Black fungal extremes - CBS

Ru s t l e r e t a l.Table 1. Conversion of different nitriles by whole cells and cell extracts of E. oligosperma R1.Activity of whole cellsActivity of cell extractsCompound U/mg of dry weight Relative activity U/mg of total protein Relative ActivityBenzonitrile 0.115 100 0.48 100Phenylacetonitrile 0.021 18 0.11 223-Cyanopyridine 0.14 124 1 2084-Cyanopyridine 0.27 232 2.46 512Acrylonitrile 0.058 50 0.58 121The specific activities were calculated from the experiments shown in Fig. 3 based on the decrease of the nitrile concentrations and related to the dry weight of the cells orthe protein concentration in the experiments with cell extracts, respectively.the conversion of 4-cyanopyridine up to 2.5 U/mg of protein werecalculated (Table 1). In the literature generally protein contents of39–56 % (expressed as % of dry weight) have been described fordifferent yeast species (Verduyn 1991). An average of 50 % proteincontent (related to the dry weight) was used for the comparison ofthe turn-over-rates of cell extracts and resting cells. Thus, it wascalculated that the cell extracts converted 3-, and 4-cyanopyridineas well as acrylonitrile with up to 5-times higher specific activitiesthan the resting cells. This suggested that at least in the case ofrapidly converted substrates the uptake of the nitriles has a limitingeffect on the in vivo metabolism of organic nitriles by the yeast cells.Furthermore, also the comparison of the relative activities of thewhole cells and the cell extracts with different nitriles suggested asignificant influence of the cell membrane on the nitrile metabolismbecause the whole cells converted the cyanopyridines andacrylonitrile in comparison to benzonitrile with decreased relativeactivities (Table 1). This was especially evident for the substratesbenzonitrile and acrylonitrile, because crude extracts convertedacrylonitrile faster than benzonitrile, while this was the opposite inthe whole cell system.Conversion of different nitriles by cell extractsThe substrate specificity of the nitrile hydrolysing activity wasdetermined with cell extracts and compared to other fungal nitrileconverting enzymes (Table 2). These experiments demonstratedthat cell extracts from E. oligosperma R1 converted in addition tobenzonitrile and the cyanopyridines various methyl-, chloro-, andhydroxy-substituted benzonitriles and phenylacetonitriles. The cellextracts converted in general all isomers of a given substitutedbenzonitrile or phenylacetonitrile. Nevertheless, it became evidentthat in most cases the meta-substituted isomers were converted withthe highest relative activities. In most cases the meta-substitutedbenzonitriles were even faster converted than benzonitrile. Incontrast, the ortho-substituted isomers were generally convertedwith the lowest relative activities.Phenylacetonitriles which carried a larger substituent at theα-position than a hydrogen atom (such as mandelonitrile and2-phenylpropionitrile) were converted with significantly reducedconversion rates. The aliphatic substrate acrylonitrile was convertedwith high relative activities (121 % compared to benzonitrile).The conversion of the nitriles resulted in almost all experiments(with the exceptions of 2-chlorobenzonitrile, 4-cyanopyridine, andacrylonitrile as substrates) in the formation of one clearly prominentproduct which represented (according to its signal intensity duringthe HPLC analysis) more than 95 % of the products formed (Table2). These products were identified by the comparison with authenticstandards by their retention times and in situ UV/VIS spectra asthe corresponding acids. In the cases of 4-cyanopyridine andacrylonitrile two products were formed in a ratio of about 9:1.The conversion of 2-chlorobenzonitrile resulted in two signals ina ratio of 7:3. However, also for these substrates it was shown byusing authentic standards that the prominent products were thecorresponding acids. The minor products were identified accordingto their retention times as the corresponding amides.The conversion of 2-cyanopyridine and 2-hydroxybenzonitriledid not result in the formation of detectable amounts of products.However, in the case of 2-cyanopyridine the conversion of thesubstrate to picolinic acid and picolinic amide had already beenobserved in the growth experiment described before (see Fig. 2).The cell extracts were also incubated with several amides, whichwould be intermediately formed if the cells would harbour a nitrilehydratase. The experiments showed that picolinic amide andnicotinic amide were not converted and isonicotinic amide only withextremely low activities to the corresponding acid. This indicatedonly a rudimental amidase activity and gave strong evidence thatthe nitriles were indeed converted by a nitrilase activity.Conversion of 2-hydroxy-3-butenenitrile by wholecells of E. oligosperma R1It was previously shown that resting cells of E. oligosperma R1were able to convert nitriles at pH values ≥1.5 (Rustler & Stolz2007). This could allow the conversion of α-hydroxynitriles, whichare unstable at neutral pH-values but stabilised under acidicconditions by resting cells of E. oligosperma R1. The conversionof acrylonitrile by whole cells and cell extracts demonstrated thegeneral ability of E. oligosperma R1 to convert aliphatic nitriles withhigh relative reaction rates. Therefore, 2-hydroxy-3-butenenitrilewas synthesised as substrate (see material and method section)and incubated in a reaction mixture containing 0.1 M Na-citratephosphatebuffer (pH 4) and resting cells of E. oligosperma R1. Theanalysis of the reaction mixture demonstrated a disappearance ofalmost 50 % of the initial amount of 2-hydroxy-3-butenenitrile withinthe first 80 min of the reaction and the formation of one product (Fig.4). In contrast, no significant decrease of 2-hydroxy-3-butenenitrilewas observed in a control experiment without cells. Only tracesof acroleine (2-propenal, acrylaldehyde) which would be formedby the chemical decomposition of 2-hydroxy-3-butenenitrile weredetected in the reaction mixtures with and without cells. Thus, it wasconcluded that 2-hydroxy-3-butenenitrile was almost completelystabilised by the acidic reaction buffer and that the resting cellsindeed converted the nitrile. The product formed from 2-hydroxy-3-butenenitrile by the cells was identified according to its retention time(R t=3.45 min) and UV/VIS spectrum in comparison with a chemicallysynthesised authentic standard as 2-hydroxy-3-butenoic acid.170