Kinetic properties and stability of glucose dehydrogenase from Bacillus amyloliquefaciens SB5 and its potential for cofactor regeneration
© Pongtharangkul et al. 2015
Received: 24 October 2015
Accepted: 27 October 2015
Published: 4 November 2015
Glucose dehydrogenases (GluDH) from Bacillus species offer several advantages over other NAD(P)H regeneration systems including high stability, inexpensive substrate, thermodynamically favorable reaction and flexibility to regenerate both NADH and NADPH. In this research, characteristics of GluDH from Bacillus amyloliquefaciens SB5 (GluDH-BA) was reported for the first time. Despite a highly similar amino acid sequence when comparing with GluDH from Bacillus subtilis (GluDH-BS), GluDH-BA exhibited significantly higher specific activity (4.7-fold) and stability when pH was higher than 6. While an optimum activity of GluDH-BA was observed at a temperature of 50 °C, the enzyme was stable only up to 42 °C. GluDH-BA exhibited an extreme tolerance towards n-hexane and its respective alcohols. The productivity of GluDH obtained in this study (8.42 mg-GluDH/g-wet cells; 1035 U/g-wet cells) was among the highest productivity reported for recombinant E. coli. With its low K M-value towards glucose (5.5 mM) and NADP+ (0.05 mM), GluDH-BA was highly suitable for in vivo applications. In this work, a recombinant solvent-tolerant B. subtilis BA overexpressing GluDH-BA was developed and evaluated by coupling with B. subtilis overexpressing an enzyme P450 BM3 F87V for a whole-cell hydroxylation of n-hexane. Significantly higher products obtained clearly proved that B. subtilis BA was an effective cofactor regenerator, a valuable asset for bioproduction of value-added chemicals.
Keywordsgdh Glucose 1-dehydrogenase Cofactor regeneration Bacillus amyloliquefaciens
Bio-based chemical production has been depicted as a promising approach to a sustainable chemical manufacturing. Biocatalytic oxidoreductions hold great potential for industrial production of enentiopure chemicals and pharmaceuticals. Most oxidoreductase enzymes, however, require prohibitively expensive nicotinamide cofactors (NADH or NADPH). To date, the use of NADPH-dependent catalysts has been severely limited by an absence of efficient methods to recycling the cofactor. An efficient cofactor regeneration system is, therefore, one of the most critical requirements prior to a commercialization of biocatalytic oxidoreductions. Thus far, biological approaches using either isolated enzyme or whole-cell biocatalyst seem to exhibit the highest potential for commercial applications (de Wildeman et al. 2007).
Glucose dehydrogenase (GluDH; EC 18.104.22.168) catalyzes the oxidation of β-d-glucose to β-D-glucono-1,5-lactone with simultaneous reduction of the cofactor NAD(P)+ to NAD(P)H. The enzyme occurs in a variety of organisms such as Bacillus megaterium, Bacillus subtilis, Gluconobacter suboxydans, Halobacterium mediterranei, Thermoplasma acidophilum, and Sulfolobus solfataricus. GluDHs from different organisms show diverse biochemical properties (e.g. activity and stability) and preference towards the cofactors (NAD+ and NADP+). Amino acid sequence alignment indicated that NAD(P)+-dependent GluDHs from the Bacillus species, belonging to the extended superfamily of short-chain dehydrogenases/reductases (SDR) (Nishiya et al. 2004), have more than 80 % homology (Xu et al. 2007). GluDH from B. subtilis (GluDH-BS) has been used extensively for in vivo NAD(P)H regeneration (Zhu et al. 2006; Schewe et al. 2008; Zhang et al. 2009, 2011; Richter et al. 2010), mainly because of its dual cofactor specificity and ease of expression in a commonly used Escherichia coli hosts.
Despite its successful application in a bioproduction of epoxyhexane (Siriphongphaew et al. 2012), in this study, GluDH from Bacillus amyloliquefaciens SB5 (GluDH-BA) was studied in detail in order to evaluate its full potential in biocatalysis. A GluDH-encoding gene from B. amyloliquefaciens SB5 has been cloned and expressed in both E. coli and B. subtilis. The kinetics and biochemical properties of the purified enzyme were evaluated. To our knowledge, this is the first report on characteristics of GluDH from B. amyloliquefaciens. Moreover, for an in vivo application, a recombinant B. subtilis 168 overexpressing GluDH-BA (referred as B. subtilis BA) was evaluated as a whole-cell cofactor regenerating biocatalyst for hydroxylation of n-hexane by coupling with B. subtilis overexpressing P450 BM3 F87V.
Chemicals and media
All chemicals used were of analytical grade and commercially available from Sigma-Aldrich (USA). Luria–Bertani (LB) and Tryptic soy broth (TSB) were used for cultivation and storage of working culture. For solid media, 1.5 % Bacto Agar (Lab M, Lancashire, UK) was added. Ampicillin (Amp) at 50 μg/μl and tetracycline (Tet) at 20 μg/μl were used for selection and cultivation of recombinant E. coli and B. subtilis, respectively.
Strains, plasmids and culture conditions
B. amyloliquefaciens SB5 was previously isolated from a petroleum contaminated soil using an enrichment technique with benzene as a screening agent. Its identity was confirmed using bioMerieux api™ 20E and 50CHB/E test strips (bioMerieux, Marcy L’Etoile, France) and 16s rDNA sequencing. The strain was deposited to TISTR Microbiological Resources Centre (Pathumthani, Thailand) under the accession number TISTR2086. Bacillus subtilis 168 (BGSC 1A1) was kindly provided by Bacillus Genetic Stock Center (BGSC, Columbus, OH). Recombinant B. subtilis 3C5N (pHBGA), referred as B. subtilis B-7, was developed previously by Siriphongphaew et al. (2012).
Construction of recombinant plasmids for an expression of glucose 1-dehydrogenase in E. coli
Primers used in this study
Purification of GluDH-BA
The entire purification process was performed at 4 °C. Escherichia coli BL21(DE3) harboring the plasmid pET23b-gdh-ba was cultivated in an auto-induction medium (containing 0.5 g/L glucose, 6 g/L glycerol, 2 g/L lactose, 10 g/L peptone, 5 g/L yeast extract, 5 g/L sodium chloride, 6 g/L Na2HPO4 and 3 g/L KH2PO4) at 37 °C, 200 rpm for 6 h and then at 20 °C, 200 rpm for 14 h. Cells were harvested, resuspended in phosphate buffer (pH 8) and broken with Bead beater-1 (0.25 g of 1-mm ϕ glass bead per ml; 90 s per cycle at 4300 rpm; 5 cycles; chilled on ice for 1 min between each cycle) (Biospec, OK, USA). After centrifugation at 11,337×g, 4 °C for 30 min, the supernatant was collected and purified using PrepEase® Histidine-Tagged Protein Purification Kits – High Specificity according to the recommended protocol (usb, OH, USA). The purified enzyme was diluted (5×) with Citrate-Phosphate-Borate or CPB buffer (pH 6) and then concentrated using a spin filter with MWCO of 10 kDa prior to a storage at −80 °C. The protein concentration was determined by the Bradford assay (Bradford 1976) using bovine serum albumin as a standard. SDS-PAGE was carried out on a 4–12 % Bis–Tris Gel (NuPAGE Novex, Thermo Scientific, IL, USA) at a constant current (15 mA/gel). PageRuler™ Prestained Protein Ladder (Thermo Scientific, IL, USA) was used as a molecular mass marker. After electrophoresis, the gel was stained with Coomassie brilliant blue R-250 (Laemmli 1970) and de-stained by soaking in the methanol:acetic acid solution.
Construction of recombinant plasmids and expression of glucose 1-dehydrogenase in B. subtilis
Bacillus subtilis 168, previously reported to exhibit an organic solvent-tolerant property (Siriphongphaew et al. 2012), was selected as a host for development of a whole-cell cofactor regenerator. The gdh fragments were amplified from the plasmid pJET-gdh-bs and pJET-gdh-ba using primers containing restriction sites for BsrGI and KpnI (Table 1; primers no. 3, 6 and 7). After a double digestion with BsrGI and KpnI, the fragments were inserted into a Bacillus expression vector pHP2N. Plasmid pHP2N was constructed based on a plasmid backbone of E. coli-Bacillus shuttle vector pHY300PLK (TaKaRa, Shiga, Japan) with an insertion of a strong P2 promoter from a commercial expression plasmid pNCMO2 (TaKaRa, Shiga, Japan). The resulting plasmids, named as pHGBS and pHGBA, were introduced into B. subtilis 168 by electroporation using a protocol described previously (Siriphongphaew et al. 2012). Successful transformation was checked by a colony PCR as well as a restriction analysis. The recombinant B. subtilis 168 overexpressing GluDH-BS and GluDH-BA were referred as B. subtilis BS and B. subtilis BA, respectively.
Crude enzyme preparation
Crude enzyme extract was prepared from B. subtilis BS and B. subtilis BA as follow. Cells were cultivated in LBtet at 37 °C, 200 rpm for 16 h, harvested by centrifugation at 11,337×g for 10 min, washed twice and re-suspended in CPB buffer to an optical density (600 nm) of 10. Cells were broken with Bead beater as previously described and supernatant was collected after centrifugation at 11,337×g for 10 min.
GluDH activity assay
GluDH activity was assayed by measuring the absorbance of NAD(P)H at 340 nm. Unless specified otherwise, the reaction was performed in 50 mM Tris–Cl buffer (pH 8) with 0.5 mM NAD(P)+ and 50 mM glucose at 37 °C. The concentration (U/ml) was calculated using the millimolar extinction coefficient (ε 340) of 6.22. One unit (U) of GluDH activity was defined as the formation of 1 μmole of NAD(P)H per minute and the specific activity was reported in terms of U/g-CDW as well as U/mg-protein.
Effect of pH and temperature on activity and stability of purified GluDH-BA and intracellular GluDH-BA
Effect of pH on specific activity of purified GluDH-BA was determined by measuring the activity at 37 °C using CPB buffer with a pH range from 5 to 10. The effect of pH on enzyme stability was determined from the residual activity (%) after incubating the purified enzyme in the CPB buffer with a specified pH at 30 °C for 6 h. The stability of the enzyme inside the whole-cell biocatalyst was determined in a similar manner except that the cell suspension (OD600 = 10) was incubated in the specified buffer for 6 h before being used for preparation of crude extract. For comparison, the stability of intracellular GluDH-BA was compared with those of crude GluDH-BS and crude GluDH-BA prepared from B. subtilis BS and B. subtilis BA, respectively.
Effect of temperature on activity and stability of the purified GluDH-BA was evaluated using CPB (pH 6) over the temperature range of 10–60 °C. The effect of temperature on stability was determined from the residual activity after incubating the enzyme at a specified temperature for 6 h.
Effect of organic solvents on stability of GluDH-BS and GluDH-BA
Effect of various organic solvents (DMSO, acetone, ethanol, n-butanol, n-hexane, 1-hexanol and 2-hexanol) on stability of the purified GluDH-BS and GluDH-BA were evaluated by incubating the enzyme with either 10 or 50 % (v/v) of the specified organic solvent at 30 °C for 1 h. The GluDH activity was then assayed using CPB (pH 6) at 37 °C. Relative activity (%) was calculated based on a control in which water was added instead of the organic solvent.
Apparent K M and V max value of the purified GluDH-BA were determined using various substrate concentrations (10-250 mM of glucose) at a fixed concentration of the acceptor (0.5 mM of NAD+ or NADP+). Double reciprocal plot was used to determine K M and V max and only the concentrations that did not cause substrate inhibition were used. The initial rate was determined by linear regression as a slope of the linear plot between NAD(P)H formation and time.
Application of B. subtilis BA as a whole-cell cofactor regenerator
All experiments were performed in duplicate. Statistical analysis including ANOVA and multiple comparisons (Tukey’s test) were performed using Minitab (Release 15, State College, PA, USA).
Strain and amino acid sequence
B. amyloliquefaciens SB5 has been deposited to Thailand Institute of Scientific and Technological Research or TISTR (Pathumthani, Thailand) with the accession number TISTR2086. An amino acid sequence of its gdh gene has been submitted to the GenBank nucleotide sequence database (NCBI) under an accession number JQ305165.
Amino acid sequence and structural analysis of GluDH from B. amyloliquefaciens SB5
GluDH activity of cell-free extracts prepared from wild-type strains and their recombinants
B. subtilis 168
0.99 ± 0.44
0.008 ± 0.001
B. subtilis 3C5N
0.22 ± 0.05
0.002 ± 0.000
B. amyloliquefaciens SB5
15.8 ± 1.90
0.166 ± 0.085
B. subtilis B-7b
0.02 ± 0.02
0.0002 ± 0.0002
B. subtilis BSc
6.84 ± 0.59
0.020 ± 0.003
B. subtilis BAd
1601 ± 146
6.24 ± 0.39
Kinetic constants of purified GluDH from B. amyloliquefaciens SB5 comparing to those reported in the literature
K M value (mM)
Turnover number (1/s)a
Specific activity (U/mg-protein)
Fujita et al. (1977)
Adachi et al. (1980)
Giardina et al. (1986)
Yamamoto et al. (1990)
Lysinibacillus sphaericus G10
Ding et al. (2011)
Bacillus amyloliquefaciens SB5
0.25 ± 0.03
0.05 ± 0.004
5.5 ± 0.28
70 ± 0.3
123 ± 0.5
From structural analysis, most of the variable amino acids are located on rather highly flexible regions (Fig. 3b). Interestingly, there were cases where a mutation at the flexible regions improved the thermostability of an enzyme (Reetz et al. 2006). In order to evaluate the effect of amino acid variation between GluDH-BS and GluDH-BA on their activity and stability, we compared these variations with previous mutagenesis studies. Only P45A mutation has been previously reported to enhance activity and stability of GluDH-BS, even though the location of this amino acid is further away from the active site and subunit interface (Vázquez-Figueroa et al. 2007). Also, a single mutation of amino acids outside the active site channel (i.e. F155Y and E170 K) in GluDH-BS has been shown to affect both activity and stability significantly (Vázquez-Figueroa et al., 2007). From these evidences, it is highly likely that the differences in the sequences, even though at amino acids on the surface or remote from the catalytic center, can lead to the activity and thermostability alteration. To further our understanding of the structure–function relationship, mutational analysis of other positions is now in progress.
Expression and purification of GluDH-BA
Effect of pH on the activity and stability of GluDH-BA
Despite a widespread use of Tris–Cl buffer (pH 7.2–9.0) and potassium phosphate buffer (pH 5.0–8.0) for GluDH activity assay (Fujita et al. 1977; Boontim et al. 2004; Weckbecker and Hummel 2005), in this research, buffers with a wider pH range such as Britton-Robinson buffer (pH 2.6–11.8) and Citrate–Phosphate-Borate or CPB buffer (pH 2.0–12.0) were tested and compared with a commonly used Tris–Cl in order to eliminate the effect of buffer types in a study on the effect of pH. All three buffers were evaluated at pH 8, an optimum pH generally reported for GluDH activity assay, using GluDH-BA. While Britton-Robinson buffer resulted in a significantly lower GluDH activity (58 % of that observed in Tris–Cl buffer), CPB buffer was found to be comparable to Tris–Cl (not significantly different at α = 0.05) and therefore was suitable for a study on the effect of pH. Moreover, as Tris–Cl was reported to interfere with the Bradford dye used for protein assay (Stoll and Blanchard 1990), CPB was used throughout the rest of this study.
% Relative in specific activity of crude GluDH-BS determined after incubation in CPB (pH 8, 30 °C) for a specified period
Time of exposure (h)
% Relative of specific activityA
100 ± 0a
73.8 ± 14.9ab
41.2 ± 15.5b
1.3 ± 0.6c
0 ± 0c
For bioconversion of toxic chemicals, tolerance of the genetically engineered host towards such chemicals was highly critical (Schewe et al. 2008; Siriphongphaew et al. 2012). When compared with E. coli DH5α, B. subtilis168 exhibited significantly higher tolerance towards toxic chemicals (Siriphongphaew et al. 2012) and therefore was selected for development of a whole-cell cofactor regenerator in this study. To investigate the effect of extracellular pH on the stability of intracellular GluDH-BA, cells of a recombinant B. subtilis BA were incubated in CPB with a specified pH for 6 h and the specific activity of their cell-free extracts were then compared with that of a control (a cell-free extract prepared from the cells without an exposure). As expected, the specific GluDH activity remained unchanged regardless of the extracellular pH that the cells were exposed to (Fig. 6).
Effect of temperature on the activity and stability of GluDH-BA
Effect of organic solvents on the stability of GluDH-BS and GluDH-BA
Stability of purified GlcDH-BS and GlcDH-BA in various organic solvents
% Relative activitya
10 % v/v
50 % v/v
10 % v/v
50 % v/v
87 ± 3
98 ± 3
100 ± 0
99 ± 1
80 ± 2
1 ± 1
99 ± 1
1 ± 1
86 ± 3
1 ± 1
100 ± 1
19 ± 16
0 ± 0.5
0 ± 0
2 ± 0
1 ± 1
89 ± 0
99 ± 1
100 ± 0
100 ± 0
56 ± 10
94 ± 0
68 ± 4
69 ± 1
95 ± 1
93 ± 2
Unlike GluDH-BS which equally preferred NAD+ and NADP+ as a cofactor, a purified GluDH-BA exhibited higher preference towards NADP+ (Table 3), similar to GluDHI, GluDHII and GluDHIWG3 from B. megaterium (Mitamura et al. 1989; Yamamoto et al. 1990). In comparison with GluDH-BS (42.9 mM), a significantly lower K M-value towards glucose (5.5 mM) was observed for GluDH-BA, suggesting its ability to immediately react with a small amount of glucose present in vivo. Faster NAD(P)H regeneration can be achieved at lower glucose concentration for the same amount of GluDH. Therefore, a high expression level may not be required, making GluDH-BA suitable for enzymatic cascading. Moreover, for an in vivo application in a whole-cell biocatalyst, an enzyme with low K M for NAD(P)+ would be most appropriate due to the low NAD(P)+ concentration within the cell. Nonetheless, the reaction rate would depend on the turnover number of the enzyme. For this reason, GluDH from Sulfolobus solfataricus, despite having a low K M for NAD(P)+, is probably not suitable for in vivo applications due to its low turnover number (Table 3).
For applications in a form of crude lysate and purified enzyme, an enzyme with high K M (possibly for glucose but not necessary for NAD+) and high kcat would be most preferable. Such kinetic properties would allow for an operation under high substrate concentration (glucose) and therefore fast reaction rate (dictated by the kcat) could be obtained. GluDH with low K M towards glucose, including GluDH from S. solfataricus, is more likely to suffer from glucose inhibition than other GluDHs. Nonetheless, for industrial and synthesis purposes, kinetic parameters alone should not be used to identify which enzyme is suitable for catalysis. Stabilities and inhibitions should also be considered. Since GluDHs are widely used as cofactor regeneration enzyme, it would be useful to have a wide range of GluDHs to select from. This is because certain variants may be inhibited or incapable in a certain NAD(P)H-consuming reaction.
Application as a whole-cell cofactor regenerator for hydroxylation of n-hexane
In a recombinant B. subtilis BA, GluDH-BA was expressed at a high level (6.24 U/mg-total protein) comparable to the GluDH activity obtained in E. coli when using the pET system (7.18 U/mg-total protein; Richter et al. 2010) or that expressed in B. subtilis under a constitutive strong promoter P43 (4.9 U/mg-total protein; Zhu et al. 2006). In this study, B. subtilis BA was evaluated as a whole-cell cofactor regenerator in a coupling whole-cell system for hydroxylation of n-hexane.
Permeabilization by a toluene treatment (at 1 % v/v) was frequently employed in bioconversion to guarantee access of substrate and cofactor into the cells (Cánovas et al. 2005; Zhang et al. 2009). Unfortunately, toluene treatment resulted in an absolute loss of indigenous cofactors inside the cells and extracellular supplementation of NAD(P)+ was necessary. Permeabilized B. subtilis B-7 failed to catalyze the hexane hydroxylation even when 0.2 mM NADP+ was supplemented extracellularly, agreeing with an extremely low GluDH activity observed previously (Table 2). Interestingly, an incorporation of a non-permeabilized whole-cell B. subtilis BA could effectively restore the bioconversion (Fig. 8).
In this study, NAD(P)-dependent GluDH from B. amyloliquefaciens SB5 (GluDH-BA) has been successfully cloned and expressed in both E. coli and B. subtilis. Several strains of B. amyloliquefaciens have been widely regarded as a member of ‘plant growth promoting rhizobacteria’ or PGPR (Idriss et al. 2007). The mechanisms by which PGPR can exert a positive effect on plant growth include the solubilization of insoluble inorganic phosphate compounds (such as tricalcium phosphate and hydroxyapatite) via an action of organic acids synthesized by them. Considering the fact that gluconic acid (formed via an action of glucose dehydrogenase) was reported to be the most common agent for mineral phosphate solubilization, it was not surprising that B. amyloliquefaciens with a high level of GluDH activity has been identified as one of the most powerful inorganic phosphate solubilizers (Rodriguez and Fraga 1999). High level of GluDH activity observed in B. amyloliquefaciens SB5 (Table 2) agreed well with those findings and the phosphate solubilizing property of B. amyloliquefaciens SB5 is now being investigated in detail.
Comparing to GluDHs reported from genus other than Bacillus, the specific activity of the purified GluDH-BA (123 U/mg-protein) was comparable to that reported for G. suboxydans (Adachi et al. 1980), but lower than that of L. sphaericus G10 (Ding et al. 2011) and S. solfataricus (Giardina et al. 1986) (Table 3). Nonetheless, it should be noted that a specific activity value highly depends on several factors (i.e. buffer, pH and substrate concentration) and therefore may not be a good parameter for comparison. Despite a highly similar sequence with GluDH-BS, GluDH-BA exhibited significantly higher specific activity (4.7-fold) and stability when pH was higher than 6. GluDH-BA also exhibited higher tolerance towards organic solvents, especially hexanols.
Amino acid sequence alignment and structural analysis of both GluDHs revealed several novel potential amino acid positions [e.g. position 16 in the region of NAD(P)+-binding motif and position 95 in the active site channel] for further improvement of the enzyme activity and stability via site-directed mutagenesis. The 3D structure of GluDH IV from B. megaterium (Nishioka et al. 2012) revealed that amino acid position 16 was a part of the outer layer of the active site channel located at least 16 Å away from the reactive center. The difference in size and chemical properties of an amino acid at this position may account for the difference in enzyme activity and stability. Despite the fact that a single mutation of amino acids outside the active site channel in GluDH-BS affected both activity and stability significantly (Vázquez-Figueroa et al. 2007), similar phenomenon can be observed as a result of accumulated mutations at scattered locations as well (e.g. an enzyme-surface mutant Q42E, P45A and N46A in B. subtilis; Vázquez-Figueroa et al. 2008).
High specific activity as well as low K M-value towards glucose and NADP+ of GluDH-BA suggested its potential for in vivo applications. While the high specific activity allows for an efficient cofactor recycling even with a low level of expression (exerting lower metabolic burden onto the cells and making it more suitable for enzymatic cascading), the low K M-value, on the other hand, allows the enzyme to immediately react with a small amount of glucose and NADP+ present in vivo. As GluDH-BA was successfully co-expressed along with the enzyme P450 BM3 in our previous study (Siriphongphaew et al. 2012), in this study, a whole-cell B. subtilis overexpressing GluDH-BA alone was evaluated as a cofactor regenerator in a coupling whole-cell system instead. Coupling whole-cell system offers several advantages including process flexibility as well as an opportunity to supplement additional cofactor externally. The enzymes-of-interest can be expressed in their most appropriate hosts and the enzyme activity level required for optimal bioconversion can be adjusted easily by adjusting the amount of each whole-cell biocatalyst used. Moreover, as a whole-cell biocatalyst generally maintains its cytoplasmic pH near neutrality via several acid and alkaline pH homeostasis (Booth 1985; Padan et al. 2005; Baker-Austin and Dopson 2007), the intracellular enzyme (GluDH-BA in our case) is well protected from an extreme pH outside the cells. This allows for longer use period of the biocatalyst as well as higher process flexibility (i.e. wider range of possible reactions, ease of process control). Although B. subtilis BA has been proven as an effective cofactor regenerator for a coupling whole-cell system in this study, for practical applications, it should be investigated further on its ability to be recycled or stored as a frozen cell pellet.
PC and TP carried out the molecular cloning experiments and enzyme purification. NS and TP conducted the enzyme characterization studies. PP performed the protein structural analysis work. TP drafted the manuscript with ideas contributed by PP, KH and WP. All authors read and approved the final manuscript.
This work was supported by the Thailand Research Fund (TRF) under a Project Number MRG5580048 to TP with WP as her mentor. A gene encoding P450 BM3 F87V was kindly provided by Prof. Jun Ogawa from Division of Applied Life Sciences, Kyoto University, Japan.
The authors declare that they have no competing interests.
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