Aminated Single-walled Carbon Nanotubes as Carrier for Covalent Immobilization of Penylalanine Ammonia-lyase

A new and efficient immobilized form of phenylalanine ammonia-lyase (PAL) was obtained by covalent linkage onto amino functionalized single-walled carbon nanotubes (SwCNTNH2) as carrier. The catalytic properties of the resulted nanostructured biocatalyst (SwCNTNH2-PAL) were tested in the kinetic resolution of racemic 2-amino-3-(thiophen-2-yl)propanoic acid 1 by ammonia elimination and in the enantiotope selective addition of ammonia onto (E)-3-(thiophen-2-yl)acrylic acid 2. SwCNTNH2-PAL was a durable biocatalyst in batch mode for ammonia elimination from 1 (>85% of original activity after 7 cycles) and in ammonia addition to 2 (>70% of original activity after 3 cycles in 6 M NH3 , pH 10.0). The ammonia addition onto 2 was also studied in a continuous-flow microreactor packed with SwCNTNH2-PAL (2 M NH3, pH 10.0, 15 bar) in the 30-80 °C temperature range. No significant loss of PAL activity was observed over 72 h in the microreactor up to 60 °C. Productivity of SwCNTNH2-PAL at 30 °C was significantly higher in the enantiotope selective ammonia addition performed in a packed-bed reactor operated in continuousflow mode (rflow =2.41 mmol min -1 g-1) than in the reaction performed in batch system (rbatch = 1.38 mmol min -1 g-1).

for stereoselective biotransformation. Application of immobilized enzymes in miniaturized or microfluidic reactors for biotransformations offers multiple advantages such as higher efficiency of the process and improved operational stability of the biocatalysts [22,23].
In our previous study [19], phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) was immobilized by covalent linkage onto carboxylated single-walled carbon nanotubes (SwCNT COOH ) to prepare PcPAL biocatalysts with improved properties for continuous-flow applications. In this study we performed the covalent immobilization of the same enzyme on aminated single walled carbon nanotubes (SwCNT NH2 ) with the aim to further improve the enzyme stability for the ammonia addition reaction. The SwCNT NH2 -PAL obtained in this way was tested as biocatalyst for stereoselective biotransformations, both in batch mode and in continuous-flow reactors.

Materials 2.1.1 Enzyme
Phenylalanine ammonia-lyase from parsley (Petroselinum crispum, PcPAL) was obtained after overexpression in E. coli and purification using a previously reported protocol [14].

Equipment
Transmission electron microscopy (TEM) was performed using an automatic H-7650 TEM, Hitachi (Tokyo, Japan) with accelerating voltage 40-120 kV, zoom 200x-600000x, by dipping a holey-carbon TEM grid into a suspension of the immobilized enzyme. Sample was imaged in the TEM operating at 120kV using an Olympus KeenView G2 camera.
Elemental analyses were carried out with a Vario Micro Cube analyzer, Elementer Analysensysteme GmBH (Langeselbold, Germany).
The quantitative protein assay based on Bradford method was performed on an Agilent 8453 UV-Vis spectrophotometer (Santa Clara, CA, USA).
The enzymatic reactions in batch mode were shaken in a Titramax 1000 instrument.
The conversions for batch reactions were calculated using the 1 H-NMR spectra in [D 4 ]-acetic acid as solvent at 25 °C, recorded on Ascend 400 or Ascend 600 NMR spectrometers, Bruker (Karlsruhe, Germany) operating at 400 and 600 MHz, respectively [19].
The amination reaction of the single-walled carbon nanotubes was performed at 600 W power at 50 bar pressure, 300 °C for 1 h in a CEM Microwave-assisted Reactor, CEM Corporation (Matthews, USA).

Functionalization of SwCNT with amino groups
The functionalization of carbon nanotubes was carried out using a modified method by Mirzarakhmetova et al. [26]. The reaction mixture contained SwCNT (400 mg), urea (400 mg) and dimethyl formamide (DMF, 5 mL). The reaction was carried out in a microwave assisted reactor at 300 °C for 1 h. After the reaction the modified SwCNT was washed with DMF, methanol, CH 2 Cl 2 , and dried. Elemental analysis data shown an ~ 4.8% NH 2 content for the aminated single walled carbon nanotubes (SwCNT NH2 ). According to TEM analysis, the geometry of the parent SwCNT (ID = 0.8-1.6 nm, OD 1-2 nm, Length = 5-30 µm) did not changed significantly by amine functionalization.

Covalent binding of PcPAL onto SwCNT NH2
SwCNT NH2 (20 mg) was incubated with a solution of glycerol diglycidyl ether (GDE, 32.4 mg, 0.2 mmol) in CH 2 Cl 2 (5 mL) under shaking at 1350 rpm at room temperature overnight, with occasional sonication to avoid bundled SwCNT formation (Fig. 1, step i). The sample was filtered on a membrane filter and then washed with CH 2 Cl 2 (3×1 mL). To the resulted modified SwCNT NH2 , a solution of PcPAL (2.0 mg in 1 mL of 0.1 M Tris buffer, pH 8.8) was added and the mixture was shaken at room temperature at 1350 rpm, overnight ( Fig. 1, step ii). The formed biocatalyst was filtered off on a membrane filter and washed with distilled water (3×10 mL). The amount of immobilized PcPAL was determined by comparison of the mass of PcPAL in the solution before the immobilization (2.0 mg in 1 mL solution) and after immobilization (0.06 mg, in the unified filtrates) by spectrophotometric assay using the Bradford method.

Ammonia elimination from 1 catalyzed by SwCNTNH 2 -PAL
Into the solution of racemic 2-amino-3-(thiophen-2-yl)propanoic acid (1, 4.7 μmol, 0.8 mg) in Tris buffer (1 mL, 0.1 M, pH 8.8) was added SwCNT NH2 -PAL biocatalyst (6 mg). After preforming the reaction at room temperature for 17 h under shaking (1250 rpm), the reaction mixture was filtered on a membrane filter and the filtrate was concentrated. 1 H-NMR spectra of the residue, after removal of the solvent, were recorded and used to calculate conversions as previously reported [19]. The recovered SwCNT NH2 -PAL was washed with Tris buffer (1 mL, 0.1 M, pH 8.8) and after filtration it was reused under the same reaction conditions.

Ammonia addition to 2 with SwCNTNH 2 -PAL in batch mode
Into the solution of 3-(thiophen-2-yl)acrylic acid (2, 4.5 μmol, 0.7 mg) in ammonia solution (2 M, 1 mL, pH set to 10 with CO 2 ), the SwCNT NH2 -PAL biocatalyst (6 mg) was added and the reaction was performed for 17 h at room temperature under shaking (1350 rpm). The reaction mixture was filtered and the filtrate was concentrated. 1 H-NMR spectra of the residue, after removal of the solvent, were recorded and used to calculate conversions as previously reported [19]. Before reuse under the same reaction conditions, the recovered SwCNT NH2 -PAL was washed with Tris buffer (1 mL, 0.1 M, pH 8.8), conditioned by shaking for 2 h in phosphate buffer (1 mL, 0.1 M, pH = 6) and washed with Tris buffer (1 mL, 0.1 M, pH 8.8) again.

Ammonia addition to 3-(thiophen-2-yl)-acrylic acid 2 with SwCNT NH2 -PAL in continuous-flow packed-bed microreactor
The continuous-flow experiments were performed in a thermostated SynBioCart microreactor (30 mm × 3 mm ID PTFE SynBioCart columns filled with 110 mg of SwCNT NH2 -PAL in a PEEK SynBioCart column holder closed with PTFE membrane filter of 0.22 µm pore size) attached to the pump module of an Agilent LC 1150 HPLC system and a back-pressure regulator valve (VICI AG, JR-BPR1). Prior to perform the reaction, the microreactor was washed with 2 M ammonia solution (pH set to 10 with CO 2 ) at 0.5 mL min -1 flow rate for 20 min.
The experiments were performed by pumping the solution of 3-(thiophen-2-yl)acrylic acid (2, 4.5 mM) in 2 M ammonia solution (pH set to 10 with CO 2 ) through the thermostated SwCNT NH2 -PAL-filled column at various temperatures (25-80 °C) at a flow rate of 0.1 mL min -1 . Samples (10 μL) were withdrawn at every 10 min (after reaching the stationary state, usually 30 min) and analyzed by an UV spectrophotometer (added to 990 μL of 2 M ammonia solution, at 290 nm). The experiments at room temperature (25 °C) were conducted without backpressure regulation, the experiments between 30 and 80 °C were performed with backpressure (set to 15 bar).
All experiments were performed with the same SwCNT NH2 -PAL-filled column in the temperature range 25-60 °C. Intermittently, the immobilized PAL-filled column was stored at 4 °C (after washing with distilled water, at 0.5 mL min -1 for 30 min). In tests above 60 °C, new SwCNT NH2 -PAL-filled columns were used at each temperature.

Results and Discussion
The aim of this study was to create covalently immobilized PAL biocatalyst with even higher activity and reusability in the ammonia addition reaction onto arylacrylates than was observed for SwCNT COOH -PAL [19]. To achieve this goal SwCNT was functionalized first with amino groups to give a support (SwCNT NH2 ) suitable for direct bisepoxide-functionalization and subsequent covalent attachment of PcPAL (SwCNT NH2 , see Fig. 1). According to our estimation assuming zig-zag conformation for the linkers, the resulted linker in SwCNT NH2 -PcPAL was about 0.7 nm shorter than the previously applied linker in SwCNT COOH -PAL [19]. Furthermore, this shorter linker was expected to be more stable because it did not contain hydrolysable amide bond. The catalytic properties of the resulted novel immobilized form of PcPAL (SwCNT NH2 -PAL) were evaluated in the ammonia elimination from 2-amino-3-(thiophen-2-yl) propanoic acid 1 and in the ammonia addition onto 3-(thiophen-2-yl)acrylic acid 2 as test reactions ( Fig. 3 and Table 1).

Covalent immobilization of PcPAL onto SwCNT NH2
The method applied for covalent attachment of PcPAL onto SwCNT COOH [19] was simplified for the covalent immobilization of the enzyme onto functionalized SwCNT NH2 . First, SwCNT NH2 was activated by reaction with a large excess of glycerol diglycidyl ether (GDE), followed by attachment of PcPAL onto the surface of SwCNT NH2 via the GDE-based linker (Fig. 1). Immobilization yield (97±1.5% of the PcPAL was bound to the bisepoxide-activated SwCNT NH2 resulting in 88 μg protein/mg biocatalyst) as well as the stability of covalently immobilized enzyme (Fig. 2 showing TEM data before and after 12 cycles) were high and reproducible.

Biocatalytic behavior of SwCNT NH2 -PAL in stereoselective biotransformations of 2-amino-3-(thiophen-2-yl) propanoic acid 1 and 3-(thiophen-2-yl)acrylic acid 2 in batch mode
The novel SwCNT NH2 -PAL preparation was tested as biocatalyst at room temperature in the ammonia elimination from 2-amino-3-(thiophen-2-yl)propanoic acid 1 and in the ammonia addition to 3-(thiophen-2-yl)acrylic acid 2 performed in batch mode (Fig. 3). The kinetic resolution of the racemic 1 catalyzed by SwCNT NH2 -PAL occurred with high conversions (close to the theoretically possible 50% conversion). Moreover, the biocatalyst retained most of its initial activity and could be reused several times (Table 1). In the case of ammonia addition to 2 requiring much harsher conditions (6 M ammonia, pH 10) the new SwCNT NH2 -PAL form was significantly more active (64.2% conversion, see Table 1) than SwCNT COOH -PAL (36.9% conversion [19]). Moreover, SwCNT NH2 -PAL was also much more durable, losing more than 80% of its original activity only after 6 cycles (Table 1), in contrast to SwCNT COOH -PAL which inactivated completely after 3 cycles [19]. In ammonia solutions the enhanced durability of SwCNT NH2 -PAL compared to SwCNT COOH -PAL was expected because the linker of SwCNT NH2 -PAL contained non-hydrolyzable ether and alkylamine bonds besides the C-C bonds. Contrarily, it was indicated that an amide bond -being present in the linker of SwCNT COOH -PAL -could undergo hydrolysis under various conditions [27] or transamidation with other amines even under mild conditions by enzyme catalysis [28].
As it was known that ammonia addition onto arylacrylates can be performed at lower ammonia concentrations [19,29], the B A reaction was tested with 2 in 3 M and 2 M ammonia solutions as well. At the lower ammonia concentrations SwCNT NH2 -PAL exhibited significantly higher stability (Fig. 4). In 2 M ammonia at pH 10, more than 82% of the initial activity was retained even after 12 cycles. The unusual shape of the inactivation curves in recycling study with a plateau followed by rapid inactivation (Fig. 4) initiated a hypothesis. It could be assumed that at high concentration of ammonia, the small NH 3 molecules penetrated gradually into the protein and replaced the structural water. Further, it could be assumed that such replacements happened at first in the region close to the surface of the enzyme which resulted in non-detrimental changes in the PcPAL structure. The rapid deactivation happened when deeper penetration of NH 3 molecules into the enzyme destroyed the active structure.
Thus, it could be assumed that penetration of ammonia into the surface layer of protein might be reversed by conditioning the enzyme in slightly acidic medium. This possibility was tested in the recycling of SwCNT NH2 -PAL from 6 M ammonia medium used for ammonia addition to 2 (Fig. 5). Thus, in order to regain the activity and increase the durability of the immobilized PcPAL the biocatalyst was washed and kept in phosphate buffer (pH = 6) for 2 h at room temperature under shaking at 1250 rpm when the enzyme's apparent deactivation was observed. Because the enzyme regained its initial activity for the ammonia addition reaction to 2 the initial hypothesis has been validated (Fig. 5).
Based on these results, a modified washing/reconditioning protocol was applied for recycling of SwCNT NH2 -PAL from 6 M ammonia medium (pH =10). The modified protocol involved acidic reconditioning after each ammonia addition reaction as described above. In this way the enzyme preserved more than 90% of its initial activity even after 25 cycles (Fig. 6). This means at least five-fold durability enhancement [from five cycles (see Fig. 4) to at least 25 cycles (Fig. 6)].

Investigation of the ammonia addition reaction to 3-(thiophen-2-yl)acrylic acid 2 catalyzed by SwCNT NH2 -PAL in a continuous-flow packed-bed microreactor
A further way of process intensification is the application of the biocatalysts in continuous-flow microreactors [30]. Application of continuous-flow microreactors can significantly enhance the operational stability of immobilized biocatalysts [23]. Thus, ammonia addition to 2 was studied in a biocompatible, SwCNT NH2 -PAL-packed-bed PTFE microreactor column (SynBioCart) operated in continuous-flow mode (Fig. 7). The continuous-flow system comprised the biocatalyst-filled microreactor in the column thermostat of a HPLC system equipped with a back-pressure regulator. Our previous study with SwCNT COOH-PAL-filled continuousflow microreactor indicated that application of proper backpressure (15 bar) was necessary to avoid apparent enzyme activity loss due to bubble formation [19]. Thus, in this study all experiments above room temperature were performed with back-pressure regulation (15 bar).
To evaluate the degree of activity enhancement of SwCNT NH2 -PAL in the ammonia addition reaction to 2 the specific reaction rate of the biotransformation was determined at 30°C in continuous-flow and in batch modes as defined by Eq. (1) and Eq. (2), respectively [30], [P]: product concentration (μmol mL -1 ), f: flow rate (mL min -1 ), m e : mass of immobilized biocatalyst (g); and r batch : specific reaction rate in a shake or stirred batch system, n p : amount of the product (μmol), t: the reaction time (min). The specific reaction rate is an applicable measure to compare the biocatalyst's productivity under various conditions. For a comparison between the productivity of an immobilized biocatalyst in a continuous-flow reaction and in the same reaction in batch mode, the specific reaction rates should be investigated at the same degree of conversions since the rate of product formation is not a linear function of conversions [30]. The conversion of 64% (being far enough from the >90% equilibrium conversion [24]) was chosen for comparison of the batch and of the continuous-flow mode ammonia addition reactions to 2 catalyzed by SwCNT NH2 -PAL in 2 M ammonia (pH 10, at 30 °C). As expected, the specific reaction rate in the continuous-flow reactor (r flow =2.63μmol min -1 g -1 ) was significantly higher than that in the batch reaction (r batch = 1.38 μmol min -1 g -1 ). Notably, the specific reaction rate for SwCNT NH2 -PAL in the continuousflow reactor was also higher than for SwCNT NH2 -PAL under similar conditions (r flow =2.39 μmol min -1 g -1 ) [19].
The enhanced stability and higher specific activity of SwCNT NH2 -PAL compared to the previously studied SwCNT COOH -PALs [19] could be rationalized by analysis of the functional group density of the two carriers. From the weight proportion of NH 2 (4.8 w/w%) in SwCNT NH2 an average distance of 0.75 nm could be calculated between the amino functions on the surface of SwCNT NH2 (Fig. 1). Considering the elongated spheroid shape of homotetrameric PcPAL with axes of ~8.5 nm and ~17 nm, there is a high possibility of multipoint fixation of the enzyme onto the GDE-treated SwCNT NH2 .
It is known that multipoint covalent attachment can stabilize the immobilized enzymes [31,32]. Moreover, it was demonstrated that multipoint attachment could enhance the operational stability of immobilized enzymes in microfluidic reactor as well [33].
Thus, the six-fold lower surface density of the carboxylate functions could be calculated for the carboxylated carbon nanotube used as basic carrier for SwCNT COOH -PALs [19], which results in much lower possibility of multipoint fixation as compared to SwCNT NH2 -PAL in the present study. Moreover, the linkers are ~0.8 nm shorter in SwCNT NH2 -PAL as the ones in SwCNT COOH -PALs [19], and they do not contain hydrolysable functional groups like the amide bond within the linkers of SwCNT COOH -PALs. This may also contribute to the enhanced durability of PcPAL in the novel SwCNT NH2 -PAL biocatalyst.
The temperature dependence of the SwCNT NH2 -PALcatalyzed continuous-flow mode ammonia addition to 2 in 2 M ammonia (pH 10) in the 30-80 °C range was quite similar, to that of SwCNT COOH -PAL [19]. A local minimum of the initial conversion was observed at 50 °C ( Fig. 8) with significantly lower conversion than at 40 °C or even at 30 °C. The long term operational stability study of the SwCNT NH2 -PAL was studied in the continuous-flow mode ammonia addition to 2 in 2 M ammonia (pH 10) in the 30-80 °C temperature range (Fig. 9). This study indicated that at 50 °C and 60 °C the activity of SwCNT NH2 -PAL remained stable over 72 h and was higher than those obtained between 30 °C and 50 °C. Thus, the apparent decrease of the activity of SwCNT NH2 -PAL at 50 °C cannot be attributed to irreversible thermal inactivation. At 70 °C and 80 °C however, while the initial activity of the biocatalyst increased with temperature, the enzyme inactivated rapidly and irreversibly. (1) (2) Fig. 9 Temperature effect on the long term stability in ammonia addition to 3-(thiophen-2-yl)acrylic acid 2 (4.5 mM, 2 M ammonia, pH 10, 30 °C)

Conclusions
In this study, covalent immobilization of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) on amine -functionalized single-walled carbon nanotubes (SwCNT NH2 ) was achieved. The novel immobilized form of PcPAL (SwCNT NH2 -PAL) was applicable for the synthesis of (R)-2amino-3-(thiophen-2-yl)propanoic acid (R)-1 by kinetic resolution of the corresponding racemate as well as of its enantiomer (S)-1 by enantiotope selective ammonia addition to the achiral 3-(thiophen-2-yl)acrylic acid 2. By applying glycerol diglycidyl ether (GDE)-activated SwCNT NH2 as support material, the resulted SwCNT NH2 -PAL biocatalyst showed higher activity in the ammonia addition reaction to 2 then the previously reported PcPAL form immobilized on carboxy functionalized nanotubes (SwCNT NH2 -PALs) and could be reused more times. Further stability enhancement could be achieved by conditioning the immobilized PAL biocatalyst in a slightly acidic buffer (phosphate, pH 6) for several hours during recycling. In this way, SwCNT NH2 -PALs retained more than 90% of its initial activity even after 25 cycles. When tested in a packed-bed continuous flow microreactor, the SwCNT NH2 -PAL was stable in the ammonia addition reaction to 2, with its initial activity being fully maintained for 72 h at 60 °C.
The main factors leading to the enhanced stability are the higher linker density in SwCNT NH2 -PAL as compared to SwCNT NH2 -PALs resulting in higher probability of multipoint enzyme fixation and the absence of hydrolysable functions in the linkers.