Biotechnol Lett DOI 10.1007/s10529-015-1992-0
ORIGINAL RESEARCH PAPER
Expression, characterization of a novel nitrilase PpL19 from Pseudomonas psychrotolerans with S-selectivity toward mandelonitrile present in active inclusion bodies Huihui Sun . Wenyuan Gao . Hualei Wang Dongzhi Wei
.
Received: 2 September 2015 / Accepted: 3 November 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Objectives To identify a novel nitrilase with Sselectivity toward mandelonitrile that can produce (S)mandelic acid in one step. Results A novel nitrilase PpL19 from Pseudomonas psychrotolerans L19 was discovered by genome mining. It showed S-selectivity with an enantiomeric excess of 52.7 % when used to hydrolyse (R, S)mandelonitrile. No byproduct was observed. PpL19 was overexpressed in Escherichia coli BL21 (DE3) and formed inclusion bodies that were active toward mandelonitrile and stable across a broad range of temperature and pH. In addition, PpL19 hydrolysed nitriles with diverse structures; arylacetonitriles were the optimal substrates. Homology modelling and docking studies of both enantiomers of mandelonitrile in the active site of nitrilase PpL19 shed light on the enantioselectivity. Conclusions A novel nitrilase PpL19 from P. psychrotolerans L19 was mined and distinguished from other nitrilases as it was expressed as an active inclusion body and showed S-selectivity toward mandelonitrile.
H. Sun W. Gao H. Wang (&) D. Wei State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, People’s Republic of China e-mail: [email protected]
Keywords Active inclusion body (S)-Mandelic acid Mandelonitrile Nitrilase Pseudomonas psychrotolerans L19
Introduction Nitrilase (EC 3.5.5.X) converts nitriles to the corresponding carboxylic acids. The production of (R)mandelic acid from mandelonitrile with a high degree of enantioselectivity is important as it is used for the production of semisynthetic cephalosporins, penicillins, antitumor agents, and antiobesity agents (Banerjee et al. 2006; Baum et al. 2012; Liu et al. 2011; Wang et al. 2013b; Zhang et al. 2011). (S)Mandelic acid is also an important intermediate used for synthesizing non-steroidal anti-inflammatory drugs, such as celecoxib and deracoxib (Mateo et al. 2006). However, there is no one-step nitrilase-mediated pathway to produce (S)-mandelic acid from mandelonitrile because no S-selective nitrilase has been identified. Robertson et al. (2004) described the existence of four nitrilases showing a relatively low preference for the formation of (S)-mandelic acid (25–30 % enantiomeric excess (ee)) from (R, S)-mandelonitrile that was based on metagenomic technology. A possible approach for producing (S)-mandelic acid at high enantiomeric purity was reported by Chmura et al. (2013). The approach involved constructing ‘‘cascade
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Biotechnol Lett
reactions’’ by using a highly (S)-specific oxynitrilase from Manihot esculenta, a non-selective nitrilase from Pseudomonas fluorescens EBC191, and an amidase from Rhodococcus erythropolis. In another study, the enantioselectivity of a nitrilase from P. fluorescens EBC191 was reversed to (S)-mandelic acid by protein engineering (Sosedov and Stolz 2015). However, the ee value was \37 %. Furthermore, all the mutants generated the by-products (S)-mandelamine in different proportions. In this study, a novel S-selective nitrilase PpL19 from P. psychrotolerans L19 that could hydrolyse (R, S)-mandelonitrile to (S)-mandelic acid with an ee of 52.7 % was discovered by genome mining. It was overproduced in Escherichia coli BL21 (DE3) in an active insoluble form. The substrate specificity of PpL19 and its stability as a function of temperature and pH were investigated. Homology modelling and docking studies were also performed to investigate the enantioselectivity of nitrilase PpL19.
of nitrilases. Mandelonitrile and other chemicals were purchased from Sigma-Aldrich. Database mining Database searches of sequence data were performed using BLASTP. Nitrilase 1A10 (AAR97486.1) from an uncultured organism was chosen as the identifier to detect potential S-selective mandelonitrile specific nitrilases based on similar data mining (Robertson et al. 2004). The screening criteria were the same as described previously (Wang et al. 2013b). Nitrilase sequences showing 30–60 % amino acid identity to nitrilase 1A10 were extracted from the GenBank database. Expression of nitrilase genes in E. coli
Materials and methods
Recombinant E. coli BL21 (DE3) cells used for expressing nitrilase were cultivated in lysogeny broth (LB) containing kanamycin (50 lg/ml) at 37 °C. When the OD600 of the culture reached 0.6–0.8, IPTG was added to 0.1 mM. The induced cultures of E. coli BL21 (DE3) were further incubated for 20 h at 20 °C.
Materials
Inclusion body isolation
Nitrilase genes were synthesized by Shanghai Generay Biotech Co., Ltd. and inserted into plasmid pET28a(?). The resulting plasmids were transformed into Escherichia coli BL21 (DE3) for the expression
The induced cells were harvested by centrifugation (10,0009g, 10 min), washed twice with physiological saline, and resuspended in 10 ml lysis buffer (50 mM Tris/HCl, pH 8). Cells were disrupted by sonication on
Table 1 Activity and enantioselectivity of nitrilases mined from GenBank database Nitrilase
Organism
GenBank accession number
Amino acid identity (%)a
Specific activity (U/mg protein)b
ee value of acid (%)c
313
Variovorax sp. CF313
WP_007829732
58
6.4
89.9
GPnor51
Luminiphilus syltensis NOR5-1B
EED35210.1
41
15
96.7
316
Acidovorax sp. CF316
WP_007851333
37
1.9
92.6
6799
Desulfomonile tiedjei DSM 6799
AFM28028
36
0.34
95.7
39116
Zymoseptoria tritici IPO323
WP_039792070
36
0.28
85.5
CCS1
Jannaschia sp. CCS1
ABD56652.1
36
10
94.7
BTAil
Bradyrhizobium sp. BTAi1
WP_012044777
35
4.3
90.9
RW1
Sphingomonas wittichii RW1
WP_011951832
34
3.5
96.4
Bradyrhizobium sp. ORS 278
WP_011927383
34
1
89.7
Pseudomonas psychrotolerans L19
EHK72968.1
33
1.8
52.7
ORS278 d
PpL19 a
The amino acid identity is compared with the nitrilase 1A10 (AAR97486.1) from an uncultured organism
b
Activities and enantioselectivities of crude enzymes were determined by the method of Zhang et al. (2013)
c
The enantio-preference of nitrilase PpL19 was S-; the enantiopreference of all the other nitrilases was R-
d
Active inclusion bodies were used to determine the activity and enantioselectivity of nitrilase PpL19
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Biotechnol Lett
ice, and the lysate was centrifuged for 30 min at 10,0009g and 4 °C. The cell pellets were washed with buffer containing 50 mM Tris/HCl (pH 8), 100 mM NaCl, and 1 % (v/v) Triton X-100 at room temperature for 30 min, then in the same buffer without Triton X-100 for 30 min. The washed inclusion bodies were collected by centrifugation as above and analysed by SDS-PAGE. Assay of nitrilase activity Nitrilase activity was measured in a reaction mixture (1 ml) containing 50 mM Tris/HCl (pH 8) and nitrilase. After preincubation at 30 °C for 10 min, the reaction was initiated by rapid mixing with mandelonitrile (10 mM). The reaction was quenched by adding 0.1 ml 2 M HCl and the mixture was centrifuged. The formation and optical purity of mandelic acid in the supernatant were analysed by HPLC as described previously (Wang et al. 2013a). The Berthelot assay was also performed in some cases to measure the amount of ammonia formed in nitrilase reactions (Weatherburn 1967). All assays were performed in triplicate and the experimental error was \5 %. One unit of the enzyme activity was defined as the amount of enzyme that produced 1 lmol mandelic acid per min under the standard assay conditions.
Results Screening of recombinant nitrilase BLASTP was performed to identify nitrilases capable of enantioselectively hydrolysing mandelonitrile to (S)-mandelic acid. Ten nitrilases showing 30–60 % amino acid identities with nitrilase 1A10 from an uncultured organism (Robertson et al. 2004) were selected from the GenBank database and overexpressed in E. coli. All the nitrilases were expressed as soluble proteins with R-selectivity toward mandelonitrile, except for PpL19 from P. psychrotolerans L19 that was largely expressed in inclusion bodies and were active toward mandelonitrile and exhibited Sselectivity to produce (S)-mandelic acid with an ee value of 52.7 % (Table 1). No byproduct was observed during the hydrolysis process. A little of the nitrilase PpL19 was expressed in soluble form (Fig. 1) but no activity was observed when this was used to hydrolyse mandelonitrile (data not shown).
Homology modelling and docking studies The 3D homology model of nitrilase PpL19 was constructed using Accelrys Discovery Studio Modeler 4.1 (Sˇali and Blundell 1993), using the crystal structures of nitrilase NIT6803 (PDB accession code ˚ ) (Zhang et al. 2014) from 3WUY, resolution 3.1 A Synechocystis sp. PCC6803 and nitrilase PaNit (PDB ˚ ) (Raczynska accession code 3IVZ, resolution 1.57 A et al. 2011) from Pyrococcus abyssi as templates. The structure of PpL19 generated was improved by refining the loop conformations and assessing the compatibility of the amino acid sequence with known PDB structures. The resulting model was used to perform docking studies. Both enantiomers of mandelonitrile were docked into the model of PpL19 using C-DOCKER (Kim et al. 2009; Yeom et al. 2008). The active sites were defined as a collection of amino acid ˚ radius residues enclosed within a sphere of 5 A centered on the catalytic triad.
Fig. 1 SDS-PAGE analysis of the expression of nitrilase PpL19 in Escherichia. coli Lane 1 protein marker; lane 2 soluble fraction; lane 3 insoluble fraction; lane 4 purified nitrilase PpL19 inclusion bodies isolated from cells after washing
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Biotechnol Lett
The optimum temperature for activity was 30 °C and PpL19 retained 80 % of its maximum activity even at
60 °C (Fig. 2). This result differs from most watersoluble nitrilases whose activity decrease sharply [55 °C (Sun et al. 2015; Wang et al. 2013b). The temperature-activity curve indicated that the enzyme was particularly stable at 20 °C and retained 50 % of its initial activity after 40 h (Fig. 2c). pH 8 was optimal for the hydrolysis reaction (Fig. 2b) and PpL19 [ 80 % activity between pH 7 and 9. Arylacetonitrilases usually have a rather narrow optimum pH, at neutral or slightly alkaline values (Zhang et al.
Fig. 2 Effects of temperature and pH on nitrilase PpL19 activity and stability. a Enzyme activity was measured at 20–60 °C in 100 mM Tris/HCl buffer (pH 8). Activity at 40 °C (the optimum temperature) was taken as 100 % (2.8 U/mg). b Enzyme activity was determined with mandelonitrile as substrate at pHs from 5.2 to 10.28 at 30 °C. Activity at pH 8 (the optimum pH) was taken as 100 % (2.7 U/mg). c Purified enzyme was incubated in Tris/HCl buffer at different
temperatures and the residual activity with mandelonitrile as substrate was measured at various time points. The activity at time = 0 was taken as 100 % (2.8, 2.8, 2.8 U/mg for 20, 30 and 40 °C, respectively). d Purified enzyme was incubated at different pHs, and the residual activity was measured at various time points. The activity at time = 0 was taken as 100 % (2.7, 2.7 and 2.7 U/mg for pH 7.6, 8 and 8.6, respectively). Experiments were performed in triplicate
Expression of PpL19 in inclusion bodies was at a high level, and the pure inclusion bodies contained [90 % nitrilase PpL19 with a molecular mass of 37 kDa by SDS-PAGE (Fig. 1), which is in agreement with the size predicted from the amino acid sequence. Characterization of nitrilase PpL19
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Biotechnol Lett Table 2 Substrate specificity of purified PpL19 Substrate
Relative activity (%)
Benzonitrile
64 ± 0.5
3-Hydroxyphenylpropionitrile
49 ± 10
2-Cyanopyridine 3-Cyanopyridine
28 ± 0.24 29 ± 0.5
Phenylacetonitrile
404 ± 12
Mandelonitrile
100 ± 21
a-Methylphenylacetonitrile
88 ± 10
2-Chloromandelonitrile
20 ± 1
Acrylonitrile
0.52 ± 0
3-Hydroxypropionitrile
0.21 ± 0
Succinonitrile
0.01 ± 0
1,2-Phenylenediacetonitrile
4.2 ± 0.22
2-Methylglutaronitrile
9.6 ± 0.3
3-Hydroxyglutaronitrile
0.05 ± 0
Fumaronitrile
0.72 ± 0.05
Nitrilase activity toward various nitriles (10 mM) was measured under standard assay conditions. The activity toward mandelonitrile was defined as 100 % = 2.8 U/mg
Fig. 3 Structure of PpL19. a The structure of PpL19 modelled using Discovery Studio. b-strands are in yellow and a-helices in red. Loops and other secondary structures are in green. The conserved catalytic triad (E48, K130, C164) is shown as sticks
2011). PpL19 nitrilase showed high stability at pH 7.6 (Fig. 2d). Substrate specificity Nitrilase PpL19 had a broad substrate spectrum and hydrolysed a large number of structurally-related nitriles (Table 2). PpL19 preferentially hydrolyse arylacetonitriles and the enzyme exhibited the highest activity toward phenylacetonitrile. Significant activities were also observed with heterocyclic nitriles, such as 2-cyanopyridine and 3-cyanopyridine, and the nitrilase could also hydrolyse aromatic nitriles such as benzonitrile and 3-hydroxyphenylpropionitrile. Considering the distinctive S-selectivity of PpL19 toward mandelonitrile, its enantioselectivity toward other arylacetonitriles was also determined. PpL19 showed S-selectivity toward a-methylphenylacetonitrile, giving a product with 61 % ee. Notably, PpL19 displayed R-selectivtity toward 2-chloromandelonitrile, a derivative of mandelonitrile (ee 70 %). The
and coloured based on element types. b The solvent-accessible surface of PpL19. Positive and negative residues are shown in blue and red, respectively. The catalytic area is in purple
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Biotechnol Lett
Fig. 4 Modelled structure of the active sites of PpL19 with (S)-mandelonitrile and (R)-mandelonitrile. Distances between (S)mandelonitrile or (R)-mandelonitrile and Cys-164 are marked with cyan and magenta dashed lines, respectively
Table 3 Catalytic properties of nitrilase PpL19 in synthesis of (S)-(?)-mandelic acid compared with other reported nitrilases Enzyme
Conversion (%)
ee (%)
Reference
1A8
14
26
Robertson et al. (2004)
1A9
13
30
Robertson et al. (2004)
1A10
16
25
Robertson et al. (2004)
5B17
15
27
Robertson et al. (2004)
PpL19
100
52.7
This study
distinct enantioselectivity toward phenylacetonitrile with substituents at different positions may be due to steric hindrance or electronic effect. Homology modelling and docking studies As Fig. 3a showed, the conserved catalytic residues E48, K130 and C164, proposed to be the catalytic triad of nitrilase, have similar orientations and locations in the nitrilase model to these properties in nitrilase crystal structures. A surface cavity was predicted to be the binding pocket with C164 lying at the bottom of the cavity (Fig. 3b).
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When (S)- and (R)-mandelonitrile were docked into the active sites of PpL19 (Fig. 4), there was a notable difference in the distance between the different enantiomers and the catalytic site; the cyano group of (S)-mandelonitrile and the sulfur of C164 were ˚ ) than with (R)-mandelonitrile closer together (3.751 A ˚ (3.833 A). This difference in distance might explain the enantio-preference for mandelonitrile exhibited by nitrilase PpL19.
Discussion Biocatalytic processes are widely used in the pharmaceutical and fine chemicals industries and offer advantages over conventional chemical catalysts in safety and environmental aspects. Nitrilase-mediated biotransformation processes are used to produce optically active a-hydroxyl carboxylic acids, such as (R)-(-)-mandelic acid (Sun et al. 2015; Wang et al. 2013a, b). However, only a few nitrilases exhibiting relatively low S-selectivity toward mandelonitrile have been reported (Robertson et al. 2004; Sosedov
Biotechnol Lett
and Stolz 2015). Screening for enzymes with high Sselectivity is important to meet the requirements of industrial applications. A novel S-selective nitrilase, PpL19, from P. psychrotolerans L19 was identified here by genome mining. PpL19 could enantioselectively hydrolyse (R, S)-mandelonitrile into (S)-mandelic acid with an ee value of 52.7 % and no byproduct was observed. Nitrilase PpL19 showed the highest conversion and enantioselectivity among the reported nitrilases that can produce (S)-mandelic acid (Table 3). However, the selectivity and activity of PpL19 is too low to meet the requirements of industrial production; further modification of PpL19 to improve its selectivity and activity would be necessary to make it suitable for manufacturing processes. To understand how nitrilase PpL19 interacts with the substrates (R)- and (S)-mandelonitrile, a structural model was created. When (R)- and (S)-mandelonitrile were docked into it, the cyano carbon atoms of both pointed directly toward the catalytic triad, but there were remarkable differences in the catalytic distance: a shorter distance was observed for the S-enantiomer, which would result in a higher enzyme activity. Different hydrolysis rates of R- and S-enantiomer substrates result in enzymatic enantio-preference (Zhang et al. 2014). In conclusion, a novel nitrilase PpL19 from P. psychrotolerans L19, S-selective toward mandelonitrile, was identified and expressed in active inclusion bodies. PpL19 exhibited a broad substrate range. Further study will focus on engineering PpL19 to improve its performance in the hydrolysis of mandelonitrile to produce optically pure (S)-mandelic acid. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21406068/B060804), China Postdoctoral Science Foundation funded project (No. 2014M560308) and National Major Science and Technology Projects of China (2012ZX09304009).
References Banerjee A, Kaul P, Banerjee UC (2006) Purification and characterization of an enantioselective arylacetonitrilase from Pseudomonas putida. Arch Microbiol 184:407–418 Baum S, Williamson DS, Sewell T, Stolz A (2012) Conversion of sterically demanding a, a-disubstituted phenylacetonitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191. Appl Environ Microbiol 78:48–57
Chmura A, Rustler S, Paravidino M, Rantwijk FV, Stolz A, Sheldon RA (2013) The combi-CLEA approach: enzymatic cascade synthesis of enantiomerically pure (S)mandelic acid. Tetrahedron 24:1225–1232 Kim JS, Tiwari MK, Moon HJ, Jeya M, Ramu T, Oh DK, Kim IW, Lee JK (2009) Identification and characterization of a novel nitrilase from Pseudomonas fluorescens Pf-5. Appl Microbiol Biotechnol 83:273–283 Liu ZQ, Dong LZ, Cheng F, Xue YP, Wang YS, Ding JN, Zheng YG, Shen YC (2011) Gene cloning, expression, and characterization of a nitrilase from Alcaligenes faecalis ZJUTB1. J Agric Food Chem 21:11560–11570 Mateo C, Chmura A, Rustler S, Rantwijk FV, Stolz A, Sheldon RA (2006) Synthesis of enantiomerically pure (S)-mandelic acid using an oxynitrilase-nitrilase bienzymatic cascade: a nitrilase surprisingly shows nitrile hydratase activity. Tetrahedron 17:320–323 Raczynska JE, Vorgias CE, Antranikian G, Rypniewski W (2011) Crystallographic analysis of a thermoactive nitrilase. J Struct Biol 173:294–302 Robertson DE, Chaplin JA, DeSantis G et al (2004) Exploring nitrilase sequence space for enantioselective catalysis. Appl Environ Microbiol 70:2429–2436 Sˇali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234:779–815 Sosedov O, Stolz A (2015) Improvement of the amides forming capacity of the arylacetonitrilase from Pseudomonas fluorescens EBC191 by site-directed mutagenesis. Appl Microbiol Biotechnol 99:2623–2635 Sun HH, Gao WY, Fan HY, Wang HL, Wei DZ (2015) Cloning, purification and evaluation of the enzymatic properties of a novel arylacetonitrilase from Luminiphilus syltensis NOR5-1B: a potential biocatalyst for the synthesis of mandelic acid and its derivatives. Biotechnol Lett 38:1655–1661 Wang HL, Sun HH, Wei DZ (2013a) Discovery and characterization of a highly efficient enantioselective mandelonitrile hydrolase from Burkholderia cenocepacia J2315 by phylogeny-based enzymatic substrate specificity prediction. BMC Biotechnol 13:14 Wang HL, Sun HH, Gao WY, Wei DZ (2013b) Efficient production of (R)-o-chloromandelic acid by recombinant Escherichia coli cells harboring nitrilase from Burkholderia cenocepacia J2315. Org Proc Res Dev 18:767–773 Weatherburn MW (1967) Phenol-hypochlorite reaction for determination of ammonia. Anal Chem 39:971–974 Yeom SJ, Kim HJ, Lee JK, Kim DE, Oh DK (2008) An amino acid at position 142 in nitrilase from Rhodococcus rhodochrous ATCC 33278 determines the substrate specificity for aliphatic and aromatic nitriles. Biochem J 415:401–407 Zhang ZJ, Xu JH, He YC, Ouyang LM, Liu YY (2011) Cloning and biochemical properties of a highly thermostable and enantioselective nitrilase from Alcaligenes sp. ECU0401 and its potential for (R)-(-)-mandelic acid production. Bioprocess Biosyst Eng 34:315–322 Zhang LJ, Yin B, Wang C, Jiang SQ, Wang HL, Yuan YA, Wei DZ (2014) Structural insights into enzymatic activity and substrate specificity determination by a single amino acid in nitrilase from Syechocystis sp. PCC6803. J Struct Biol 188:93–101
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ORIGINAL RESEARCH PAPER
Expression, characterization of a novel nitrilase PpL19 from Pseudomonas psychrotolerans with S-selectivity toward mandelonitrile present in active inclusion bodies Huihui Sun . Wenyuan Gao . Hualei Wang Dongzhi Wei
.
Received: 2 September 2015 / Accepted: 3 November 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Objectives To identify a novel nitrilase with Sselectivity toward mandelonitrile that can produce (S)mandelic acid in one step. Results A novel nitrilase PpL19 from Pseudomonas psychrotolerans L19 was discovered by genome mining. It showed S-selectivity with an enantiomeric excess of 52.7 % when used to hydrolyse (R, S)mandelonitrile. No byproduct was observed. PpL19 was overexpressed in Escherichia coli BL21 (DE3) and formed inclusion bodies that were active toward mandelonitrile and stable across a broad range of temperature and pH. In addition, PpL19 hydrolysed nitriles with diverse structures; arylacetonitriles were the optimal substrates. Homology modelling and docking studies of both enantiomers of mandelonitrile in the active site of nitrilase PpL19 shed light on the enantioselectivity. Conclusions A novel nitrilase PpL19 from P. psychrotolerans L19 was mined and distinguished from other nitrilases as it was expressed as an active inclusion body and showed S-selectivity toward mandelonitrile.
H. Sun W. Gao H. Wang (&) D. Wei State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, People’s Republic of China e-mail: [email protected]
Keywords Active inclusion body (S)-Mandelic acid Mandelonitrile Nitrilase Pseudomonas psychrotolerans L19
Introduction Nitrilase (EC 3.5.5.X) converts nitriles to the corresponding carboxylic acids. The production of (R)mandelic acid from mandelonitrile with a high degree of enantioselectivity is important as it is used for the production of semisynthetic cephalosporins, penicillins, antitumor agents, and antiobesity agents (Banerjee et al. 2006; Baum et al. 2012; Liu et al. 2011; Wang et al. 2013b; Zhang et al. 2011). (S)Mandelic acid is also an important intermediate used for synthesizing non-steroidal anti-inflammatory drugs, such as celecoxib and deracoxib (Mateo et al. 2006). However, there is no one-step nitrilase-mediated pathway to produce (S)-mandelic acid from mandelonitrile because no S-selective nitrilase has been identified. Robertson et al. (2004) described the existence of four nitrilases showing a relatively low preference for the formation of (S)-mandelic acid (25–30 % enantiomeric excess (ee)) from (R, S)-mandelonitrile that was based on metagenomic technology. A possible approach for producing (S)-mandelic acid at high enantiomeric purity was reported by Chmura et al. (2013). The approach involved constructing ‘‘cascade
123
Biotechnol Lett
reactions’’ by using a highly (S)-specific oxynitrilase from Manihot esculenta, a non-selective nitrilase from Pseudomonas fluorescens EBC191, and an amidase from Rhodococcus erythropolis. In another study, the enantioselectivity of a nitrilase from P. fluorescens EBC191 was reversed to (S)-mandelic acid by protein engineering (Sosedov and Stolz 2015). However, the ee value was \37 %. Furthermore, all the mutants generated the by-products (S)-mandelamine in different proportions. In this study, a novel S-selective nitrilase PpL19 from P. psychrotolerans L19 that could hydrolyse (R, S)-mandelonitrile to (S)-mandelic acid with an ee of 52.7 % was discovered by genome mining. It was overproduced in Escherichia coli BL21 (DE3) in an active insoluble form. The substrate specificity of PpL19 and its stability as a function of temperature and pH were investigated. Homology modelling and docking studies were also performed to investigate the enantioselectivity of nitrilase PpL19.
of nitrilases. Mandelonitrile and other chemicals were purchased from Sigma-Aldrich. Database mining Database searches of sequence data were performed using BLASTP. Nitrilase 1A10 (AAR97486.1) from an uncultured organism was chosen as the identifier to detect potential S-selective mandelonitrile specific nitrilases based on similar data mining (Robertson et al. 2004). The screening criteria were the same as described previously (Wang et al. 2013b). Nitrilase sequences showing 30–60 % amino acid identity to nitrilase 1A10 were extracted from the GenBank database. Expression of nitrilase genes in E. coli
Materials and methods
Recombinant E. coli BL21 (DE3) cells used for expressing nitrilase were cultivated in lysogeny broth (LB) containing kanamycin (50 lg/ml) at 37 °C. When the OD600 of the culture reached 0.6–0.8, IPTG was added to 0.1 mM. The induced cultures of E. coli BL21 (DE3) were further incubated for 20 h at 20 °C.
Materials
Inclusion body isolation
Nitrilase genes were synthesized by Shanghai Generay Biotech Co., Ltd. and inserted into plasmid pET28a(?). The resulting plasmids were transformed into Escherichia coli BL21 (DE3) for the expression
The induced cells were harvested by centrifugation (10,0009g, 10 min), washed twice with physiological saline, and resuspended in 10 ml lysis buffer (50 mM Tris/HCl, pH 8). Cells were disrupted by sonication on
Table 1 Activity and enantioselectivity of nitrilases mined from GenBank database Nitrilase
Organism
GenBank accession number
Amino acid identity (%)a
Specific activity (U/mg protein)b
ee value of acid (%)c
313
Variovorax sp. CF313
WP_007829732
58
6.4
89.9
GPnor51
Luminiphilus syltensis NOR5-1B
EED35210.1
41
15
96.7
316
Acidovorax sp. CF316
WP_007851333
37
1.9
92.6
6799
Desulfomonile tiedjei DSM 6799
AFM28028
36
0.34
95.7
39116
Zymoseptoria tritici IPO323
WP_039792070
36
0.28
85.5
CCS1
Jannaschia sp. CCS1
ABD56652.1
36
10
94.7
BTAil
Bradyrhizobium sp. BTAi1
WP_012044777
35
4.3
90.9
RW1
Sphingomonas wittichii RW1
WP_011951832
34
3.5
96.4
Bradyrhizobium sp. ORS 278
WP_011927383
34
1
89.7
Pseudomonas psychrotolerans L19
EHK72968.1
33
1.8
52.7
ORS278 d
PpL19 a
The amino acid identity is compared with the nitrilase 1A10 (AAR97486.1) from an uncultured organism
b
Activities and enantioselectivities of crude enzymes were determined by the method of Zhang et al. (2013)
c
The enantio-preference of nitrilase PpL19 was S-; the enantiopreference of all the other nitrilases was R-
d
Active inclusion bodies were used to determine the activity and enantioselectivity of nitrilase PpL19
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Biotechnol Lett
ice, and the lysate was centrifuged for 30 min at 10,0009g and 4 °C. The cell pellets were washed with buffer containing 50 mM Tris/HCl (pH 8), 100 mM NaCl, and 1 % (v/v) Triton X-100 at room temperature for 30 min, then in the same buffer without Triton X-100 for 30 min. The washed inclusion bodies were collected by centrifugation as above and analysed by SDS-PAGE. Assay of nitrilase activity Nitrilase activity was measured in a reaction mixture (1 ml) containing 50 mM Tris/HCl (pH 8) and nitrilase. After preincubation at 30 °C for 10 min, the reaction was initiated by rapid mixing with mandelonitrile (10 mM). The reaction was quenched by adding 0.1 ml 2 M HCl and the mixture was centrifuged. The formation and optical purity of mandelic acid in the supernatant were analysed by HPLC as described previously (Wang et al. 2013a). The Berthelot assay was also performed in some cases to measure the amount of ammonia formed in nitrilase reactions (Weatherburn 1967). All assays were performed in triplicate and the experimental error was \5 %. One unit of the enzyme activity was defined as the amount of enzyme that produced 1 lmol mandelic acid per min under the standard assay conditions.
Results Screening of recombinant nitrilase BLASTP was performed to identify nitrilases capable of enantioselectively hydrolysing mandelonitrile to (S)-mandelic acid. Ten nitrilases showing 30–60 % amino acid identities with nitrilase 1A10 from an uncultured organism (Robertson et al. 2004) were selected from the GenBank database and overexpressed in E. coli. All the nitrilases were expressed as soluble proteins with R-selectivity toward mandelonitrile, except for PpL19 from P. psychrotolerans L19 that was largely expressed in inclusion bodies and were active toward mandelonitrile and exhibited Sselectivity to produce (S)-mandelic acid with an ee value of 52.7 % (Table 1). No byproduct was observed during the hydrolysis process. A little of the nitrilase PpL19 was expressed in soluble form (Fig. 1) but no activity was observed when this was used to hydrolyse mandelonitrile (data not shown).
Homology modelling and docking studies The 3D homology model of nitrilase PpL19 was constructed using Accelrys Discovery Studio Modeler 4.1 (Sˇali and Blundell 1993), using the crystal structures of nitrilase NIT6803 (PDB accession code ˚ ) (Zhang et al. 2014) from 3WUY, resolution 3.1 A Synechocystis sp. PCC6803 and nitrilase PaNit (PDB ˚ ) (Raczynska accession code 3IVZ, resolution 1.57 A et al. 2011) from Pyrococcus abyssi as templates. The structure of PpL19 generated was improved by refining the loop conformations and assessing the compatibility of the amino acid sequence with known PDB structures. The resulting model was used to perform docking studies. Both enantiomers of mandelonitrile were docked into the model of PpL19 using C-DOCKER (Kim et al. 2009; Yeom et al. 2008). The active sites were defined as a collection of amino acid ˚ radius residues enclosed within a sphere of 5 A centered on the catalytic triad.
Fig. 1 SDS-PAGE analysis of the expression of nitrilase PpL19 in Escherichia. coli Lane 1 protein marker; lane 2 soluble fraction; lane 3 insoluble fraction; lane 4 purified nitrilase PpL19 inclusion bodies isolated from cells after washing
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The optimum temperature for activity was 30 °C and PpL19 retained 80 % of its maximum activity even at
60 °C (Fig. 2). This result differs from most watersoluble nitrilases whose activity decrease sharply [55 °C (Sun et al. 2015; Wang et al. 2013b). The temperature-activity curve indicated that the enzyme was particularly stable at 20 °C and retained 50 % of its initial activity after 40 h (Fig. 2c). pH 8 was optimal for the hydrolysis reaction (Fig. 2b) and PpL19 [ 80 % activity between pH 7 and 9. Arylacetonitrilases usually have a rather narrow optimum pH, at neutral or slightly alkaline values (Zhang et al.
Fig. 2 Effects of temperature and pH on nitrilase PpL19 activity and stability. a Enzyme activity was measured at 20–60 °C in 100 mM Tris/HCl buffer (pH 8). Activity at 40 °C (the optimum temperature) was taken as 100 % (2.8 U/mg). b Enzyme activity was determined with mandelonitrile as substrate at pHs from 5.2 to 10.28 at 30 °C. Activity at pH 8 (the optimum pH) was taken as 100 % (2.7 U/mg). c Purified enzyme was incubated in Tris/HCl buffer at different
temperatures and the residual activity with mandelonitrile as substrate was measured at various time points. The activity at time = 0 was taken as 100 % (2.8, 2.8, 2.8 U/mg for 20, 30 and 40 °C, respectively). d Purified enzyme was incubated at different pHs, and the residual activity was measured at various time points. The activity at time = 0 was taken as 100 % (2.7, 2.7 and 2.7 U/mg for pH 7.6, 8 and 8.6, respectively). Experiments were performed in triplicate
Expression of PpL19 in inclusion bodies was at a high level, and the pure inclusion bodies contained [90 % nitrilase PpL19 with a molecular mass of 37 kDa by SDS-PAGE (Fig. 1), which is in agreement with the size predicted from the amino acid sequence. Characterization of nitrilase PpL19
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Biotechnol Lett Table 2 Substrate specificity of purified PpL19 Substrate
Relative activity (%)
Benzonitrile
64 ± 0.5
3-Hydroxyphenylpropionitrile
49 ± 10
2-Cyanopyridine 3-Cyanopyridine
28 ± 0.24 29 ± 0.5
Phenylacetonitrile
404 ± 12
Mandelonitrile
100 ± 21
a-Methylphenylacetonitrile
88 ± 10
2-Chloromandelonitrile
20 ± 1
Acrylonitrile
0.52 ± 0
3-Hydroxypropionitrile
0.21 ± 0
Succinonitrile
0.01 ± 0
1,2-Phenylenediacetonitrile
4.2 ± 0.22
2-Methylglutaronitrile
9.6 ± 0.3
3-Hydroxyglutaronitrile
0.05 ± 0
Fumaronitrile
0.72 ± 0.05
Nitrilase activity toward various nitriles (10 mM) was measured under standard assay conditions. The activity toward mandelonitrile was defined as 100 % = 2.8 U/mg
Fig. 3 Structure of PpL19. a The structure of PpL19 modelled using Discovery Studio. b-strands are in yellow and a-helices in red. Loops and other secondary structures are in green. The conserved catalytic triad (E48, K130, C164) is shown as sticks
2011). PpL19 nitrilase showed high stability at pH 7.6 (Fig. 2d). Substrate specificity Nitrilase PpL19 had a broad substrate spectrum and hydrolysed a large number of structurally-related nitriles (Table 2). PpL19 preferentially hydrolyse arylacetonitriles and the enzyme exhibited the highest activity toward phenylacetonitrile. Significant activities were also observed with heterocyclic nitriles, such as 2-cyanopyridine and 3-cyanopyridine, and the nitrilase could also hydrolyse aromatic nitriles such as benzonitrile and 3-hydroxyphenylpropionitrile. Considering the distinctive S-selectivity of PpL19 toward mandelonitrile, its enantioselectivity toward other arylacetonitriles was also determined. PpL19 showed S-selectivity toward a-methylphenylacetonitrile, giving a product with 61 % ee. Notably, PpL19 displayed R-selectivtity toward 2-chloromandelonitrile, a derivative of mandelonitrile (ee 70 %). The
and coloured based on element types. b The solvent-accessible surface of PpL19. Positive and negative residues are shown in blue and red, respectively. The catalytic area is in purple
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Fig. 4 Modelled structure of the active sites of PpL19 with (S)-mandelonitrile and (R)-mandelonitrile. Distances between (S)mandelonitrile or (R)-mandelonitrile and Cys-164 are marked with cyan and magenta dashed lines, respectively
Table 3 Catalytic properties of nitrilase PpL19 in synthesis of (S)-(?)-mandelic acid compared with other reported nitrilases Enzyme
Conversion (%)
ee (%)
Reference
1A8
14
26
Robertson et al. (2004)
1A9
13
30
Robertson et al. (2004)
1A10
16
25
Robertson et al. (2004)
5B17
15
27
Robertson et al. (2004)
PpL19
100
52.7
This study
distinct enantioselectivity toward phenylacetonitrile with substituents at different positions may be due to steric hindrance or electronic effect. Homology modelling and docking studies As Fig. 3a showed, the conserved catalytic residues E48, K130 and C164, proposed to be the catalytic triad of nitrilase, have similar orientations and locations in the nitrilase model to these properties in nitrilase crystal structures. A surface cavity was predicted to be the binding pocket with C164 lying at the bottom of the cavity (Fig. 3b).
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When (S)- and (R)-mandelonitrile were docked into the active sites of PpL19 (Fig. 4), there was a notable difference in the distance between the different enantiomers and the catalytic site; the cyano group of (S)-mandelonitrile and the sulfur of C164 were ˚ ) than with (R)-mandelonitrile closer together (3.751 A ˚ (3.833 A). This difference in distance might explain the enantio-preference for mandelonitrile exhibited by nitrilase PpL19.
Discussion Biocatalytic processes are widely used in the pharmaceutical and fine chemicals industries and offer advantages over conventional chemical catalysts in safety and environmental aspects. Nitrilase-mediated biotransformation processes are used to produce optically active a-hydroxyl carboxylic acids, such as (R)-(-)-mandelic acid (Sun et al. 2015; Wang et al. 2013a, b). However, only a few nitrilases exhibiting relatively low S-selectivity toward mandelonitrile have been reported (Robertson et al. 2004; Sosedov
Biotechnol Lett
and Stolz 2015). Screening for enzymes with high Sselectivity is important to meet the requirements of industrial applications. A novel S-selective nitrilase, PpL19, from P. psychrotolerans L19 was identified here by genome mining. PpL19 could enantioselectively hydrolyse (R, S)-mandelonitrile into (S)-mandelic acid with an ee value of 52.7 % and no byproduct was observed. Nitrilase PpL19 showed the highest conversion and enantioselectivity among the reported nitrilases that can produce (S)-mandelic acid (Table 3). However, the selectivity and activity of PpL19 is too low to meet the requirements of industrial production; further modification of PpL19 to improve its selectivity and activity would be necessary to make it suitable for manufacturing processes. To understand how nitrilase PpL19 interacts with the substrates (R)- and (S)-mandelonitrile, a structural model was created. When (R)- and (S)-mandelonitrile were docked into it, the cyano carbon atoms of both pointed directly toward the catalytic triad, but there were remarkable differences in the catalytic distance: a shorter distance was observed for the S-enantiomer, which would result in a higher enzyme activity. Different hydrolysis rates of R- and S-enantiomer substrates result in enzymatic enantio-preference (Zhang et al. 2014). In conclusion, a novel nitrilase PpL19 from P. psychrotolerans L19, S-selective toward mandelonitrile, was identified and expressed in active inclusion bodies. PpL19 exhibited a broad substrate range. Further study will focus on engineering PpL19 to improve its performance in the hydrolysis of mandelonitrile to produce optically pure (S)-mandelic acid. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21406068/B060804), China Postdoctoral Science Foundation funded project (No. 2014M560308) and National Major Science and Technology Projects of China (2012ZX09304009).
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