonitrile had not been analysedin the studies by Kaplan et al. (2006a, c) but had been previouslydescribed for another fungal nitrilase from F. oxysporum f. sp melonis(Goldlust & Bohak 1989). However, the enzyme of F. oxysporum f.sp melonis converted acrylonitrile (in comparison to benzonitrile assubstrate) with significantly lower relative activities. Thus it can beconcluded that the nitrile converting activity from E. oligosperma R1appears to accept a slightly wider range of substrates than otherfungal nitrilases.The formation of amides as by-products of nitrilase catalyzedreactions had already been observed with various nitrilases frombacteria, plants, and fungi, e.g. from Rhodococcus ATCC 39484,Pseudomonas fluorescens EBC191, Arabidopsis thaliana, and F.oxysporum f. sp. melonis, and A. niger K10 (Goldlust & Bohak 1989,Stevenson et al. 1992, Effenberger & Osswald 2001, Osswald et al.2002, Šnajdrová et al. 2004, Kiziak et al. 2005, Mateo et al. 2006,Fernandes et al. 2006, Kaplan et al. 2006b, c, Rustler et al. 2007). Itwas proposed that the formation of these by-products is based on anatypical cleavage of the tetrahedral intermediate formed during thereaction which might be triggered by electron withdrawing effects ofdifferent substituents (Fernandes et al. 2006). The nitrilase activityfrom E. oligosperma R1 produced the largest relative amounts ofamides during the induction experiments from 2-cyanopyridine(Fig. 2; ratio acid: amide about 7:4) and during the experimentswith cell extracts (Table 2) from 2-chlorobenzonitrile (ratio acid:amide about 7:3). Furthermore some amides (< 10 molar % of totalproducts) were also formed during the turn-over of 4-cyanopyridineand 3-chlorobenzonitrile. These results follow the emerging trendfor the relationship between substrate structure and the degreeof amide formation for fungal nitrilases. Thus, also P. multicolorCCF 2244 and the purified nitrilase from A. niger K10 producedrelatively large amounts of amides from chlorobenzonitriles andcyanopyridines (Kaplan et al. 2006b, c). From the available datait appears that the nitrilase from E. oligosperma R1 is regarding toits tendency for amide formation situated somehow intermediatebetween the enzymes from F. solani O1 (less amide formation)and those from P. multicolor CCF 2244 and A. niger K10 (strongertendency for amide formation).In conclusion it might be summarised that the recent work inthe group of L. Martinková (Kaplan et al. 2006a,b,c; Vejvoda etal. 2008) about the nitrile converting systems of filamentous fungitogether with our study about the black yeast E. oligosperma R1(Rustler & Stolz, 2007, this manuscript) suggest that evolutionaryrather different fungi synthesise nitrilases which clearly resembleeach other regarding their induction system and substratespecificity. These fungal systems might be of some relevancebecause of the high specific activities that can be obtained underoptimal induction conditions and also the high specific activities ofthe purified enzymes with their preferred substrates. In addition,it was shown in the present study that the acid tolerance of fungiallows their utilisation as whole cell catalysts for the conversion ofnitriles that are stabilised under acidic conditions. This observationmight significantly enhance the importance of fungal nitrilases forbiotransformation reactions.173
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Mechanisticand structural studies on Rhodococcus ATCC39484 nitrilase. Biotechnologyand Applied Biochemistry 15: 283–302.Vejvoda V, Kaplan O, Bezouška K, Pompach P, Šulc M, Cantarella M, Benada O,Uhnáková B, Rinágelová A, Lutz-Wahl S, Fischer L, Křen V, Martínková L(2008). Purification and characterization of a nitrilase from Fusarium solani O1.Journal of Molecular Catalysis B: Enzymatic. 50: 99–106.Verduyn C (1991). Physiology of yeasts in relation to biomass yields. Antonie vanLeeuwenhoek 60: 325–353.174
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Studies in Mycology 61 (2008)Black
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Studies in MycologyThe Studies in M
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PREFACEThe terms "black fungi" or "
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available online at www.studiesinmy
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Dr o u g h t m e e t s a c i dTable
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Dr o u g h t m e e t s a c i d2003/
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Dr o u g h t m e e t s a c i din Pe
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Dr o u g h t m e e t s a c i d99100
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Dr o u g h t m e e t s a c i dAnamo
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Dr o u g h t m e e t s a c i dFig.
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Dr o u g h t m e e t s a c i dFig.
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Dr o u g h t m e e t s a c i dconve
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Dr o u g h t m e e t s a c i dal. 2
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Za l a r e t a l.Table 1. List of a
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Za l a r e t a l.Table 1. (Continue
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Za l a r e t a l.Fig. 2. Consensus
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Za l a r e t a l.Fig. 4. Macromorph
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Za l a r e t a l.Fig. 5. Aureobasid
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Za l a r e t a l.Fig. 7. Aureobasid
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Za l a r e t a l.the type species o
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Za l a r e t a l.Vishniac HS, Onofr
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Yu r l o va e t a l.Table 1. Strain
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Yu r l o va e t a l.112ОС2Fig. 1.
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Yu r l o va e t a l.RESULTSFourteen
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Yu r l o va e t a l.100908070605040
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Yu r l o va e t a l.AcetateMelanin(
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Fat t y-a c i d-m o d if y i n g e
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Va u p o t i č e t a l.cerevisiae)
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Va u p o t i č e t a l.Fig. 3. Mor
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Va u p o t i č e t a l.HMG-CoA red
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Pl e m e n i ta s e t a l.entire en
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Pl e m e n i ta s e t a l.glycerol
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Pl e m e n i ta s e t a l.Fig. 1. T
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Pl e m e n i ta s e t a l.diversity
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Ti n e a n i g r aFig. 1. Map showi
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Ti n e a n i g r aand Polynesia (Ue
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On o f r i e t a l.Table 1. Test pa
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On o f r i e t a l.Analyses of resp
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On o f r i e t a l.Fig. 6. Percenta
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On o f r i e t a l.Fig. 11. Percent
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On o f r i e t a l.2005). The abili
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A r o c k-i n h a b i t i n g a n c
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Page 192 and 193: homogentisate 165, 171, 173Hortaea
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