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.

Background

Nitrilases enable hydrolysis of nitriles to be performed under mild conditions and often in a stereo- or regioselective manner. These enzymes have thus great potential in organic synthesis but drawbacks such as instability, low activity or low selectivity lessen their practical use [1,2]. These limits may be overcome by searching for new nitrilases or improving known ones. Recently, the former approach has often made use of database mining [1-6].

According to GenBank search, not only bacteria, which have been intensively exploited as a source of nitrilases since the 1980s, but also filamentous fungi harbour a large number of nitrilase genes [7]. Apart from the teleomorph/anamorph pair Gibberella/Fusarium, the Aspergillus genus is a rich source of these enzymes, which exhibit low homology to bacterial nitrilases and thus may differ in their catalytic properties.

We have recently purified and characterized the first nitrilase in the Aspergillus genus, namely from the Aspergillus niger K10 strain [8], which was selected by nitrilase activity screening in filamentous fungi. In this study, the gene encoding this enzyme was amplified, cloned and sequenced and the protein deduced from gene sequencing was found to be highly homologous with a number of putative nitrilases in Aspergillus and Penicillium.

The natively expressed enzyme exhibited high specific activities towards (hetero) aromatic nitriles and was fairly stable under operational conditions for its use in nitrile hydrolysis [9]. Here, to potentiate its industrial utility, we expressed this enzyme in Escherichia coli. Heterologous expression has not been reported for any fungal nitrilases, as far as we know. On the other hand, a number of nitrilases from bacteria [1-4,6,10-13], and from the plant Arabidopsis thaliana [14,15] have been expressed in E. coli, as well as several cyanide hydratases from fungi [16].

Recombinant E. coli cells harbouring the A. niger gene produced the active enzyme (Nit-ANigRec). However, this enzyme differed in its catalytic properties from the wild-type enzyme that was purified from A. niger K10 (Nit-ANigWT). The quaternary structures of Nit-ANigRec and Nit-ANigWT were also different. Nitrilases and cyanide hydratases are proteins with unique structural properties, being able to exist in a number of different homooligomeric species - dimers, short homo-oligomeric spirals and extended helices [12,17-19]. The occurrence of these structural types in Nit-ANigRec and Nit-ANigWT was compared, indicating lower tendency of the former enzyme to form long helices. The homogeneity of this enzyme was enhanced by maturing (formation of species differing in molecular weight) during storage of the refolded enzyme, followed by size exclusion chromatography. The resulting protein appeared to be useful for analytical centrifugation and electron microscopy studies. It is also promising for nitrilase crystallization, which is thought to be impaired in enzymes forming the aforementioned helices [12].

A deeper insight into three-dimensional structures of nitrilases is impaired by missing crystal structures. The previous models of nitrilases from Pseudomonas fluorescens [1] and Rhodococcus rhodochrous [12,20] were therefore generated by exploiting their homology with crystallized members of the nitrilase superfamily. Here we have used an analogous approach to construct the first model of a fungal nitrilase, which is distantly related to the above bacterial enzymes.

Results

Determining the Aspergillus niger K10 nitrilase sequence

Previously, the determination of the N-terminal amino acid sequence of Nit-ANigWT suggested a high similarity of this enzyme to a group of highly conserved putative nitrilases (with ≥90% amino acid identity) from the Aspergillus genus (Additional file 1). This enabled us to design degenerate primers, which were based on the N-terminal and a conserved internal sequence of two putative Aspergillus fumigatus nitrilases. Combining the sequence data obtained from amplifications using both nitrilase-specific primers and from 5'-RACE and 3'-RACE amplifications provided a complete sequence of the nitrilase gene (GenBank:ABX75546).

The amino acid sequence deduced from this nitrilase gene confirmed that Nit-ANigWT was highly similar to putative nitrilases from Aspergillus (A. clavatus, A. fumigatus, A. flavus, A. nidulans, A. oryzae, A. terreus) and Neosartorya fischeri (teleomorph of Aspergillus fischerianus). While our study of Nit-ANigWT sequencing was in progress, a sequence of a nitrilase-coding gene (GenBank:XP_001389844) from another A. niger strain (CBS 513.88) was deposited in the database. The amino acid sequence of this hypothetical protein was 99% identical to that of the enzyme being studied by us. Later, another very similar nitrilase (with 89% amino acid identity) (GenBank:XP_002562104) was sequenced in Penicillium chrysogenum. However, neither of these two nitrilases has been studied at the protein level.

Nitrilase expression, purification and refolding

The expression of the enzyme was achieved with the pOK101 and pOK102 vectors and 7 out of the 9 E. coli strains tested (see Methods), the BL21(DE3) and BL21-CodonPlus(DE3)-RIL strains being exceptions. The absence of nitrilase activity in these strains may have been caused by low transformation efficiency, endonuclease activity or limited translation due to codon bias. In all other strains, the nitrilase was expressed after IPTG addition and formed about one half of the soluble cellular proteins, as determined by SDS-PAGE (not shown). However, the nitrilase activities of the recombinant strains were not as high. Total activities of BL21 or Rosetta-gami strains were between 65 and 230 U L-1, while those of Arctic Express strains were on the average lower (34-150 U L-1). These values were at most approx. twofold higher compared to those achieved in the native producer A. niger K10 (ca. 100 U L-1) [8]. However, the time required for maximum activity yield in E. coli was only ca. 20 h, which was approx. three times less than in A. niger. Of the above E. coli strains, the highest activity of approx. 230 U L-1 was obtained in BL21-Gold(DE3) strain carrying the pOK101 plasmid (pET-30(+) containing the nit gene). Further increase in total activity to approx. 500 U L-1 was brought about by variation of cultivation parameters (IPTG concentration, induction time, temperature; data not shown) and by co-expression of pTf16 plasmid (coding for the trigger factor). However, the latter accounted for only about 10% increase in the total activity. This strain designated E. coli BL21-Gold(DE3)(pOK101/pTf16) has been used throughout further work. Nit-ANigRec was purified from this culture to near homogeneity with an approx. 2-fold increase in specific activity and 35% yield (Table 1). As expected from the nitrilase activity of the whole cells, the specific activity of the purified enzyme for benzonitrile - 0.60 U mg-1 - was significantly (two orders of magnitude) lower than that of Nit-ANigWT - 91.6 U mg-1 [8].

Table 1

Purification of recombinant nitrilase from Aspergillus niger K10

Step	Total protein,mg	Specific activity,U mg -1	Total activity,U	Yield,%	Purification,fold
Cell-free extract	444.4	0.29	130.5	100	-
Q-Sepharose	159.5	0.39	61.5	43.2	1.34
Sephacryl S-200	76.7	0.60	46.1	35.1	2.07

Enzyme activity was assayed with 25 mM benzonitrile (see Methods for details).

As the above results suggested the possibility of incorrect protein folding, the protein was fully denatured in 6 M guanidine-HCl and 2 M TCEP (tris-carboxyethylphosphine), and refolded in vitro. However, the best refolding conditions (see Methods) selected by screening the commercial iFOLD 1 system merely led to the recovery of the initial activity and not to its improvement.

According to SDS-PAGE analysis (not shown), the apparent molecular weight of the subunit of the purified enzyme (42.7 kDa) was higher than that of Nit-ANigWT - 38.5 kDa [8]. This indicated that the latter protein underwent a post-translational modification in its native environment. In order to clarify the molecular nature of this difference, we performed N-terminal sequencing and peptide mass mapping with both Nit-ANigRec and Nit-ANigWT (Figure 1). The N-terminal sequence of both enzymes was identical, indicating that the processing occurred most probably at the C-terminus of the enzyme. Indeed, peptide mass mapping using both trypsin and Asp-N in gel digestion revealed that the C-terminal tryptic peptide Val327 - Asn356 was present in Nit-ANigRec but absent in Nit-ANigWT. More specifically, the Asp-N generated peptide Asp298-Val313 detected in the recombinant protein was shortened to Asp298-Arg310 in the native enzyme. These results provide evidence that Nit-ANigWT was shortened by 46 amino acid residues at the C-terminus, and is composed of Met1 - Arg310 of amino acid sequence coded by the corresponding nitrilase gene.

Figure 1

Summary of the sequence analysis of heterologously expressed nitrilase . Sequence analysis was performed by automated Edman degradation of nitrilase blotted onto PVDF membrane (underlined) in combination with peptide mass mapping using MALDI TOF mass spectrometry of peptides extracted after in gel digestion with trypsin (bold) or Asp-N protease (italics). The position of C-terminal truncation by 46 amino acids in the native preparation is indicated by an asterisk.

Preparation of homogeneous enzyme for structural studies

Electron microscopy study of Nit-ANigRec showed heterogeneous population of particles of different shapes from nearly isometric ones in size of about 14 nm to elongated ones reaching over 30 nm in length. Additionally, smaller particles of different shapes and some bigger clusters were also observed (Additional file 2). However, long filamentous structures typical for Nit-ANigWT [17] were not observed in this sample. Despite the limited ability of Nit-ANigRec to form the aforementioned filamentous structures, the purified enzyme was not suitable for structural studies or protein crystallography. The refolded protein (see above) also still exhibited some molecular heterogeneity as revealed by electron microscopy (Additional file 3). Though the gel-filtration chromatography fractionated the refolded enzyme as a single major peak (Mw ≅ 600 kDa), peak fronting and the appearance of a minor peak preceding the major one suggested that a small part of the enzyme aggregated into higher-molecular weight species (Figure 2A). After a 1-month storage of the major-peak protein fraction at 4°C, this aggregation occurred again, as well as a notable shift of the molecular mass of the enzyme towards lower values (about 500 kDa; Figure 2B). After removing the aggregated form, the rest of the enzyme remained rather stable, since after 10 more days of storage under the same conditions, fewer aggregates could be observed, without any further change in enzyme size (Figure 2C). Nevertheless, even after such "maturation" in enzyme quaternary structure, the enzyme was composed of a rather heterogeneous mixture of molecular forms, which were separated by gel filtration and examined by electron microscopy (Figure 2D through 2G). In the early eluting fractions the enzyme occurred in the form of short tubes and was rather heterogeneous, whereas the late eluting fractions contained the enzyme in more homogeneous forms (cf. Figure 2Dvs. 2G).

Figure 2

Effect of aging on the quaternary structure of recombinant refolded nitrilase. Gel filtration analysis on Superose 6B of freshly refolded enzyme (A), and enzyme stored for 30 and 40 days at 4°C (B and C, respectively). Fractions were collected from the last separation, and analyzed by electron microscopy using material eluted between 26 and 27 min (D), 30 and 31 min (E), 33 and 34 min (F), and 37-38 min (G). The homogeneous round-shaped particles observed in the latter fractions (G) were analyzed in an analytical ultracentrifuge using sedimentation velocity (H) and sedimentation equilibrium (I) experiments as detailed in Methods. Fitted data with residual plots showing goodness of fit are shown together with calculated continuous size distribution c(s) of sedimenting species.

Data obtained from sedimentation velocity analysis of the latter fractions (Figure 2H) suggested a rather broad mass distribution of sedimenting species with values of apparent sedimentation coefficients ranging between 10 and 30S, the majority (approx. 70%) of particles falling between 12 and 22S, in 95% confidence level. Integration of size distribution for the main particle fraction yielded a weight average sedimentation coefficient s* = 16.8 ± 2.4S (s20,w = 17.8 ± 2.0S) and a frictional coefficient ratio f/f0 = 1.42 corresponding to a moderately elongated particle. Global analysis of sedimentation equilibrium data (Figure 2I) resulted in weight average particle mass of 564 ± 5 kDa in 95% confidence level, with almost no observable tendency to aggregate in the time course of the experiment (as judged from the residual plot of fit analysis). Based on the value of sedimentation coefficient, frictional coefficient ratio and observed particle mass, the size and shape of the majority of particles was estimated as 20 × 10 ± 5 nm and this correlates with electron microscopy (Figure 2G). Taking into account the theoretical molar mass of nitrilase monomer is 40 kDa, we can conclude that majority of observed nitrilase oligomers was composed of 14 ± 2 nitrilase subunits, as deduced from a combination of data from SDS electrophoresis, gel filtration, electron microscopy and analytical ultracentrifugation, although higher oligomers were still present in significant amount.

In agreement with the results obtained by other techniques, DLS (dynamic light scattering) measurements (Table 2) confirmed the gradual decrease in size in the above protein fractions starting with 22.8 nm oligomers (Mw > 1 MDa) down to particles having a diameter of about 14.8 nm (Mw ≅ 370 ± 50 kDa). All fractions analysed by DLS (Table 2) are polydisperse with a polydispersity index (PdI) in the range 0.23-0.30 with equal data quality. Fractions with the lowest PdI correspond to the second half of the gel filtration peak. The smallest particle size and the highest homogeneity make these fractions most suitable for further analyses including prospective protein crystallization attempts.

Table 2

Measurement of size and heterogeneity of recombinant refolded nitrilase in fractions separated by gel filtration (see Figure 2) using dynamic light scattering (DLS)

Elution time, min	Diameter, nm	Mw, kDa	PdI
32	22.8	1000	0.30
34	19.7	710	0.24
36	17.0	500	0.29
38	14.5	350	0.23
40	14.8	370	0.23

PdI = polydispersity index

Homology modelling and molecular dynamics

A BLAST search identified five proteins with relevant known structures: the NitFhit protein from Caenorhabditis elegans (pdb-code 1EMS) [21]; hypothetical protein Ph0642 from Pyrococcus horikoshii (1J31) [22]; Nit3 protein from Saccharomyces cerevisiae (a member of branch 10 of the nitrilase superfamily, pdb-code 1F89) [23]; the pyrimidine degrading enzyme from Drosophila melanogaster (2VHH) [24] and mouse nitrilase-2 (2W1V) [25] with corresponding identities of 22, 23, 20, 21 and 22%. Although the percentage of identity is at the lower threshold for homology modelling, 3D alignment with the SHEBA plug-in in YASARA showed a high conservation of secondary structure elements among the selected templates, thus supporting the attempt to at least obtain a useful low-resolution homology model. The C-terminal part (residues 316-356) was modelled based on the crystal structure of kinesin from Rattus norvegicus (2KIN, 29% of identity) [26] using residues 136-183 as a template, as this part has been lost in hydrolases. Figure 3 shows a structure-based multiple sequence alignment of nitrilase from Aspergillus niger with selected templates and sequences of previously published homology models of nitrilases from R. rhodochrous J1 [12,20] (identity 38%) and Pseudomonas fluorescens [1] (26%). Secondary structure is given as assigned by Procheck [27]. Figure 4A shows a view of the enzyme with the catalytic domain on the left and the active site in the domain center. The three long loops at the entrance of the active site are interesting features of the modelled structure. Loops including the residues that correspond to 236-252 and 55-64 between β2 and α2 and 236-252 between β10-β12 (coloured magenta in Figure 4A) in the primary sequence were found in just one template structure, 2VHH, but these residues were not resolved in the crystal structure [24]. A loop corresponding to 196-207 between β8 and α6 (yellow in Figure 4A) was not found in any template structure (Additional file 4). The nitrilase from Rhodococcus rhodochrous J1 [12] presents similar residues at the corresponding primary sequence positions, and similar external loops in its homology model. The nitrilase from Pseudomonas fluorescens lacks the insertion at the position similar to 196-207 but it has one additional loop between β14 and α7.

Figure 3

Multiple sequence alignment. (A) Multiple sequence alignment of A. niger K10 with template structures 1EMS [21], 1J31 [22], 1F89 [23], 2VHH [24] and 2W1V [25] (letters in upper case) and sequences of homologous nitrilases (letters in lower case) from Rhodococcus rhodochrous J1 (RrJ1) [12] and from Pseudomonas fluorescens (Pf-5) [1]. Clustal W scheme is used for marking similar residues. Amino acids from catalytic triad are strongly conserved; they are marked by red arrow and enclosed in blue rectangles. Secondary structures for template 1EMS and A. niger K 10 nitrilase model are shown above and under the aligned sequences, respectively, with numeration as in [12]. (B) Sequence alignment of A. niger K 10 with 2KIN [26] was used for modelling the C-terminal part. Secondary structure as assigned by Procheck [27] is shown for A. niger K 10 and for 2KIN above and under the aligned sequences, respectively.

Figure 4

Homology model of nitrilase (A) and active site amino acids (B) with docked benzonitrile. Loops formed by residues 55-64 and 236-252 are coloured magenta and loop formed by residues 196-207 is yellow. The catalytic domain is on the left side. Active site amino acids with docked benzonitrile (magenta) after 2 ns of molecular dynamics simulation (B). Hydrogen atoms are omitted. The catalytic triad is represented by Glu 48, Lys 130 and Cys 165. The only hydrogen bond (yellow dotted line) is created by hydrogen atom of Lys 130 and nitrogen of benzonitrile.

A docking attempt in AutoDock, using benzonitrile as the substrate, found a position in the centre of the enzyme with the lowest binding energy, and thus the highest affinity. This position involved the predicted triad of active residues (Figure 4B; Additional file 4), demonstrating the basic correctness of the modelled structure.

The geometrical parameters of spiral structures obtained from electron microscopy were used to draft a plausible multimeric arrangement. The electron micrographs corresponded in size and general shape to helical segments made up of dimers (Figure 5A and 5C). Hereby, taking into account the size and shape of the monomeric model, approximately 16 subunits would be organized in a spiral or helical arrangement and 8 dimers would form one helical turn that could be extended in both directions (Figure 5B and 5D). Similar loops are found at the C-surface in the helical-like form of the nitrilase from R. rhodochrous [12]. 14-16 subunits in the multimeric structure can be assumed for the aliphatic nitrilase from R. rhodochrous K22 [28] which has an identity of 42% with the nitrilase from A. niger K10.

Figure 5

Nitrilase multimer. (A) and (C) panels are images from electron microscope, (B) and (D) - overlay of top view and side view, respectively, of constructed multimer and image from electron microscope.

Comparison of reaction optima, substrate specificity, selectivity and stability of the heterologously expressed nitrilase and the nitrilase isolated from the native organism

The optimal reaction conditions of Nit-ANigRec and Nit-ANigWT were different. Nit-ANigRec exhibited a lower temperature optimum (38 vs. 45°C) when assayed after 10-min reaction time. Its activity decreased to 55 and 4% at 45 and 50°C, respectively, while Nit-ANigWT retained significant activity up to 55°C. The pH-range of Nit-ANigRec (ca. pH 5.5-9.5) was slightly shifted towards lower values compared to that of Nit-ANigWT (ca. pH 6-10).

Nit-ANigRec, incubated for 1 h at 40, 45 and 50°C, exhibited a residual activity > 80, 36 and 1.3%, respectively. Its stability was thus lower than that of Nit-ANigWT, which still exhibited 59, 24 and 6% of the maximum activity after 1-h incubation at 45, 50 and 55°C, respectively. The effects of various additives on Nit-ANigRec activity were similar to those reported for the Nit-ANigWT [8]. p-Hydroxymercuribenzoate, Hg2+, Ag+ and Al3+ ions completely abolished the activity of both preparations.

The relative activities of Nit-ANigWT decreased in the order 4-cyanopyridine > benzonitrile > 3-chlorobenzonitrile > 4-chlorobenzonitrile > phenylacetonitrile > 3-cyanopyridine > 2-cyanopyridine >> 2-phenylpropionitrile. In contrast, the best substrate of Nit-ANigRec was 2-cyanopyridine, followed by 3-cyanopyridine and 3-chlorobenzonitrile (Table 3). Thus 2-cyanopyridine was the only substrate for which Nit-ANigRec exhibited a similar or higher activity (9 U mg-1 of protein at 38°C after 10-min reaction time or 26 U mg-1 of protein 45°C after 1-min reaction time) compared to Nit-ANigWT (7 U mg-1 of protein at 45°C after 10-min reaction time). Other substrates were transformed at very low rates or not transformed at all. In Nit-ANigRec, the production of amide by-product was most significant with 2-cyanopyridine (23% amide in total product). Nevertheless, this was much less than with Nit-ANigWT, which gave a product consisting of up to 88% picolinamide. From the other substrates tested, the recombinant nitrilase produced only a maximum of 5% amide in the total product, in contrast to the enzyme isolated from the native organism producing high amounts of amides also from 4-chlorobenzonitrile and 4-cyanopyridine (Table 3).

Table 3

Substrate specificity and chemoselectivity of purified nitrilase isolated from A. niger K10 (Nit-ANigWT) and heterologously expressed nitrilase (Nit-ANigRec)

Substrate	Relative activity, %		Amide, molar % of total product
	Nit-ANigWT	Nit-ANigRec	Nit-ANigWT	Nit-ANigRec
Benzonitrile	27	4.9	9	0
2-Chlorobenzonitrile	0	0	-	-
3-Chlorobenzonitrile	10	3.7	3	5
4-Chlorobenzonitrile	8.4	0.2	80	0
2-Cyanopyridine	2.4	100	88	23
3-Cyanopyridine	4.6	12.9	6	2
4-Cyanopyridine	100	0.8	36	0
Phenylacetonitrile	4.9	0.2	0	0
2-Phenylpropionitrile	traces	0	0	0

Enzyme activity was assayed as described in Methods. The specific activities of Nit-ANigWT and Nit-ANigRec for their best substrates 4-cyanopyridine (306 U mg-1 at 45°C) and 2-cyanopyridine (9.0 U mg-1 at 38°C), respectively, were taken as 100%. Data represent the mean of four independent measurements with relative standard deviation values <5%.

All the compounds tested as potential stabilizers of the nitrilase (sugars, sugar alcohols, albumin, glycine) improved the Nit-ANigRec stability to a significant extent during either incubation at 38°C or repeated freezing/thawing cycles (Table 4). Without any stabilizer, the enzyme retained about 36 and 60% of its initial activity, respectively, but full activity was preserved in the presence of 1% glycine as the most powerful stabilizer. The combined action of freezing/thawing (20 cycles) and 3-h incubation at 45°C decreased nitrilase activity by >90% (to 0.057 U mg-1 protein). However, the same treatment in the presence of glycine, D-sorbitol, xylitol or glucose (5% each) allowed a 4-5-fold higher enzyme activity recovery (data not shown). A mixture of glycine and ammonium sulfate proved to be most efficient, enhancing the final activity by nearly a factor of 14 compared to the control without stabilizer.

Table 4

Effect of potential stabilizers on nitrilase activity

Compound (concentration)	Residual activity, %
	Method A	Method B
None	36.3	60
Glycine (1%, w/v)	100	100
Sucrose (10%, w/v)	56.1	64.8
D-Glucose (10%, w/v)	73.7	92.7
Trehalose (10%, w/v)	48.5	89.7
D-Sorbitol (10%, w/v)	59.1	93.9
Xylitol (10%, w/v)	32.7	85.4
D-myo-inositol (10%, w/v)	57.3	97.0
D-Glycerol (10%, w/v)	54.4	86.6
Bovine serum albumin (0.1%, w/v)	56.7	87.0
Bovine serum albumin (1%, w/v)	64.9	80.6

Potential stabilizers were pre-incubated with the enzyme (0.28 mg of protein mL-1) for 180 min (method A) or added to enzyme solutions of the same protein concentration, which were then frozen and thawed 10 times (method B). The enzyme activity was assayed as described in Methods. The specific activity in the presence of 0.1% (w/v) glycine (0.70 and 0.67 U mg-1 protein in method A and B, respectively) was used as the reference value. Data represent the mean of four independent measurements with relative standard deviation values <5%.

Discussion

A large number of putative nitrilase and cyanide hydratase sequences are contained within the whole genomic sequences of fungi. As far as we know, none of the sequenced fungal nitrilases which were predicted to act on organic nitriles have been characterized, contrary to the situation with the fungal cyanide hydratases. Likewise, no sequence data have been available for the characterized nitrilases from Fusarium solani IMI196840 [29] and Fusarium oxysporum f. sp. melonis [30]. Only recently have partial amino acid sequences been identified in the nitrilases from Fusarium solani O1 [17] and Fusarium solani IMI196840 [31], the latter enzyme being probably different from that previously characterized in the same strain [29].

The putative nitrilases of the Aspergillus genus can be roughly divided into two groups, which share a relatively low degree of amino acid identity (30-40%) [7]. One of these groups is closely related to cyanide hydratases (with ca. 60-85% amino acid identity) and the A. niger K10 nitrilase was shown to be a member of this group. The high tendency of this enzyme to form amides from nitriles [8] is in accordance with its evolutionary relationship to cyanide hydratases, the reaction product of which is formamide [16,32].

The heterologous expression of the enzyme in E. coli BL21-Gold(DE3)(pOK101/pTf16) led to a notable increase in enzyme productivity (25.8 U L-1 h-1) under optimized conditions, which was fifteen times higher than in the native producer (approx. 1.7 U L-1 h-1). The potential to synthesize the active enzyme may be even higher in the heterologous producer as indicated by the high ratio of nitrilase to other cellular proteins. However, the output of Nit-ANitRec production was lessened by the low specific activity of the enzyme (at least when using benzonitrile as substrate). When the productivity of Nit-ANigRec and Nit-ANigWT was compared using the preferred substrate of the former enzyme, 2-cyanopyridine, that of the heterologous host was three orders of magnitude higher than that of the native producer.

In comparison with Nit-ANigWT, Nit-ANigRec produced a lower percentage of amide in total product from all substrates tested. With 2-cyanopyridine, the major products of the reaction were different, that is picolinic acid (77% of total product) and picolinamide (>80% of total product) with the Nit-ANigRec and Nit-ANigWT, respectively. Picolinic acid is an intermediate in the production of pharmaceuticals such as local anaesthetics. Nitrilases with satisfactory activities towards 2-cyanopyridine have rarely been reported. The best activity for this compound (approx. 1 U mg-1 protein) was reported in the thermostable nitrilase from Bacillus pallidus Dac521 [33]. This was much less than the activity determined for Nit-ANigRec (9 U mg-1 protein at 38°C).

Nit-ANigRec was less stable than the Nit-ANigWT but this drawback could be overcome by using some low-molecular-weight compounds or bovine serum albumin. These compounds, known collectively as osmolytes, have been recognized as efficient agents in protein stabilization [34]. Of the compounds tested, glycine (1%) was most efficient for the A. niger K10 nitrilase. Glycine and related compounds (sarcosine, betaine) were described as powerful agents able to protect proteins against thermal unfolding [34,35].

Nitrilases forming spiral structures differ from their nonspiral-forming homologs by two insertions of between 12 and 14 amino acids, and a C-terminal extension of up to 35 amino acids [36]. Recently, detailed structural reconstructions using electron microscopy and molecular modelling reported that the formation of spiral helices in the natively produced nitrilases may be related to the removal of 39 C-terminal amino acids from the wild-type protein [12]. This post-translational modification was postulated to be due to autocatalytic activity of this enzyme. The approx. 4-kDa difference in molecular weights of Nit-ANigRec and Nit-ANigWT suggested that a similar-sized peptide was cleaved in the latter enzyme. This assumption was verified by mass spectroscopic analysis, indicating missing cleavage of 46 amino acid residues at the C-terminus of Nit-ANigRec. The R. rhodochrous nitrilase consisting of full-length subunits was unable to form filamentous structures, which were reported for the post-translationally modified enzyme [12], and also for cyanide hydratase [19,37] and cyanide dihydratase [38]. In accordance with these observations, Nit-ANigWT was to a large extent composed of tube-like structures [17], while Nit-ANigRec exhibited a limited tendency to this arrangement.

The reason for the differences in catalytic properties (substrate specificity, reaction optima, amide formation, stability) between Nit-ANigRec and Nit-ANigWT is not clear but most probably it can be ascribed to differences in the post-translational processing of the two forms of the enzyme, and its subsequent effects on the folding, subunit interaction, and oligomerization of the enzyme. A recent mutational analysis revealed a number of effects caused by deletions or mutations in the C-terminal portion of arylacetonitrilase from Pseudomonas fluorescens EBC191 [39]. In this enzyme, the C-terminal deletions of up to 32 amino acids did not cause any differences in the catalytical properties. However, longer deletions of 47 to 67 amino acids resulted in reduction of enzymatic activity, increased formation of amide, and in changes in the enantiomeric selectivity. The effects caused by C-terminal deletions could be reversed by the addition of the corresponding sequences from another nitrilase [39]. It appears difficult to determine what is the relation of the above changes to those caused by 46 amino acid difference observed in the fungal nitrilase described here, and this issue certainly deserves detailed investigations in the future. It remains also unclear if missing post-translational modification is the primary event leading to partial enzyme misfolding, or if this misfolding negatively affected the autocatalytic cleavage of the enzyme.

The changes in catalytic behaviour could be also caused by differences in quaternary structure between Nit-ANigRec and Nit-ANigWT. Similarly, a small increase in activity was associated with fibre formation in cyanide dihydratase in Bacillus pumilus [36].

Attempts to express the enzyme in a eukaryotic host (Yarrowia lipolytica; D. Brady et al., personal communication) did not bring about any positive effect on the enzyme activity, which was barely detectable in the yeast cells. The effect of chaperone co-expression in E. coli was not very efficient in the heterologous expression of this enzyme either, though previous experiments suggested the importance of chaperones for the correct folding of proteins of the nitrilase superfamily. Chaperones were co-purified with nitrilases from Bacillus pallidus [33], Pseudomonas fluorescens [40] and A. niger [8] and also played an important role in folding D-carbamoylase [41]. In vitro re-folding of the enzyme from A. niger was also tested as a potential tool to improve its specific activity but did not prove successful. It appears that re-folding is not necessary for heterologous production of fungal cyanide hydratases or nitrilases as a number of them were produced as fully functional enzymes in E. coli [e.g., [12,16,19]].

As far as we know, little or nothing has been reported on the differences between heterologously expressed nitrilases and nitrilases isolated from the native organisms. This is probably because a number of known nitrilases have been purified and characterized either purely from the heterologous host or purely from the native producer. The enzyme with the highest homology to A. niger nitrilase, cyanide hydratase from A. nidulans (with 86% amino acid identity), was only examined with a single substrate (HCN) [16] and not compared with the purified enzyme from the wild-type producer as far as we know. Even if both enzyme forms were available in bacterial nitrilases [e.g., [12,42]], the enzymatic properties have rarely been compared under the same conditions. Therefore, potential differences between the nitrilases isolated from the native organisms and the heterologously expressed nitrilases may have gone unnoticed. Differences in biochemical properties of different nitrilase species may reflect partial misfolding of individual subunits, different post-translational modifications, but the diversity of the enzymes in terms of structural variants (dimers, short spirals, filaments) may be also important in this respect.

Conclusions

In conclusion, heterologous expression of a fungal nitrilase operating on organic nitriles was achieved in this study for the first time. The enzyme differed from that produced by the wild-type strain A. niger K10 in subunit molecular weight due to a missing post-translational modification (46 C-terminal amino acid cleavage). Nevertheless, it may still be useful for some biocatalytic applications, and even gained some advantages over the enzyme isolated from the native organism, such as a higher hydrolytic activity for 2-cyanopyridine. Purification of the enzyme from the heterologous host was straightforward, enabling tens of mg of the purified protein to be obtained for structural and activity studies. The refolded enzyme underwent changes in its oligomeric structure during storage and was finally fractionated to give a structurally almost homogeneous protein potentially useful for crystallographic nitrilase studies, which have so far been largely impaired by specific quaternary arrangement of these enzymes.

Methods

DNA manipulations

Total RNA was isolated from A. niger K10 using RNeasy Plant Mini Kit (Qiagen) and used to synthesize cDNA using SuperScript II Reverse Transcriptase (Invitrogen) and anchored oligo d(T)23 VN primer (NEB). Partial cDNA of the nitrilase gene was amplified using the degenerate forward primer NITR_NTERMFW01, 5'-AAY GCI GAR CCI GGI TGG TTY GA-3', derived from the N-terminal fragment sequence NAEPGWFD of Aspergillus fumigatus nitrilases [GenBank:EDP55254, XP_756085] and degenerate reverse primer NITR_INT2RE01, 5'-CAT RTA RTG ICC ICC RAA RTC IGC-3', derived from the internal peptide fragment sequence ADFGGHYM from the same A. fumigatus nitrilases. The PCR product (about 0.9 kb) obtained after 35 cycles of PCR on a Mastercycler personal cycler (Eppendorf) with proofreading DNA Polymerase Pfu Turbo (Stratagene) was cloned into the pBluescript SK+ vector (Stratagene) and sequenced using automated DNA sequencer (ABI PRISM 3130xl) according to the manufacturer's protocols.

To obtain 5' and 3' cDNA end sequences, a BD SMART RACE cDNA Amplification Kit (Clontech) was employed. 5'-RACE and 3'-RACE amplifications were performed using 30 cycles of PCR with BD Advantage 2 DNA Polymerase (Clontech). A common universal primer (UPM) was used for the amplifications of cDNA ends together with two distinct gene-specific primers: 5'-RACE reverse primer, NITR_RACE_FW01, 5'-CCG CGT CGG CCA CCT CAA CTG CTG GGA G-3', and 3'-RACE forward primer, NITR_RACE_RE01, 5'-CGT GGA CCT GCT CGC CCA AAG AGG CTG C-3', respectively. The resulting 5'-RACE and 3'-RACE PCR products (approx. 0.6 and 0.7 kb, respectively) were cloned and sequenced as described above.

In order to amplify the DNA fragment coding for the nitrilase sequence, forward primer 5'-GCC ATA TGG CAC CMG TCT TRA AGA AGT ACA A-3', (M = A or C; R = A or G), and reverse primer, 5'-GCA AGC TTT TAC TAG TTC TCC GAA TCC ACG GT-3' were used. The corresponding PCR product obtained after 30 cycles of PCR with Taq-DNA polymerase (Clontech) was ligated into the cloning vector pCR 2.1 TOPO (Invitrogen), cloned in One Shot® TOP10 E. coli Competent Cells (Invitrogen) and sequenced as described above.

Database searches were performed using the BLASTX and BLASTP programme [43,44]. Alignment of amino acid sequences was performed using ClustalW software [45].

For expression, the NdeI-HindIII fragment was ligated into the corresponding sites of the vectors pET-30a(+) (Novagen) and pRSET B (Invitrogen). The resulting vectors pOK101 and pOK102 were transformed into selected strains of E. coli (see below). Alternatively, E. coli was transformed with plasmid pOK101 and plasmid pTf16 (Takara) containing the tig gene which encodes the trigger factor.

Microbial cultures and plasmids

A. niger K10 was grown as described previously [8]. E. coli strains were grown in LB broth at 28°C (strains BL21(DE3), BL21-Gold(DE3), BL21-Gold(DE3)pLys, BL21-CodonPlus(DE3)-RIPL, BL21-CodonPlus(DE3)-RIL (Stratagene), Rosetta-gami 2 (DE3) (Novagen)) or 14°C (strains Arctic Express(DE3), Arctic Express(DE3)-RP and Arctic Express(DE3)-RIL (Stratagene)). Cultures of strains harbouring plasmid pOK101, pOK102 or both plasmids pOK101 and pTf16 (Takara) were performed with kanamycin (50 μg mL-1), ampicillin (150 μg mL-1) or ampicillin (150 μg mL-1) and chloramphenicol (20 μg.mL-1), respectively, in addition to selected antibiotics, with resistance to them encoded in the host chromosome, i.e. chloramphenicol (35 μg mL-1; strain BL21-Gold(DE3)pLys), tetracycline (12.5 μg mL-1; strain BL21-Gold (DE3), both chloramphenicol and tetracycline (strains BL21-CodonPlus(DE3)-RIPL and BL21-CodonPlus(DE3)-RIL, Rosetta-gami 2(DE3)), or gentamycin (20 μg.mL-1; Arctic Express strains). The expression of nitrilase was monitored after induction with IPTG (1 mM) by nitrilase activity assay using whole cells (see below) and by 12% SDS-PAGE [46] followed by Coomassie staining.

Enzyme purification

Nit-ANigRec was purified from the culture of E. coli BL21-Gold(DE3)(pOK101/pTf16). The culture was grown under the following optimized conditions: arabinose (2 g L-1) and IPTG (0.5 mM) addition to cultures with OD610 of 0.6 and 1.1, respectively, and cultivation temperature shift from 37 to 26°C after induction with IPTG. The cells were harvested at OD610 ≈ 8.7 (16 h after IPTG addition) and disrupted by sonication. After removing cell debris by centrifugation (13,000× g, 4°C, 30 min), the supernatant proteins were eluted through a Hi-Prep 16/10 Q FF column (Amersham Biosciences), with a linear gradient of NaCl (0.15-1 M) in Tris/HCl buffer (50 mM, pH 7.6). Active fractions were pooled, concentrated using an Amicon Ultra-4 unit (cut-off 10 kDa; Milipore) and injected into a Hi-Prep 16/60 Sephacryl S-200 column. The proteins, eluted with Tris/HCl buffer (50 mM, pH 7.6, 150 mM NaCl), were pooled, concentrated, analyzed by SDS-PAGE as described above and stored at -80°C.

The refolded enzyme was purified in two steps consisting in Q-Sepharose HP chromatography and gel filtration on Superose 6 Prep Grade. The sample was injected into a Q-Sepharose HP column (1.6 × 11.5 cm) pre-equilibrated with Tris/HCl buffer (50 mM, pH 7.5; 29 mM NaCl, 1 mM NaN3). Proteins were eluted with a linear gradient of NaCl (0.029-1 M) in Tris/HCl buffer (50 mM, pH 7.5; 29 mM NaCl, 1 mM NaN3), concentrated and injected into a Superose 6 Prep Grade column (1 × 25 cm). Proteins were eluted with Tris/HCl buffer (50 mM, pH 7.5; 150 mM NaCl). Those with a molecular weight of approx. 600 kDa (major peak) were collected and stored for one month at 4°C. Then the gel filtration was repeated, the fractions (eluted as the major peak of approx. 500 kDa) pooled, concentrated and stored at 4°C for a further 10 days. Gel filtration was repeated with this sample and the active fractions analyzed separately for enzyme activity, by analytical centrifugation and by electron microscopy.

Nit-ANigWT was purified as described previously [8].

Protein concentration was determined according to Bradford [47] using bovine serum albumin as the standard.

Refolding

Refolding conditions were screened using iFOLD Protein Refolding System 1 (Novagen). The purified enzyme (1 mg; 0.1 mL of Tris/HCl buffer, pH 7.6) was mixed with 1 mL of 6 M guanidine hydrochloride in phosphate-buffered saline (PBS) consisting of 10 mM sodium phosphate buffer (pH 7.4; 150 mM NaCl) supplemented with 1 mM of tris(2-carboxyethyl)phosphine hydrochloride and 0.03% N-lauryl sarcosine. Each of the refolding agent mixtures available in iFOLD Protein Refolding System 1 was transferred into a well of a Greiner BioOne UV Star microplate and 0.010 mL of enzyme solution added into each well. After a 20-h incubation of the microplate with periodic shaking, optical density at 340 nm was determined using a Safire microplate reader (TECAN). Samples with the lowest level of precipitation were assayed for nitrilase activity.

The refolding conditions selected via the above screening were used on a larger scale (1-10 mg protein). The purified enzyme (1-10 mg in Tris/HCl buffer, pH 7.5) was denatured as described above in a total volume of 10 ml. The sample was sonicated and mixed with 90 ml of 50 mM Tris buffer, pH 7.5, containing 100 mM NaCl, 20% glycerol, 12.5 mM methyl-β-D-cyclodextrin and 1 mM NaCl (buffer C12 according to iFOLD Protein Refolding System 1; approx. 10 mL mg-1 protein). Thereafter, the refolding mixture was dialyzed against 2 L of 50 mM Tris/HCl buffer (pH 7.5, 29 mM NaCl, 1 mM NaN3). After 4 h at 4°C, the dialysis buffer was changed for 2 L of fresh buffer, and dialysis was left to proceed overnight. The refolded nitrilase was recovered using Q-Sepharose and Superose 6 chromatographies as described above.

Mass spectrometry analysis

Peptide mass fingerprinting of fragments obtained by tryptic or Asp-N digestion of Nit-ANigRec and Nit-AnigWT was as decribed previously [8].

Determination of N-terminal amino acid sequence

The N-terminal sequences of Nit-ANigRec and Nit-ANigWT were analyzed as described previously [8].

Enzyme assays

The nitrilase activity was determined with 25 mM benzonitrile as described previously [8] with slight modification. If not stated otherwise, reaction temperature was 30 or 38°C with recombinant cells or Nit-ANigRec, respectively, instead of 45°C with Nit-AnigWT. Substrate specificity was determined using 25 mM of various nitriles as substrates under the above conditions. The substrates and reaction products were analyzed by HPLC.

Analytical HPLC

Benzonitrile, its analogues, phenylacetonitrile, 2-phenylpropionitrile and the corresponding reaction products (acids, amides) were analyzed using a Chromolith Flash RP-18 (Merck; 25 mm × 4.6 mm) in a mobile phase consisting of acetonitrile : water : H3PO4, 200 : 799 : 1 (flow-rate 2 mL.min-1; 35°C). Heterocyclic nitriles and their products were analyzed as described previously [9].

Analytical ultracentrifugation

Sedimentation velocity and sedimentation equilibrium experiments were performed using a ProteomeLab XL-I analytical ultracentrifuge (Beckman Coulter) using an An50Ti rotor, and dual absorbance and laser interference optics. Before the experiment, 0.5 mL samples of nitrilase diluted to 0.4 mg.mL-1 were dialyzed for 20 h against 2 L of 50 mM Tris-HCl pH 7.5 with 150 mM NaCl and 1 mM NaN3, and the dialysis buffer was used as a reference and sample dilution buffer. The sedimentation velocity experiment was conducted at 15,000 rpm and 20°C using an epon double-sector cell (Beckman Coulter). Sample (400 μL) and dialysate (430 μL) were loaded into the sample and reference cells, respectively. Based on buffer composition and nitrilase amino acid sequence using the program SEDNTERP [48], buffer density and nitrilase partial specific volume were estimated to be 1.00585 g.mL-1 and 0.7331 mL.g-1, respectively. Absorbance scans were performed at 280 nm with 5 min intervals using a spacing of 0.003 cm in continuous scan mode and were analyzed with the program SEDFIT [49,50]. A continuous size-distribution for non-interacting discrete species model was calculated and the sedimentation coefficient value determined by integration. Sedimentation equilibrium ultracentrifugation runs were performed with nitrilase concentrations of 0.4, 0.2 and 0.1 mg.mL-1 and at speeds of 3,000, 3,500, 4,000, 4,500, 5,000, 5,500 and 6,000 rpm in a six-channel epon cell for 16 h at 20°C with 110 and 130 μL of sample and reference, respectively. Absorbance data was collected at 280 nm by averaging 20 scans with radial increments of 0.001 cm in step scan mode. The sedimentation equilibrium experiments were globally analyzed with the program SEDPHAT version 6.21 [50,51]. The size and shape of the sedimenting species was predicted using the Teller method in the program SEDNTERP.

Dynamic light scattering

The particle size distribution of the nitrilase solution from individual fractions obtained by gel filtration was assessed using the dynamic light scattering method (Malvern Instruments, ZEN3600) in a low volume glass cuvette (45 μL) at 18°C with an appropriate enzyme concentration in 50 mM TRIS, 150 mM NaCl, 1 mM NaN3, pH 7.5, sample volume 30 μL. The particle diameter values corresponding to the maxima of peaks in the mass distribution are reported. Molecular weight estimations were made using an empirical mass vs. size calibration curve of the instrument software (Dispersion Technology Software 5.03, Malvern Instrument).

Electron microscopy

Protein complexes were negatively stained (2% uranyl acetate) on glow discharge activated carbon coated grids [52]. Samples were viewed under a Philips CM100 electron microscope at 80 kV. Digital images were recorded using MegaView II slow scan camera at primary magnification of 64,000× resulting in pixel size of 0.98 nm.

The search for homologous structures was done with BLAST [43,44]. Structures with the highest identity were extracted from the Protein Data Bank and used as templates for modelling. A structure-based multiple sequence alignment was done with the T-Coffee server [53] and manually corrected on the basis of consensus secondary structure prediction [54]. Three-dimensional models consisting of all non-hydrogen atoms were constructed with the package Modeller 9.1 [55] and validated with ProSA [56].

YASARA [57] was used for visualization, molecular dynamics simulation (MD) and building the multimeric structure. MD was run in water with Yamber2 force field, with a periodic boundary, in an NPT ensemble (temperature set at 298 K, constant pressure and constant number of particles in the cell). Substrate docking of substrate was done using AutoDock 4.0 [58].

Authors' contributions

LM, KB and OK designed research, LM and KB wrote the major parts of the paper. OK, OP, ABV, AR and OŠ carried out the molecular genetic studies. RE and NK designed the homology model. Structural studies were carried out by KB, DK and OV (re-folding, analytical ultracentrifugation), OB (electron microscopy), JD and JD (dynamic light scattering). OK and AM carried out biochemical characterization. JF analyzed and interpreted the gene sequence. VK, MC, KS and MK critically revised the manuscript draft. All authors read and approved the final manuscript.

Additional file 1

Alignment of DNA sequences of fungal nitrilase and cyanide hydratase genes (pdf file).

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Additional file 2

Electron micrograph of Nit-ANigRec before re-folding (tif file).

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Additional file 3

Electron micrograph of Nit-ANigRec after re-folding (tif file).

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Additional file 4

PDB-file of the refined homology model of nitrilase subunit, prepared in YASARA.

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