INTRODUCTION

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Nitrile compounds are discharged into the environment as industrial waste water, agricultural chemicals, etc. (23). Many microorganisms can use nitriles as a source of carbon and/or nitrogen for growth. Asano et al. have isolated various nitrile-degrading microorganisms from soil (2, 5, 24) and clarified that nitriles are converted to carboxylic acids either by a combination of nitrile hydratase (1, 3, 6) and amidase (4) or by nitrilase (8). These enzymes have been extensively evaluated from the viewpoint of chemical industry: the industrial production of acrylamide, nicotinamide, and 5-cyanovalelamide are typical examples (5, 6, 16, 26). Despite the importance of the nitrile-hydrolyzing enzymes in industry, information about their distribution in microorganisms is quite limited, and their physiological role has never been well understood since they have been screened only from nitrile-degrading microorganisms.

We have been studying aldoxime-degrading enzymes and isolated various aldoxime-degrading microorganisms, e.g., Bacillus sp. strain OxB-1 (7) and Rhodococcus sp. strain YH3-3 (12), from soil. The isolated strains metabolized aldoximes through nitriles into the corresponding carboxylic acid by a combination of a novel aldoxime dehydratase and nitrile-hydrolyzing enzymes (7, 12) (Fig. 1). The novel aldoxime dehydratase was purified and characterized from Bacillus sp. strain OxB-1 (7, 15). The enzyme from Rhodococcus sp. strain YH3-3 was applied to the enzymatic synthesis of nitriles from aldoximes under a mild condition (12, 13). A nitrile hydratase responsible for aldoxime metabolism was purified and characterized from Rhodococcus sp. strain YH3-3, and its properties were compared with the known nitrile hydratases (14).

View larger version (7K): [in this window] [in a new window]   FIG. 1.   Microbial metabolism of aldoximes. Bacillus sp. strain OxB-1 metabolizes PAOx (R = PhCH2) to form PAN, which is successively hydrolyzed to PAA by the action of nitrilase (7). On the other hand, Rhodococcus sp. strain YH3-3 metabolizes PyOx (R = 3-pyridyl) as follows: the aldoxime is dehydrated to form CyPy, which is converted to NAm by nitrile hydratase, and the NAm is successively hydrolyzed to NA by amidase (12).

To elucidate the generality of the relationship of aldoxime dehydratase and nitrile-hydrolyzing enzymes in microorganisms, we examined the distribution of both enzyme activities with the intact cells of a variety of aldoxime- or nitrile-degrading microorganisms, monitoring the decrease of the aldoximes, and we studied the relationship among the enzymes.

	MATERIALS AND METHODS

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Chemicals. Meat and malt extracts were obtained from Kyokuto (Tokyo, Japan). Polypepton and yeast extract were purchased from Nippon Seiyaku (Tokyo, Japan). High-pressure liquid chromatography (HPLC) columns ODS-80Ts (4.6 by 150 mm) and Hibar LiChrosorb-NH₂ (4.0 by 250 mm) were from Tosoh Corp. (Tokyo, Japan) and Kanto Chemicals (Tokyo, Japan), respectively. Aldoximes were synthesized as described previously (7, 12). All other chemicals were from commercial sources and used without further purification.

Microorganisms and culture media. A total of 975 strains from the following type culture collections were used: the Institute of Molecular and Cellular Biosciences (IAM), University of Tokyo, Tokyo, Japan; the Institute of Fermentation (IFO), Osaka, Japan; the Japan Collection of Microorganisms (JCM), Tokyo, Japan; the National Collections of Industrial Food and Marine Bacteria (NCIMB), Aberdeen, Scotland; the National Collections of Type Cultures and Pathogenic Fungi (NCTC) London, United Kingdom; the American Type Culture Collection (ATCC); and our own laboratory (TPU). These included 407 strains of 45 genera of bacteria, 133 strains of 11 genera of actinomyces, 333 strains of 22 genera of yeasts, and 102 strains of 37 genera of fungi.

Enzyme assay and definition of units. Microbial degradation of aldoxime was qualitatively analyzed by thin-layer chromatography (TLC). After the strain was cultured in the medium containing PAOx or PyOx, 1 µl of the culture was spotted onto silica gel TLC plates (Kieselgel 60; Merck) and then developed with 20% (vol/vol) ethyl acetate in hexane. The remaining aldoxime in the medium was visualized with iodine vapor.

	RESULTS

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Distribution of PAOx and PyOx dehydration activity in bacteria and actinomyces. We selected PAOx and PyOx as a model compound of alkylaldoxime and arylaldoxime, respectively. Bacterial and actinomycetal strains in our stock cultures were aerobically grown in the medium containing PAOx or PyOx, and the ability to degrade the aldoximes was screened by qualitative measurement of aldoxime disappearance in the culture medium by using TLC assay. Among the 540 strains tested, 37 strains were able to degrade PAOx, and this activity was distributed across 7 genera, while 96 strains, distributed across 30 genera, could degrade PyOx. Although some aerobically grown facultative anaerobes could degrade PyOx, none of the statically grown cells could degrade PAOx nor PyOx. We measured not only the aldoxime dehydration activities but also the PAN- and CyPy-hydrolyzing activities in the cells of PAOx- or PyOx-degrading strains. Since all of the aldoxime-degrading strains contained both aldoxime dehydratase and nitrile-hydrolyzing enzymes, nitriles formed by the dehydration of aldoximes were subsequently converted to amides and/or acids by the action of the latter enzymes. We therefore analyzed the dehydration activity by measuring the consumption of aldoxime in the assay. Nitrile-hydrolyzing activity was estimated by measuring the rate of consumption of nitrile, which was equal to the sum of nitrile hydratase and nitrilase activities. As shown in Tables 1 and 2, all of the strains grown with PAOx or PyOx exhibited the aldoxime dehydration activities, together with nitrile-hydrolyzing enzyme activities. The 21 strains marked with a number symbol (#) exhibited both PAOx and PyOx dehydration activities.

Distribution of aldoxime dehydration activity in fungi. We next screened for fungal strains which degrade PAOx or PyOx. PAOx-degrading activity was widely distributed in various genera. Of the 102 strains, 52 among 28 genera were able to degrade PAOx, whereas PyOx-degrading activity was seen in only a limited number of fungal strains; it was seen in 11 strains among 3 genera. Tables 3 and 4 show both of the enzyme activities in all of the active strains. Some active fungal strains degraded PAN accumulating PAAm, suggesting that the strains hydrolyzed PAN by the combinatorial action of nitrile hydratase and amidase, whereas PyOx-degrading fungi converted CyPy into NA without forming NAm.

Distribution of aldoxime dehydration activity in yeasts. Aldoxime degradation activity in yeasts is rare. We screened 333 strains, including 2 species of Brettanomyces, 29 species of Candida, 3 species of Cryptococcus, 3 species of Debaryomyces, 1 species of Endomycopsis, 1 species of Hanseniaspora, 24 species of Hanenula, 3 species of Kloeckera, 1 species of Lipomyces, 19 species of Pichia, 3 species of Rhodosporidium, 10 species of Rhodotorula, 32 species of Saccharomyces, 1 species of Saccharomycodes, 1 species of Saccharomycopsis, 3 species of Schizosaccharomyces, 1 species of Sporidiobolus, 4 species of Sporobolomyces, 12 species of Torulopsis, 3 species of Trichosporon, and 1 species of Zygosaccharomyces. Of these, two were active in degrading PAOx: Candida methanolica TPU 1217 and Pichia miso TPU 1306 showed PAOx dehydration activity at 1.31 and 0.9 (U/liter of culture), coexisting with a PAN-hydrolyzing activity at 0.5 and 0.2 (U/liter of culture), respectively, when they were grown with 0.05% of PAOx. It was also shown that both the strains degraded PAOx by a successive combination of aldoxime dehydration and nitrile-hydrolyzing enzymes. P. miso degraded PAN accumulating PAAm, while C. methanolica degraded it without the formation of PAAm. On the other hand, no yeast strain could degrade PyOx.

Aldoxime dehydration enzyme activity in nitrile-degrading microorganisms. Nineteen microorganisms (13 species, 7 genera) which had been isolated as nitrile degraders were grown with aldoximes, such as PAOx and PyOx, or nitriles, such as PAN and CyPy, and the aldoxime dehydration and nitrile-hydrolyzing activities were measured. As shown in Table 5, all of the nitrile degraders thus examined showed either PAOx or PyOx dehydratase activities. A high PAOx dehydration activity was seen in alkylnitrile-degrader, e.g., Rhodococcus sp. strain N-774 (23), Corynebacterium sp. strain C-5 (22), R. rhodochrous J-1 (3, 19), and R. erythropolis BG-16 (Y. Asano, T. Yasuda, Y. Tani, and H. Yamada, Abstr. Ann. Meet. Japan Soc. Ferment. Technol., 1981). No aldoxime dehydratase activity was seen when the strains were grown without aldoximes or nitriles, although PAN- or CyPy-hydrolyzing activity was detected in the cells.

	DISCUSSION

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Aldoximes are considered to be intermediates in the biosynthesis of certain biologically active compounds such as indoleacetic acid, cyanogenic glucosides, and glucosinolates in plants (17, 20, 21); however, very little is known about aldoxime-degrading enzymes: indoleacetaldoxime (IAOx) hydrolyase (EC 4.2.1.29), catalyzing a specific dehydration reaction of IAOx to form indoleacetonitrile, has been detected in limited genera of fungi and higher plants (20, 21), and the enzyme has only been partially purified from Gibberella spp. (17). In our previous report, we isolated various aldoxime-degrading bacteria from soil by several-months' acclimation and clarified that the isolated strains degraded aldoximes by the combination of aldoxime dehydratase and nitrile-hydrolyzing enzymes (7, 12). Rhodococcus sp. strain YH3-3, isolated in a medium containing PyOx as a sole source of nitrogen, degrades PyOx to NA by a combination of aldoxime dehydratase, nitrile hydratase, and amidase (12) (Fig. 1), whereas Bacillus sp. strain OxB-1 degrades PAOx to PAA by the action of aldoxime dehydratase and nitrilase (7) (Fig. 1), although it could not utilize PAOx as a nitrogen or carbon source due to the toxicity of PAOx. In this report, we discovered that the novel aldoxime dehydratases acting on the aldoximes are distributed among a variety of microorganisms, together with nitrile-hydrolyzing enzymes. We also screened for the aldoxime dehydratase activity from the nitrile degraders hitherto known and clarified that all of the nitrile degraders contained not only nitrile-hydrolyzing enzymes but also aldoxime dehydratase by culturing with aldoxime. None of the active strains showed aldoxime dehydration activity when they were grown in the medium without aldoximes, suggesting that aldoxime dehydratase was inducibly formed in these strains. Although a number of studies had been done on the nitrile-hydrolyzing enzymes, their induction mechanisms remained unknown. Nitrile hydratases of P. chlororaphis B23 and R. rhodochrous J-1 are strongly induced by amides and their derivatives (26), although that of Rhodococcus sp. strain N-774 (23) is formed constitutively. Nitrilases are inducibly formed (2, 9, 10, 18, 24, 25, 27), except that the enzyme is constitutively produced in Acinetobacter sp. strain AK 226 (27). In the present study we found that aldoximes act as good inducers not only for aldoxime dehydratase but also for the nitrile-hydrolyzing enzymes. From the results presented here, we cannot clarify whether aldoxime dehydratase and nitrile-hydrolyzing enzymes are regulated separately or not in these strains. We have recently discovered that the genes for aldoxime dehydratase and nitrilase coexisted as a gene cluster in the genome of Bacillus sp. strain OxB-1 (15). It is important to genetically clarify the general relationship between aldoxime dehydratase and nitrile-hydrolyzing enzymes in the aldoxime- and nitrile-degrading strains.

A total of 31 bacterial and 9 fungal aldoxime and nitrile degraders showed both the PAOx and PyOx dehydration activities, together with PAN and CyPy hydrolyzing activities. It is not clear whether the differences between PAOx and PyOx dehydration activities observed in the strains were simply due to the substrate specificity of aldoxime dehydratase or to the possible existence of two or more aldoxime dehydratases having different substrate specificities. Purification and characterization of aldoxime dehydratase and nitrile-hydrolyzing enzymes from the strains will solve this problem.

It has been reported that about 70 strains of microorganisms were isolated to degrade nitrile compounds, and they were distributed across 37 species of 19 genera (6, 24). However, they appear frequently in limited genera, i.e., Arthrobacter, Bacillus, Rhodococcus, Fusarium, since the most common screening protocols involve enrichment isolation in the media containing nitriles as a carbon or a nitrogen source: this is inevitably skewing positive clones to groups of readily culturable microorganisms. By culturing the strains with aldoxime, we showed here that the occurrence of nitrile hydratase in various species of facultative anaerobes and fungi. Although it is reported that Rhodococcus sp. strain AK32 has only nitrilase (27), culturing the strain with PAOx showed that the strain also contains nitrile hydratase and amidase in addition to nitrilase. These findings encourage us to screen further cryptic nitrile-hydrolyzing enzymes in microorganisms by culturing with aldoxime.