Organic Acid to Nitrile: A Chemoenzymatic Three-Step Route.

Various widely applied compounds contain cyano-groups, and this functional group serves as a chemical handle for a whole range of different reactions. We report a cyanide free chemoenzymatic cascade for nitrile synthesis. The reaction pathway starts with a reduction of carboxylic acid to aldehyde by carboxylate reductase enzymes (CARs) applied as living cell biocatalysts. The second - chemical - step includes in situ oxime formation with hydroxylamine. The final direct step from oxime to nitrile is catalyzed by aldoxime dehydratases (Oxds). With compatible combinations of a CAR and an Oxd, applied in one-pot two-step reactions, several aliphatic and aryl-aliphatic target nitriles were obtained in more than 80% conversion. Phenylacetonitrile, for example, was prepared in 78% isolated yield. This chemoenzymatic route does not require cyanide salts, toxic metals, or undesired oxidants in contrast to entirely chemical procedures.

Laccases and Tyrosinases in Organic Synthesis.

Laccases (Lac) and tyrosinases (TYR) are mild oxidants with a great potential in research and industry. In this work, we review recent advances in their use in organic synthesis. We summarize recent examples of Lac-catalyzed oxidation, homocoupling and heterocoupling, and TYR-catalyzed ortho-hydroxylation of phenols. We highlight the combination of Lac and TYR with other enzymes or chemical catalysts. We also point out the biological and pharmaceutical potential of the products, such as dimers of piceid, lignols, isorhamnetin, rutin, caffeic acid, 4-hydroxychalcones, thiols, hybrid antibiotics, benzimidazoles, benzothiazoles, pyrimidine derivatives, hydroxytyrosols, alkylcatechols, halocatechols, or dihydrocaffeoyl esters, etc. These products include radical scavengers; antibacterial, antiviral, and antitumor compounds; and building blocks for bioactive compounds and drugs. We summarize the available enzyme sources and discuss the scalability of their use in organic synthesis. In conclusion, we assume that the intensive use of laccases and tyrosinases in organic synthesis will yield new bioactive compounds and, in the long-term, reduce the environmental impact of industrial organic chemistry.

Plant Nitrilase Homologues in Fungi: Phylogenetic and Functional Analysis with Focus on Nitrilases in Trametes versicolor and Agaricus bisporus.

Fungi contain many plant-nitrilase (NLase) homologues according to database searches. In this study, enzymes NitTv1 from Trametes versicolor and NitAb from Agaricus bisporus were purified and characterized as the representatives of this type of fungal NLase. Both enzymes were slightly more similar to NIT4 type than to NIT1/NIT2/NIT3 type of plant NLases in terms of their amino acid sequences. Expression of the synthetic genes in Escherichia coli Origami B (DE3) was induced with 0.02 mM isopropyl ß-D-1-thiogalactopyranoside at 20 °C. Purification of NitTv1 and NitAb by cobalt affinity chromatography gave ca. 6.6 mg and 9.6 mg of protein per 100 mL of culture medium, respectively. Their activities were determined with 25 mM of nitriles in 50 mM Tris/HCl buffer, pH 8.0, at 30 °C. NitTv1 and NitAb transformed ß-cyano-L-alanine (ß-CA) with the highest specific activities (ca. 132 and 40 U mg-1, respectively) similar to plant NLase NIT4. ß-CA was transformed into Asn and Asp as in NIT4 but at lower Asn:Asp ratios. The fungal NLases also exhibited significant activities for (aryl)aliphatic nitriles such as 3-phenylpropionitrile, cinnamonitrile and fumaronitrile (substrates of NLase NIT1). NitTv1 was more stable than NitAb (at pH 5-9 vs. pH 5-7). These NLases may participate in plant-fungus interactions by detoxifying plant nitriles and/or producing plant hormones. Their homology models elucidated the molecular interactions with various nitriles in their active sites.

Genetic and Functional Diversity of Nitrilases in Agaricomycotina.

Nitrilases participate in the nitrile metabolism in microbes and plants. They are widely used to produce carboxylic acids from nitriles. Nitrilases were described in bacteria, Ascomycota and plants. However, they remain unexplored in Basidiomycota. Yet more than 200 putative nitrilases are found in this division via GenBank. The majority of them occur in the subdivision Agaricomycotina. In this work, we analyzed their sequences and classified them into phylogenetic clades. Members of clade 1 (61 proteins) and 2 (25 proteins) are similar to plant nitrilases and nitrilases from Ascomycota, respectively, with sequence identities of around 50%. The searches also identified five putative cyanide hydratases (CynHs). Representatives of clade 1 and 2 (NitTv1 from Trametes versicolor and NitAg from Armillaria gallica, respectively) and a putative CynH (NitSh from Stereum hirsutum) were overproduced in Escherichia coli. The substrates of NitTv1 were fumaronitrile, 3-phenylpropionitrile, ß-cyano-l-alanine and 4-cyanopyridine, and those of NitSh were hydrogen cyanide (HCN), 2-cyanopyridine, fumaronitrile and benzonitrile. NitAg only exhibited activities for HCN and fumaronitrile. The substrate specificities of these nitrilases were largely in accordance with substrate docking in their homology models. The phylogenetic distribution of each type of nitrilase was determined for the first time.

Biocatalytic production of mandelic acid and analogues: a review and comparison with chemical processes.

The aim of this study is to summarize the current progress in the design of biocatalytic processes applicable for the production of optically pure mandelic acids and their analogues. These compounds are used as building blocks for pharmaceutical chemistry and as chiral resolving agents. Their enzymatic syntheses mainly employed nitrile hydrolysis with nitrilases, ester hydrolysis, ammonolysis or esterification with lipases or esterases, and ketone reduction or alcohol oxidation with dehydrogenases. Each of these methods will be characterized in terms of its product concentrations, enantioselectivities, and the types of catalysts used. This review will focus on the dynamic kinetic resolution of mandelonitrile and analogues by nitrilases resulting in the production of high concentrations of (R)-mandelic acid or (R)-2-chloromandelic acid with excellent e.e. Currently, there is no comparable process for (S)-mandelic acids. However, the coupling of the S-selective cyanation of benzaldehyde with the enantioretentive hydrolysis of (S)-mandelonitrile thus obtained is a promising strategy. The major product can be changed from (S)-acid to (S)-amide using nitrilase mutants. The competitiveness of the biocatalytic and chemical processes will be assessed. This review covers the literature published within 2003-2017.

Recent advances and challenges in the heterologous production of microbial nitrilases for biocatalytic applications.

The aim of this study is to review the current state of and highlight the challenges in the production of microbial nitrilases as catalysts for the mild hydrolysis of industrially important nitriles. Together with aldoxime dehydratase, the nitrile-hydrolyzing enzymes (nitrilase, nitrile hydratase) are key enzymes in the aldoxime-nitrile pathway which is widely distributed in bacteria and fungi. The availability of nitrilases has grown significantly over the past decade due to the use of metagenomic and database-mining approaches. Databases contain plenty of putative enzymes of this type, whose overproduction may improve the spectrum and the industrial utility of nitrilases. By exploiting this resource, the number of experimentally verified nitrilases has recently increased to several hundred. We especially focus on the efficient heterologous expression systems that are applicable for the overproduction of wild-type nitrilases and their artificial variants. Biocatalyst forms with industrial potential are also highlighted. The potential industrial applications of nitrilases are classified according to their target products (α-hydroxy acids, α- and ß-amino acids, cyano acids, amides). The emerging uses of nitrilases and their subtypes (cyanide hydratases, cyanide dihydratases) in bioremediation is also summarized. The integration of nitrilases with other enzymes into artificial multienzymatic and chemoenzymatic pathways is considered a promising strategy for future applications.

Bringing nitrilase sequences from databases to life: the search for novel substrate specificities with a focus on dinitriles.

The aim of this study was to discover new nitrilases with useful activities, especially towards dinitriles that are precursors of high-value cyano acids. Genes coding for putative nitrilases of different origins (fungal, plant, or bacterial) with moderate similarities to known nitrilases were selected by mining the GenBank database, synthesized artificially and expressed in Escherichia coli. The enzymes were purified, examined for their substrate specificities, and classified into subtypes (aromatic nitrilase, arylacetonitrilase, aliphatic nitrilase, cyanide hydratase) which were largely in accordance with those predicted from bioinformatic analysis. The catalytic potential of the nitrilases for dinitriles was examined with cyanophenyl acetonitriles, phenylenediacetonitriles, and fumaronitrile. The nitrilase activities and selectivities for dinitriles and the reaction products (cyano acid, cyano amide, diacid) depended on the enzyme subtype. At a preparative scale, all the examined dinitriles were hydrolyzed into cyano acids and fumaronitrile was converted to cyano amide using E. coli cells producing arylacetonitrilases and an aromatic nitrilase, respectively.

An investigation of nitrile transforming enzymes in the chemo-enzymatic synthesis of the taxol sidechain.

Paclitaxel (taxol) is an antimicrotubule agent widely used in the treatment of cancer. Taxol is prepared in a semisynthetic route by coupling the N-benzoyl-(2R,3S)-3-phenylisoserine sidechain to the baccatin III core structure. Precursors of the taxol sidechain have previously been prepared in chemoenzymatic approaches using acylases, lipases, and reductases, mostly featuring the enantioselective, enzymatic step early in the reaction pathway. Here, nitrile hydrolysing enzymes, namely nitrile hydratases and nitrilases, are investigated for the enzymatic hydrolysis of two different sidechain precursors. Both sidechain precursors, an openchain α-hydroxy-ß-amino nitrile and a cyanodihydrooxazole, are suitable for coupling to baccatin III directly after the enzymatic step. An extensive set of nitrilases and nitrile hydratases was screened towards their activity and selectivity in the hydrolysis of two taxol sidechain precursors and their epimers. A number of nitrilases and nitrile hydratases converted both sidechain precursors and their epimers.

Cyanide hydratases and cyanide dihydratases: emerging tools in the biodegradation and biodetection of cyanide.

The purpose of this study is to summarize the current knowledge of the enzymes which are involved in the hydrolysis of cyanide, i.e., cyanide hydratases (CHTs; EC 4.2.1.66) and cyanide dihydratases (CynD; EC 3.5.5.1). CHTs are probably exclusively produced by filamentous fungi and widely occur in these organisms; in contrast, CynDs were only found in a few bacterial genera. CHTs differ from CynDs in their reaction products (formamide vs. formic acid and ammonia, respectively). Several CHTs were also found to transform nitriles but with lower relative activities compared to HCN. Mutants of CynDs and CHTs were constructed to study the structure-activity relationships in these enzymes or to improve their catalytic properties. The effect of the C-terminal part of the protein on the enzyme activity was determined by constructing the corresponding deletion mutants. CynDs are less active at alkaline pH than CHTs. To improve its bioremediation potential, CynD from Bacillus pumilus was engineered by directed evolution combined with site-directed mutagenesis, and its operation at pH 10 was thus enabled. Some of the enzymes have been tested for their potential to eliminate cyanide from cyanide-containing wastewaters. CynDs were also used to construct cyanide biosensors.

Expression control of nitrile hydratase and amidase genes in Rhodococcus erythropolis and substrate specificities of the enzymes.

Bacterial amidases and nitrile hydratases can be used for the synthesis of various intermediates and products in the chemical and pharmaceutical industries and for the bioremediation of toxic pollutants. The aim of this study was to analyze the expression of the amidase and nitrile hydratase genes of Rhodococcus erythropolis and test the stereospecific nitrile hydratase and amidase activities on chiral cyanohydrins. The nucleotide sequences of the gene clusters containing the oxd (aldoxime dehydratase), ami (amidase), nha1, nha2 (subunits of the nitrile hydratase), nhr1, nhr2, nhr3 and nhr4 (putative regulatory proteins) genes of two R. erythropolis strains, A4 and CCM2595, were determined. All genes of both of the clusters are transcribed in the same direction. RT-PCR analysis, primer extension and promoter fusions with the gfp reporter gene showed that the ami, nha1 and nha2 genes of R. erythropolis A4 form an operon transcribed from the Pami promoter and an internal Pnha promoter. The activity of Pami was found to be weakly induced when the cells grew in the presence of acetonitrile, whereas the Pnha promoter was moderately induced by both the acetonitrile or acetamide used instead of the inorganic nitrogen source. However, R. erythropolis A4 cells showed no increase in amidase and nitrile hydratase activities in the presence of acetamide or acetonitrile in the medium. R. erythropolis A4 nitrile hydratase and amidase were found to be effective at hydrolysing cyanohydrins and 2-hydroxyamides, respectively.

Cell-free reduction of carboxylic acids with secreted carboxylic acid reductase.

Mycobacterium marinum CAR (MmCAR) is one of the most widely used CARs as the key enzyme for the synthesis of aldehydes, alcohols and further products from the respective carboxylic acids. Herein, we describe the first functionally secreted 131 kDa CAR and its isolated A-domain using Komagataella phaffii and a methanol-free constitutive expression strategy. Precipitated and lyophilized MmCAR (500 µg) was isolated from the culture supernatant and showed no decrease in activity for piperonylic acid (80% conversion), even when stored for up to 3 weeks at 4°C. Lyophilized MmCAR precipitate gave 48% yield of E/Z-nonanal-4-nitrobenzoyloxime from the reduction of nonanoic acid and in-situ derivatization with O-4-nitrobenzoyl-hydroxylamine. Furthermore, K. phaffii could successfully secrete the MmCAR adenylation domain. Its activity was confirmed by the amidation of benzoic acid with n-hexylamine. Neither enzyme variant was glycosylated by the yeast. In summary, functional CAR can be secreted by K. phaffii and used for cell free conversion of carboxylic acids to various products.

Immobilization of aldoxime dehydratases on metal affinity resins and use of the immobilized catalysts for the synthesis of nitriles important in fragrance industry.

Nitriles have a wide range of uses as building blocks, solvents, and alternative fuels, but also as intermediates and components of flavors and fragrances. The enzymatic synthesis of nitriles by aldoxime dehydratase (Oxd) is an emerging process with significant advantages over conventional approaches. Here we focus on the immobilization of His-tagged Oxds on metal affinity resins, an approach that has not been used previously for these enzymes. The potential of the immobilized Oxd was demonstrated for the synthesis of phenylacetonitrile (PAN) and E-cinnamonitrile, compounds applicable in the fragrance industry. A comparison of Talon and Ni-NTA resins showed that Ni-NTA with its higher binding capacity was more suitable for the immobilization of Oxd. Immobilized Oxds were prepared from purified enzymes (OxdFv from Fusarium vanettenii and OxdBr1 from Bradyrhizobium sp.) or the corresponding cell-free extracts. The immobilization of cell-free extracts reduced time and cost of the catalyst production. The immobilized OxdBr1 was superior in terms of recyclability (22 cycles) in the synthesis of PAN from 15 mM E/Z-phenylacetaldoxime at pH 7.0 and 30 °C (100% conversion, 61% isolated yield after product purification). The volumetric and catalyst productivity was 10.5 g/L/h and 48.3 g/g of immobilized protein, respectively.

Bio-Based Valorization of Lignin-Derived Phenolic Compounds: A Review.

Lignins are the most abundant biopolymers that consist of aromatic units. Lignins are obtained by fractionation of lignocellulose in the form of "technical lignins". The depolymerization (conversion) of lignin and the treatment of depolymerized lignin are challenging processes due to the complexity and resistance of lignins. Progress toward mild work-up of lignins has been discussed in numerous reviews. The next step in the valorization of lignin is the conversion of lignin-based monomers, which are limited in number, into a wider range of bulk and fine chemicals. These reactions may need chemicals, catalysts, solvents, or energy from fossil resources. This is counterintuitive to green, sustainable chemistry. Therefore, in this review, we focus on biocatalyzed reactions of lignin monomers, e.g., vanillin, vanillic acid, syringaldehyde, guaiacols, (iso)eugenol, ferulic acid, p-coumaric acid, and alkylphenols. For each monomer, its production from lignin or lignocellulose is summarized, and, mainly, its biotransformations that provide useful chemicals are discussed. The technological maturity of these processes is characterized based on, e.g., scale, volumetric productivities, or isolated yields. The biocatalyzed reactions are compared with their chemically catalyzed counterparts if the latter are available.

Scanning aldoxime dehydratase sequence space and characterization of a new aldoxime dehydratase from Fusarium vanettenii.

The aim of this work was to map the sequence space of aldoxime dehydratases (Oxds) as enzymes with great potential for nitrile synthesis. Microbes contain an abundance of putative Oxds but fewer than ten Oxds were characterized in total and only two in fungi. In this work, we prepared and characterized a new Oxd (protein gb|EEU37245.1 named OxdFv) from Fusarium vanettenii 77-13-4. OxdFv is distant from the characterized Oxds with a maximum of 36% identity. Moreover, the canonical Oxd catalytic triad RSH is replaced by R141-E187-E303 in OxdFv. R141A and E187A mutants did not show significant activities, but mutant E303A showed a comparable activity as the wild-type enzyme. According to native mass spectrometry, OxdFv contained almost 1 mol of heme per 1 mol of protein, and was composed of approximately 88% monomer (41.8 kDa) and 12% dimer. A major advantage of this enzyme is its considerable activity under aerobic conditions (25.0 ± 4.3 U/mg for E,Z-phenylacetaldoxime at pH 9.0 and 55 °C). Addition of sodium dithionite (reducing agent) and Fe2+ was required for this activity. OxdFv favored (aryl)aliphatic aldoximes over aromatic aldoximes. Substrate docking in the homology model of OxdFv showed a similar substrate specificity. We conclude that OxdFv is the first characterized Oxd of the REE type.

Purification and characterization of heterologously expressed nitrilases from filamentous fungi.

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.

Biotransformation of benzonitrile herbicides via the nitrile hydratase-amidase pathway in rhodococci.

The aim of this work was to determine the ability of rhodococci to transform 3,5-dichloro-4-hydroxybenzonitrile (chloroxynil), 3,5-dibromo-4-hydroxybenzonitrile (bromoxynil), 3,5-diiodo-4-hydroxybenzonitrile (ioxynil) and 2,6-dichlorobenzonitrile (dichlobenil); to identify the products and determine their acute toxicities. Rhodococcus erythropolis A4 and Rhodococcus rhodochrous PA-34 converted benzonitrile herbicides into amides, but only the former strain was able to hydrolyze 2,6-dichlorobenzamide into 2,6-dichlorobenzoic acid, and produced also more of the carboxylic acids from the other herbicides compared to strain PA-34. Transformation of nitriles into amides decreased acute toxicities for chloroxynil and dichlobenil, but increased them for bromoxynil and ioxynil. The amides inhibited root growth in Lactuca sativa less than the nitriles but more than the acids. The conversion of the nitrile group may be the first step in the mineralization of benzonitrile herbicides but cannot be itself considered to be a detoxification.

Metabolism of Aldoximes and Nitriles in Plant-Associated Bacteria and Its Potential in Plant-Bacteria Interactions.

In plants, aldoximes per se act as defense compounds and are precursors of complex defense compounds such as cyanogenic glucosides and glucosinolates. Bacteria rarely produce aldoximes, but some are able to transform them by aldoxime dehydratase (Oxd), followed by nitrilase (NLase) or nitrile hydratase (NHase) catalyzed transformations. Oxds are often encoded together with NLases or NHases in a single operon, forming the aldoxime-nitrile pathway. Previous reviews have largely focused on the use of Oxds and NLases or NHases in organic synthesis. In contrast, the focus of this review is on the contribution of these enzymes to plant-bacteria interactions. Therefore, we summarize the substrate specificities of the enzymes for plant compounds. We also analyze the taxonomic and ecological distribution of the enzymes. In addition, we discuss their importance in selected plant symbionts. The data show that Oxds, NLases, and NHases are abundant in Actinobacteria and Proteobacteria. The enzymes seem to be important for breaking through plant defenses and utilizing oximes or nitriles as nutrients. They may also contribute, e.g., to the synthesis of the phytohormone indole-3-acetic acid. We conclude that the bacterial and plant metabolism of aldoximes and nitriles may interfere in several ways. However, further in vitro and in vivo studies are needed to better understand this underexplored aspect of plant-bacteria interactions.

Chemoenzymatic one-pot reaction from carboxylic acid to nitrile via oxime.

We report a new chemoenzymatic cascade starting with aldehyde synthesis by carboxylic acid reductase (CAR) followed by chemical in situ oxime formation. The final step to the nitrile is catalyzed by aldoxime dehydratase (Oxd). Full conversions of phenylacetic acid and hexanoic acid were achieved in a two-phase mode.

Heterologous expression, purification and characterization of nitrilase from Aspergillus niger K10.

BACKGROUND: Nitrilases attract increasing attention due to their utility in the mild hydrolysis of nitriles. According to activity and gene screening, filamentous fungi are a rich source of nitrilases distinct in evolution from their widely examined bacterial counterparts. However, fungal nitrilases have been less explored than the bacterial ones. Nitrilases are typically heterogeneous in their quaternary structures, forming short spirals and extended filaments, these features making their structural studies difficult. RESULTS: A nitrilase gene was amplified by PCR from the cDNA library of Aspergillus niger K10. The PCR product was ligated into expression vectors pET-30(+) and pRSET B to construct plasmids pOK101 and pOK102, respectively. The recombinant nitrilase (Nit-ANigRec) expressed in Escherichia coli BL21-Gold(DE3)(pOK101/pTf16) was purified with an about 2-fold increase in specific activity and 35% yield. The apparent subunit size was 42.7 kDa, which is approx. 4 kDa higher than that of the enzyme isolated from the native organism (Nit-ANigWT), indicating post-translational cleavage in the enzyme's native environment. Mass spectrometry analysis showed that a C-terminal peptide (Val327 - Asn356) was present in Nit-ANigRec but missing in Nit-ANigWT and Asp298-Val313 peptide was shortened to Asp298-Arg310 in Nit-ANigWT. The latter enzyme was thus truncated by 46 amino acids. Enzymes Nit-ANigRec and Nit-ANigWT differed in substrate specificity, acid/amide ratio, reaction optima and stability. Refolded recombinant enzyme stored for one month at 4°C was fractionated by gel filtration, and fractions were examined by electron microscopy. The late fractions were further analyzed by analytical centrifugation and dynamic light scattering, and shown to consist of a rather homogeneous protein species composed of 12-16 subunits. This hypothesis was consistent with electron microscopy and our modelling of the multimeric nitrilase, which supports an arrangement of dimers into helical segments as a plausible structural solution. CONCLUSIONS: The nitrilase from Aspergillus niger K10 is highly homologous (≥86%) with proteins deduced from gene sequencing in Aspergillus and Penicillium genera. As the first of these proteins, it was shown to exhibit nitrilase activity towards organic nitriles. The comparison of the Nit-ANigRec and Nit-ANigWT suggested that the catalytic properties of nitrilases may be changed due to missing posttranslational cleavage of the former enzyme. Nit-ANigRec exhibits a lower tendency to form filaments and, moreover, the sample homogeneity can be further improved by in vitro protein refolding. The homogeneous protein species consisting of short spirals is expected to be more suitable for structural studies.

Genome mining for the discovery of new nitrilases in filamentous fungi.

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.