Extremophiles (2014) 18:429–440 DOI 10.1007/s00792-014-0628-y

ORIGINAL PAPER

Purification and characterization of a thermostable aliphatic amidase from the hyperthermophilic archaeon Pyrococcus yayanosii CH1 Ling Fu • Xuegong Li • Xiang Xiao Jun Xu

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Received: 5 August 2013 / Accepted: 2 January 2014 / Published online: 16 January 2014 Ó Springer Japan 2014

Abstract Amidases catalyze the hydrolysis of amides to free carboxylic acids and ammonia. Hyperthermophilic archaea are a natural reservoir of various types of thermostable enzymes. Here, we report the purification and characterization of an amidase from Pyrococcus yayanosii CH1, the first representative of a strict-piezophilic hyperthermophilic archaeon that originated from a deep-sea hydrothermal vent. An open reading frame that encoded a putative member of the nitrilase protein superfamily was identified. We cloned and overexpressed amiE in Escherichia coli C41 (DE3). The purified AmiE enzyme displayed maximal activity at 85 °C and pH 6.0 (NaH2PO4– Na2HPO4) with acetamide as the substrate and showed activity over the pH range of 4–8 and the temperature range of 4–95 °C. AmiE is a dimer and active on many aliphatic amide substrates, such as formamide, acetamide, hexanamide, acrylamide, and L-glutamine. Enzyme activity was induced by 1 mM Ca2?, 1 mM Al3?, and 1–10 mM Mg2?, but strongly inhibited by Zn2?, Cu2?, Ni2?, and Fe3?. The presence of acetone and ethanol significantly decreased the enzymatic activity. Neither 5 % methanol nor 5 % isopropanol had any significant effect on AmiE activity (99 and 96 % retained, respectively). AmiE displayed amidase activity although it showed high sequence homology (78 % Communicated by F. Robb.

Electronic supplementary material The online version of this article (doi:10.1007/s00792-014-0628-y) contains supplementary material, which is available to authorized users. L. Fu  X. Li  X. Xiao  J. Xu (&) State Key Laboratory of Microbial Metabolism and School of Life Science and Biotechnology, State Key Laboratory of Ocean Engineering, Shanghai Jiao Tong University, Dongchuan Road 800, Shanghai 200240, China e-mail: [email protected]

identity) with the known nitrilase from Pyrococcus abyssi. AmiE is the most characterized archaeal thermostable amidase in the nitrilase superfamily. The thermostability and pH-stability of AmiE will attract further studies on its potential industrial applications. Keywords Amidase  Nitrilase superfamily  Pyrococcus yayanosii  Hyperthermophilic archaeon  Thermostability

Introduction Amidases (EC 3.5.1.4) catalyze the hydrolysis of amides to the corresponding carboxylic acids and ammonia. These enzymes are widely distributed and involved in carbon/ nitrogen metabolism in all living organisms (Sharma et al. 2009). Amidases belong either to the amidase signature family or the nitrilase superfamily (Nel et al. 2011). The former belongs to the GGSS(S/G)GS signature group containing S-cis-S-K catalytic triads (Kobayashi et al. 1997; Shin et al. 2002). The latter belongs to the nitrilase superfamily with the characteristic Glu–Lys–Cys (C-E-K) catalytic triad residues (Hung et al. 2007; Novo et al. 2002; O’Reilly and Turner 2003). In recent years, another conserved glutamate residue was reported to be essential in several members of the nitrilase superfamily for maintaining the activity, suggesting the existence of a C-E-E-K catalytic tetrad (Kimani et al. 2007; Soriano Maldonado et al. 2011; Weber et al. 2013). Enzymes of the nitrilase superfamily are classified into 13 branches, with eight of the branches having amidase or amide condensation activities (Brenner 2002; Pace and Brenner 2001; Sharma et al. 2009). Crystal structures of bacterial and archaeal enzymes in the nitrilase superfamily have been published (Hung et al. 2007; Kimani et al. 2007;

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Nel et al. 2011; Novo et al. 2002; Sakai et al. 2004). The three-dimensional structures indicate that they exist as active dimers, tetramers, hexamers, or longer oligomeric spirals, with each subunit containing a cysteine residue that is proposed to act as the catalytic nucleophile and a fourlayered abba sandwich monomeric core structure (Sakai et al. 2004). Amidases have been studied extensively because of the increasing demand for them in industrial applications, such as waste water treatment and the production of optically pure compounds (Cantarella et al. 2012; Schoemaker et al. 1997; Sharma et al. 2013; Shaw et al. 2002). A large set of amidase genes of bacterial origin have been cloned and characterized. However, there are only a few reported examples of amidases that are active at high temperatures (Baek et al. 2003b; Makhongela et al. 2007; Nawaz et al. 1996; Sharma et al. 2013; Shaw et al. 2002). In general, running industrial enzyme processes at elevated temperatures has several advantages, including a significant improvement in the rate of reaction, decreasing the possibility of microbial contamination, increasing the substrate solubility, etc. (Haki 2003). Thermophiles often produce thermostable enzymes and are thus an attractive source for finding novel enzymes (Egorova and Antranikian 2005). Hence, the exploitation of thermostable nitrilase superfamily enzymes from hyperthermophilic archaea is frequently under consideration for the synthesis of various carboxylic acids. Pyrococcus yayanosii CH1 was isolated from a sample collected at a deep-sea hydrothermal vent site. The strain CH1 can grow within a temperature range of 80–108 °C and a pressure range of 20–120 MPa, with optima at 98 °C and 52 MPa, respectively (Zeng et al. 2009). The released genome of CH1 (Xu et al. 2011) was subjected to BLAST analysis. Two hits belonging to the nitrilase superfamily, namely PYCH13550 and PYCH10400, showed low similarity to each other. PYCH10400 showed high identity ([70 %) to PH0642 from Pyrococcus horikoshii and a nitrilase from Pyrococcus abyssi. However, the real function of PYCH10400 still was unknown. In this paper, we describe the cloning, overexpression and characterization of PYCH10400 (named AmiE in this work).

Materials and methods Strains, plasmids, enzymes, and chemicals Pyrococcus yayanosii CH1, E. coli DH5a, E. coli C41 (DE3), and pET-28a (?) were obtained from laboratory stocks. Restriction enzymes were obtained from New England Biolabs (Beverly, MA, USA). DNA molecular weight markers and protein molecular markers were

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obtained from MBI (Vilnius, Lithuania). Gel Filtration Marker Kit for protein molecular weights 29,000–700,000, acetamide and other chemicals were obtained from SigmaAldrich (Taufkirchen, Germany). Bioinformatic analysis An open reading frame (ORF) in the genome of CH1 that was annotated as the amiE gene was compared with sequences in GenBank (NCBI) using the BLAST algorithm. Multiple sequence alignment was carried out with DNAMAN. Phylogenetic analysis was performed using the neighbor-joining tree algorithm with a software package for constructing evolutionary trees (MEGA, version 4.0) (Tamura et al. 2007). Trees were displayed using TreeView (Page 2002). The automated knowledge-based protein modeling server SwissModel v.3.5 was used for comparative modeling (Guex and Peitsch 1997; Konstantin et al. 2006; Schwede et al. 2003). Procheck v.3.4 for Windows NT was used for model validation (Laskowski et al. 1993). The modeled structure was analyzed using PyMOL software (Schwede et al. 2003). Plasmid construction The full-length amiE gene was amplified by PCR from CH1 genomic DNA using the following primer pair: amiEF (50 -atgccatATGAAGGTCGCCTTCGTCCAG-30 ) and amiE-R (50 -atgcaagcttCAACTCGTAGTACTCAGGTCTT CTGTCC-30 ). NdeI and HindIII restriction sites (underlined) were used to clone the amiE gene into the corresponding sites of pET-28a (?). PCR amplification was carried out using the following procedure: denaturation at 94 °C for 5 min; 30 cycles of 94 °C for 30 s, 55 °C for 45 s, 72 °C for 1 min, and a final step of 72 °C for 10 min. The PCR product was digested and cloned as the NdeI and HindIII fragment into the pET-28a (?) expression vector and transformed into E. coli DH5a. Expression and purification of AmiE The E. coli C41 (DE3) strain harboring the constructed plasmid was incubated in 1 L of LB broth with 30 lg/mL kanamycin at 37 °C for 3 h. When the OD600 reached 1.0, 0.5 mM IPTG (isopropyl-b-D-thiogalactopyranoside) was added to induce protein expression and the culture was further incubated at 30 °C. The cells were cultured in the presence of IPTG for 4 h with shaking and harvested by centrifugation at 10,0009g for 10 min. Enzyme purification was performed at room temperature. The bacterial pellet was resuspended in Binding Buffer (20 mM NaH2PO4–Na2HPO4 (pH 7.6), 150 mM

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NaCl, and 20 mM imidazole) and sonicated on ice. The cell extract was clarified by centrifugation at 10,0009g for 20 min at 4 °C. Ni Sepharose High Performance (GE Healthcare) resin was used to purify the His-tagged fusion protein. Protein purification was performed according to the manufacturer’s instructions. The protein was eluted in a solution buffer [250 mM imidazole, 150 mM NaCl, and 50 mM Na-phosphate (pH 7.6)]. Imidazole was removed using HiTrap Desalting columns (GE Healthcare) according to the manufacturer’s instructions. The purified protein was stored at 4 °C and its concentration was determined by the Bradford method using bovine serum albumin as a standard. The purity of AmiE was confirmed by 12 % (SDS-PAGE) with visualization using Coomassie Brilliant Blue R-250. The molecular mass of the recombinant amidase was determined by analytical gel filtration on a Superdex 200 (Pharmacia, Sweden) column using 19 PBS buffer. The column was calibrated with the following standards: 29 kDa (carbonic anhydrase from bovine erythrocytes), 66 kDa (albumin, bovine serum), 150 kDa (alcohol dehydrogenase from yeast), and 200 kDa (bamylase from sweet potato).

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glycine–HCl (pH 3.0), CH3COOH–CH3COONa (pH 4.0–6.0), NaH2PO4–Na2HPO4 (pH 6.0–8.0), Tris–HCl (pH 8.0–9.0), and NaOH–glycine (pH 9.0–10.0). The pH stability was determined by incubating assays in phosphate buffer for 15, 30, 45, 60, 75, 90, 105, 120 min, and measuring the residual activity. Determination of substrate specificity and kinetic constants The substrate range and specific activity were determined under standard conditions using the following series of substrates: formamide, acetamide, acrylamide, hexanamide, L-glutamine, L-asparagine, urea, benzamide, pyrazinamide, cinnamamide, nicotinamide, and salicylamide. Apparent Km values for acetamide, acrylamide, hexanamide, and L-glutamine were determined using the standard reaction assay mixture (100 lL) containing various substrate concentrations (1–30 mM). Initial reaction velocities measured at various substrate concentrations were fit to the Lineweaver–Burk transformation of the Michaelis–Menten equation. Kinetic analyses by curve fitting were performed using the Fitlinear program.

AmiE activity assay Effect of metal ions and organic solvents Amidase activity was determined by measuring the release of ammonia using the phenol-hypochlorite ammonia detection method (Weatherburn 1967). A standard reaction mixture (100 lL) contained 50 mM KH2PO4-–K2HPO4 (pH 7.6), 150 mM NaCl, 10 mM substrate, and 10 lg of enzyme. The reaction was terminated by the addition of 350 lL of reagent A (0.59 M phenol and 1 mM sodium nitroprusside). Color was developed by the addition of 100 lL of reagent B (2.0 M sodium hydroxide and 0.11 M sodium hypochlorite) after 5 min of incubation at 60 °C and absorbance was measured at 600 nm. Control reactions lacking enzyme were also performed. Standards were prepared using NH4Cl. One unit of enzyme activity was defined as the amount of enzyme that catalyzed the release of 1 lM of NH3 per min under standard assay conditions. All assays were performed in triplicate. Influence of temperature and pH on enzyme activity and stability The optimal reaction temperature for purified AmiE was determined under standard conditions over the range 4–95 °C. The thermal stability of the enzyme was determined by incubating the assays at the respective temperatures (70, 85, and 95 °C) for 15, 30, 45, 60, 75, 90, 105, 120 min, and measuring the residual activity. The optimal pH for purified AmiE was determined under standard conditions. Buffers used were 50 mM of

The effect of various metal ions (Ca2?, Cu2?, Al3?, Co2?, Fe3?, Ni2?, Zn2?, Mg2?) on the enzymatic activity was tested under standard conditions at final concentrations of 1, 5, and 10 mM. The influence of solvents, including acetone, ethanol, methanol, and isopropanol, was tested at 5, 10, and 25 % (v/v) under standard conditions. Samples containing the protein (10 lg) and metal ion/organic solvents were pre-incubated for 1 h at 4 °C and the residual activity was measured.

Results Identification of an amidase from the genomic sequence of Pyrococcus yayanosii CH1 A 786-bp ORF in the CH1 genome was identified and designated as amiE (GenBank Accession No. 10837614). The amiE gene encodes an enzyme named AmiE that belongs to the nitrilase superfamily. Phylogenetic analysis of AmiE demonstrated that amidases from archaea are located in a totally different branch when compared with those from bacteria (Fig. 1). Multiple sequence alignment of AmiE and other homologs from different sources (archaea and bacteria) that belong to the nitrilase superfamily revealed that the enzymes shared a conserved catalytic triad that can be assigned to Glu41, Lys112, and Cys145

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Fig. 1 A neighbor-joining phylogenetic tree of AmiE and closely related proteins based on conserved sequence motifs. Amino acid sequences of other enzymes were obtained from GeneBank (http://www.ncbi. nlm.nih.gov/). Sequence alignment was performed using ClustalW version 2.0, and the tree was created using MEGA version 4.0. The numbers above the bars indicate bootstrap values, with values \50 excluded

(Fig. 2). AmiE exhibited high identity ([70 %) to the homologs from archaea. AmiE exhibited high identity (82, 78, and 79 %, respectively) to the carbon–nitrogen hydrolases from the archaea Thermococcus onnurineus NA1, Pyrococcus abyssi, and Thermococcus kodakarensis KOD1. In contrast, AmiE exhibited only moderate identity to the homologs from bacteria such as Rhodothermus marinus and Pseudomonas. In addition to the conserved catalytic triad, AmiE and other homologs contained similar sequences flanking the invariant catalytic triad, regardless of their origin. A comparison of the amino acid compositions of AmiE homologs from archaea and mesophilic bacteria indicated that more hydrophobic amino acids, charged amino acids, and aromatic acids existed in archaeal amidases (data not shown). No nitrilase superfamily members from archaea were previously characterized with the exception of PH0642

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from Pyrococcus horikoshii (Sakai et al. 2004), whose structure has been solved, and a nitrilase from P. abyssi (Mueller et al. 2006). Thus, characterization of AmiE is important for investigating of the enzymology of this cluster of amidases originating from hyperthermophilic archaea. 3D model of AmiE A model of AmiE was built using the SWISS-MODEL servers. AmiE displayed high sequence homology with the nitrilase from Pyrococcus abyssi (Protein Data Bank code 3IVZ) (Raczynska et al. 2011), with a modeled residue range from amino acids 1–259 (Fig. 3a) and an identity of 78 %. In this model, [80 % of non-glycine and non-proline residues have conformational angles (u, w) in the permitted regions of the Ramachandran plot, 89.3 % fall

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Fig. 2 Multiple amino acid sequence alignment of AmiE from Pyrococcus yayanosii CH1 with homologs. 3IVZ_A: chain A, crystal structure of hyperthermophilic nitrilase from Pyrococcus abyssi GE5; YP_002308311.1: carbon–nitrogen hydrolase from Thermococcus onnurineus NA1; NP_142600.1: hypothetical protein PH0642 from Pyrococcus horikoshii OT3; YP_002583343.1: aliphatic amidase from Thermococcus sp. AM4; YP_003290612.1: nitrilase/cyanide

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hydratase and apolipoprotein N-acyltransferase from Rhodothermus marinus DSM 4252; WP_020795575.1: putative amidohydrolase from Pseudomonas sp. G5(2012); YP_003695144.1: nitrilase/cyanide hydratase and apolipoprotein N-acyltransferase from Starkeya novella DSM 506. Alignments were made using DNAMAN. The regions with black shading and white lettering indicate conserved residues. Black triangles indicate the conserved catalytic triad (Glu–Lys–Cys)

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Fig. 3 Molecular modeling of AmiE. a Ribbon diagram of the AmiE model. The b-strands and a-helices are shown in cartoon diagrams and colored according to secondary structure. Residues of the catalytic triad Glu41, Lys112, and Cys145 are shown in yellow. b Superimposition of AmiE and 3IVZ. The model (3IVZ; magentas) and AmiE (cyans) are displayed in cartoon representation. The superimposition was generated using PyMOL software. c A detailed view of the differences between the C-termini of AmiE and the template. Different residues are labeled and shown in stick form

into the most favored regions, 9.3 % fall into the additional allowed regions, 0.4 % fall into the ‘generous allowed’ region, and 0.9 % fall into the disallowed region, as defined by Procheck. In summary, one would hope to have over 90 % of the residues in these ‘‘core’’ regions. Crystal structures of various members of the nitrilase superfamily (amidases and nitrilases) show high structural homology despite low sequence conservation (Jorge et al. 2007; Kimani et al. 2007; Nel et al. 2011; Sakai et al. 2004). AmiE is comprised of 9 a-helices and 13 b-strands. AmiE can be assigned to the ab class and is predicted to have the typical abba sandwich architecture (Fig. 3a). The

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arrangements of the two sheets are in the order b12–b1– b2–b3–b4–b5 and b6–b7–b8–b9–b10–b11. The superposition of the structures of AmiE and 3IVZ shows that some residues flanking the active site are not conserved. For example, residue 114 (which is flanking the active site residue K112) in AmiE is Val, but Ile in 3IVZ. Residues 51–53 are Ser, Gly, and Asp in AmiE, but Thr, Arg, and Glu in 3IVZ (Fig. 3b). The C-terminal part of each subunit (as shown in the black box in Fig. 3a) plays an important role in protein oligomerization by extending away from the core and interacting with the other subunits. This phenomenon is

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Fig. 4 a Expression of AmiE in Escherichia coli. Lane 1 low molecular mass standards, Lane 2 culture without IPTG, Lane 3 culture with 0.1 mM IPTG, Lane 4 culture with 0.5 mM IPTG, Lane 5 culture with 1.0 mM IPTG. b SDS-PAGE gel showing the purification product from the soluble fraction of the E. coli transformant. Lane 1 low molecular mass standards, Lane 2 purified recombinant Histagged AmiE from CH1. c Gel filtration of purified AmiE (indicated with the red line). The standards are shown in blue and are as follows: 29 kDa (carbonic anhydrase from bovine erythrocytes), 66 kDa (albumin, bovine serum), 150 kDa (alcohol dehydrogenase from yeast) and 200 kDa (b-amylase from sweet potato)

similar to other reported members of the nitrilase superfamily (Raczynska et al. 2011). However, the C-terminal part of AmiE is predicted to have a different folding than the template, as shown in Fig 3c. This may be attributed to the different residues located at the 244 (Leu) and 256 (Pro) sites (Fig. 3c). These changes may account for the different activities of the two enzymes. Expression and purification of recombinant AmiE as a fusion protein from E. coli A 786-bp PCR product was inserted into the NdeI and HindIII digested pET-28a (?) expression vector. The construct was transformed into E. coli C41 (DE3) strain and gene expression was induced by IPTG. The N- and C-terminally hexa-histidine-tagged AmiE fusion protein was successfully expressed in E. coli C41 (DE3) (Fig. 4a). SDS-PAGE analysis of recombinant AmiE revealed a molecular weight of approximately 34 kDa, which was consistent with the molecular mass estimated from the deduced protein sequence (fusion with the hexa-

histidine peptide resulted in a 4-kDa increase in molecular mass). The majority of the recombinant enzyme was found in the soluble fraction. The yield of purified protein was about 15 mg from a 1-L culture of E. coli. AmiE was then purified to near homogeneity by Ni-chelation chromatography (Fig. 4b). Accordingly, it can be assumed that the native enzyme (about 70 kDa) consists of two identical subunits. This result was confirmed by gel filtration (Fig. 4c). Activity as a function of temperature and enzyme thermostability AmiE activity was determined using acetamide as a substrate with temperatures ranging 4–95 °C. Among the eight different temperatures tested, 85 °C was the optimum temperature for maximum enzyme activity with a decline in activity above or below this temperature (Fig. 5a). This value is approximately 10 °C below the optimal growth temperature of CH1 (Zeng et al. 2009). At 50 °C there was about 53 % activity compared with 85 °C. Relative activities of the purified enzyme measured at 60, 70, and 95 °C

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Fig. 5 a Effect of temperature on the enzyme activity of AmiE. Activities are expressed as percentages relative to the maximum activity at 85 °C (100 %). b Determination of AmiE thermal stability. Enzyme activity was measured over a temperature range of 4–95 °C

in NaH2PO4–Na2HPO4 (pH 6.0). For the thermal stability of AmiE, the enzyme was incubated at 70, 80, and 90 °C for 15, 30, 45, 60, 75, 90, 105, and 120 min

Fig. 6 a Effect of pH on the activity of AmiE. Activities are expressed as percentages relative to the maximum activity (100 %) in phosphate buffer (pH 6.0). b Determination of AmiE pH stability. Enzyme activity was measured in different buffers ranging pH 3.0–10.0 [glycine–HCl (pH 3.0), CH3COOH–CH3COONa (pH

4.0–6.0), NaH2PO4–Na2HPO4 (pH 6.0–8.0), Tris–HCl (pH 8.0–9.0), and NaOH–glycine (pH 9.0–10.0)]. For pH stability, the enzyme was incubated at 4 °C for 15, 30, 45, 60, 75, 90, 105, and 120 min using different buffers [NaH2PO4–Na2HPO4 (pH 6.0–8.0)]

were 80, 90, and 89 %, respectively. AmiE also showed good thermostability at 70 °C (Fig. 5b), giving extrapolated half-life (t1/2) estimates well in excess of 4 h. At 80 °C, the t1/2 value of the enzyme was 110 min. At higher temperatures, inactivation of the free enzyme was rapid, with a t1/2 value of 40 min at 90 °C. The purified enzyme could be stored for longer than 6 months at 4 °C in 50 mM phosphate buffer and 10 % glycerol without any loss of activity.

and 8.0 (NaH2PO4–Na2HPO4). Approximately 50 % of the maximal activity was observed at pH 3.0–5.0. The enzyme is effective over a pH range of 4.0–8.0. However, activity was not detected in Tris buffer, glycine–HCl (pH 3.0), or NaOH–glycine (pH 9.0–10.0). The pH stability of purified AmiE was also determined in phosphate buffer (Fig. 6b). The enzyme revealed good stability in phosphate, and amidase activity was constant for at least 2 h at pH 6.0 (NaH2PO4–Na2HPO4). Over 60 % of the maximal activity was retained at pH 7.0 and 8.0 (NaH2PO4–Na2HPO4) after 120 min.

pH optimum and pH stability The effect of pH on AmiE was examined over a pH range of 3.0–10. The purified enzyme showed highest activity at pH 6.0 (NaH2PO4–Na2HPO4) (Fig. 6a), which was lower than the optimal pH for growth of CH1 (pH 7.0). Greater than 70 % of the maximal activity was observed at pH 7.0

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Substrate specificity and kinetic constants Twelve amides were tested as substrates to study the specificity of AmiE at 85 °C and pH 6.0 (NaH2PO4–Na2HPO4).

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Table 1 AmiE substrate specificity Substrate

Relative activity (%)

Acetamide

100

Table 3 Influence of various metal ions on recombinant AmiE activity Metal ion

Relative activity (%) 1 mM

5 mM

10 mM

L-Glutamine

73

Hexanamide

38.7

CKa

Acrylamide

22.5

Ca2?

129 ± 4

108 ± 2

88 ± 2

Formamide

12.1

27 ± 6 109 ± 3

29 ± 7 104 ± 5

31 ± 2 97 ± 4

100

100

100

L-Asparagine

4.8

Cu2? Al3?

Urea

0

Co2?

94 ± 4

70 ± 7

59 ± 5

Benzamide

0

3?

Fe

94 ± 8

55 ± 2

32 ± 2

Pyrazinamide

0

Ni2?

84 ± 2

58 ± 4

69 ± 11

Nicotinamide

0

Zn2?

71 ± 2

56 ± 4

38 ± 3

Cinnamamide Salicylamide

0 0

Mg2?

104 ± 4

112 ± 2

115 ± 4

a

Table 4 Influence of various solvents (5–25 %) on AmiE activity

Table 2 AmiE kinetic parameters Substrate

Km (mM)

No metal ion added

Kcat (S-1)

Kcat/Km (mM-1 S-1)

Specific activity (U mg-1)

606.3

1,000

Acetamide

8.33

5,050.50

Acrylamide

10.86

333.33

30.69

67

Hexanamide

11.08

721.71

65.14

143

L-Glutamine

15.92

7,215.15

453.21

1,430

AmiE showed activity toward the majority of aliphatic amides tested, including formamide, acetamide, L-glutamine, hexanamide, and acrylamide (Table 1). However, AmiE did not show activity toward aromatic amides such as benzamide, nicotinamide, and pyrazinamide, which suggested that AmiE is an aliphatic amidase belonging to the second branch of the nitrilase superfamily (Sharma et al. 2009). Apparent Km values for acetamide, acrylamide, and hexanamide were determined using the Lineweaver–Burk representation of the data obtained by determining the initial rate of substrate hydrolysis. Data indicated that acetamide was clearly the favored substrate on the basis of substrate affinity when compared with the other tested substrates (Table 2). The Km (mM) values of AmiE for acetamide, acrylamide, hexanamide, and L-glutamine were determined to be 8.33, 10.86, 11.08, and 15.92. The Kcat of AmiE varies widely for different donor substrates, and the value of Kcat for acetamide was found to be much greater than for acrylamide, hexanamide, and L-glutamine. However, the specific activity of AmiE for L-glutamine was 1430 lmol mg-1 min-1, which is much greater than for the other substrates. Influence of metal ions and solvents on recombinant AmiE The effects of various metal ions on recombinant AmiE are summarized in Table 3. 1, 5, and 10 Mg2? increased the

Solvent

CK

a

Relative activity (%) 5%

10 %

25 %

100

100

100

Acetone

70 ± 14

63 ± 15

31 ± 10

Methanol Ethanol

99 ± 8 83 ± 15

86 ± 1 70 ± 5

67 ± 2 55 ± 6

Isopropanol

96 ± 16

76 ± 3

46 ± 7

a

No solvent added

activity to 104, 112, and 115 %, respectively. Enzyme activity also increased in the presence of low concentrations (1 and 5 mM) of Al3? and Ca2?, but was slightly inhibited by 10 mM Al3? and Ca2?. Zn2?, Cu2?, and Ni2? significantly inhibited AmiE activity. 10 mM Fe3? also decreased the activity to 32 %. As shown in Table 4, 5 % methanol and 5 % isopropanol displayed no significant effect on the hydrolytic activity of AmiE. However, obvious inactivation of AmiE with other solvents was observed, especially with 25 % acetone and isopropanol.

Discussion In this study, we report the characterization of an amidase named AmiE from P. yayanosii CH1, which shares structural homology with a nitrilase from P. abyssi (Raczynska et al. 2011). To the best of our knowledge, this is the first example of a detailed characterized thermostable amidase belonging to the nitrilase superfamily from archaea. The heterologous expression of genes from thermophiles in a mesophilic host strain such as E. coli and Bacillus subtilis is an alternative way to meet the

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increasing demand for highly thermostable enzymes, such as proteases and lipases, from thermophilic organisms. Recombinant AmiE was expressed as a (N and C terminal) His tag fusion protein in E. coli. The high yield of AmiE (15 mg/l) in small-scale fermentations of E. coli suggests that it can be efficiently produced upon scale-up for industrial purposes. Although members of the nitrilase superfamily share conserved catalytic triad residues, AmiE did not show any detectable activity on nitrile substrates (acetonitrile, acrylonitrile, benzonitrile, mandelonitrile, and adiponitrile). This phenomenon is similar to that previously observed for an amidase from Nesterenkonia (Nel et al. 2011). AmiE displayed amidase activity rather than the nitrilase from P. abyssi, although they showed 78 % identity. Substitution of amino acids flanking the conserved catalytic triad residues or other residues that are not conserved forming different spatial locations may account for their different activities (Pace and Brenner 2001). This will be investigated further in future experiments. AmiE showed its highest activity at 85 °C. In addition, AmiE showed good thermostability at 70 °C (Fig. 5). This feature was shared by the alanine amidase from Brevibacillus borstelensis BCS-1 (Baek et al. 2003a). Moreover, this value was higher than that for most of the amidases studied thus far (Egorova et al. 2004; Ko et al. 2010; Makhongela et al. 2007; Shen et al. 2012). The optimal pH was similar to that for the amidase from Geobacillus thermoglucosidasius AUT-01 (Cha and Chambliss 2013). Expressed thermostable AmiE exists as a dimer (Fig. 4a), which was similar to the amidase from Nesterenkonia strain AN1, which has an optimal temperature of 30 °C (Nel et al. 2011), but differs from the previously described hexameric aliphatic amidases from Helicobacter pylori (Hung et al. 2007), Pseudomonas aeruginosa (Andrade et al. 2007), and G. pallidus (Makhongela et al. 2007). However, among the three hexameric aliphatic amidases, only the amidase from G. pallidus showed heat stability. Interestingly, members belonging to the nitrilase superfamily in thermococcales all formed dimers, including PH0642 from P. horikoshi (Sakai et al. 2004) and a thermostable nitrilase from P. abyssi (Mueller et al. 2006). These results suggest that some key amino acids and structural elements may play more important roles in sustaining the heat stability rather than determining the aggregation state. The optimal pH values of other amidases studied thus far were higher than pH 7.0 (Baek et al. 2003a; Egorova et al. 2004; Ko et al. 2010; Makhongela et al. 2007; Scotto d’Abusco et al. 2001). AmiE can hydrolyze aliphatic amides and showed no activity towards aromatic amides (Table 1). Therefore, we suggest that AmiE belongs to the second branch (aliphatic amidase) of the nitrilase superfamily. The analysis of the substrates efficiently hydrolyzed by aliphatic amidases in

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the nitrilase superfamily indicates that AmiE is similar to the amidase from G. pallidus (Makhongela et al. 2007), and different from other amidases, such as the amidase from Nesterenkonia strain AN1, which displayed little activity on linear amides containing fewer than 2 and [4 (e.g., formamide, butyramide and hexanamide) carbon atoms (Andrade et al. 2007; Nel et al. 2011; Skouloubris et al. 2001). The specific activity of AmiE for acetamide is 1,000 lmol mg-1 min-1, compared with 4,140 lmol mg-1 min-1 for the amidase from G. pallidus, 3.67 lmol mg-1 min-1 for the amidase from Nesterenkonia strain AN1, and 3.8 lmol mg-1 min-1 for the amidase from Sulfolobus solfataricus (Makhongela et al. 2007; Nel et al. 2011; Scotto d’Abusco et al. 2001). Of the selected aliphatic substrates, AmiE is weakly active towards Lasparagine, acrylamide, and formamide. The preferred substrates are acetamide and glutamine. This result is also different from what is reported for aliphatic amidases from the nitrilase superfamily in bacteria. For example, the aliphatic amidase from Nesterenkonia had the highest affinity for propionamide, but showed little activity towards acetamide and glutamine (Nel et al. 2011). However, the small aliphatic amides used as experimental substrates were not likely the natural substrates for AmiE, due to their high Km values. Nitrile-degrading enzymes have been commonly observed in bacteria that can utilize nitriles or amides as sole sources of carbon and nitrogen (Banerjee et al. 2002). However, strain CH1 is a chemoorganotrophic organism, requires complex carbon sources for growth (Zeng et al. 2009), and was not able to grow on amides (including acetamide, glutamine, etc.). However, transcription of the amidase gene was observed when the strain was cultivated in broth that included complex carbon sources. Further investigation of the physiological role of AmiE in CH1 is necessary. It is known that the catalytic triad (C-E-K) is conserved in all members of the nitrilase superfamily (Pace and Brenner 2001). However, as noted in the introduction, a C-E-E-K tetrad in place of a C-E-K triad, in which an additional glutamate is hydrogen bonded to the catalytic lysine, has been found in several members of the nitrilase superfamily. We have mutated this glutamate (E119) in AmiE to aspartate. The wild-type enzyme was active against the substrates as demonstrated by ammonia production. Ammonia was also detected when acetamide was incubated with E119D mutant for 10 min at 85° C in triplicate repeats. The E119D mutant displayed 47 % of the wild-type activity using acetamide as substrate (Fig. S1). So, mutating E119 to aspartate, therefore, did not render the amidase inactive. This is different from what has been reported by Soriano Maldonado et al. 2011 and Weber et al. 2013 (Kimani et al. 2007). Moreover, the mutant

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enzyme was less stable than the wild type. Further research will be focused on the role of this residue. Moreover, members from different branches of the nitrilase superfamily showed different substrate binding and catalytic mechanisms. Thus, a more detailed study will be focused on the binding pocket–substrate interactions, which will give a better understanding of how nitrilases and amidases differ with respect to their substrate binding, catalytic mechanisms, and the functions of the individual residues. In conclusion, the apparent temperature optimum, thermostability, and pH stability of AmiE indicate that this amidase originating from the hyperthermophilic archaeon P. yayanosii CH1 might have potential industrial applications. Acknowledgments The project was supported by National Hightech R&D Program (863 Program, Grant 2012AA092103) and the State Key Laboratory of Ocean Engineering of China (Grant GKZD010045). We thank Geng Wu and Guangyu Yang for experimental assistance.

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