Robust, site-specifically immobilized phenylalanine ammonia-lyases for the enantioselective ammonia addition of cinnamic acids

Phenylalanine ammonia-lyases (PALs) catalyse the non-oxidative deamination of l-phenylalanine to trans-cinnamic acid, while in the presence of high ammonia concentration, the synthetically attractive reverse reaction occurs. Although they have been intensively studied, the wider application of PALs for the large scale synthesis of non-natural amino acids is still rather limited, mainly due to the decreased operational stability of PALs under the high ammonia concentration conditions of ammonia addition. Herein, we describe the development of a highly stable and active immobilized PAL-biocatalyst obtained through site-specific covalent immobilization onto single-walled carbon nanotubes (SWCNTs), employing maleimide/thiol coupling of engineered enzymes containing surficial Cys residues. The immobilization method afforded robust biocatalysts (by strong covalent attachment to the support) and allowed modulation of enzymatic activity (by proper selection of binding site, controlling the orientation of the enzyme attached to the support). The novel biocatalysts were investigated in PAL-catalyzed reactions, focusing on the synthetically challenging ammonia addition reaction. The optimization of the immobilization (enzyme load) and reaction conditions (substrate : biocatalyst ratio, ammonia source, reaction temperature) involving the best performing biocatalyst SWCNTNH2-SS-PcPAL was performed. The biocatalyst, under the optimal reaction conditions, showed high catalytic efficiency, providing excellent conversion (c ∼90% in 10 h) of cinnamic acid into l-Phe, and more importantly, possesses high operational stability, maintaining its high efficiency over >7 reaction cycles. Moreover, the site-specifically immobilized PcPAL L134A/S614C and PcPAL I460V/S614C variants were successfully applied in the synthesis of several l-phenylalanine analogues of high synthetic value, providing perspectives for the efficient replacement of classical synthetic methods for l-phenylalanines with a mild, selective and eco-friendly enzymatic alternative.


Site-directed mutagenesis
PcPAL mutant variants S390C, S542C, S614C, S707C were obtained through site-directed mutagenesis using the PcPAL C704S/C716S cloned in pET-19b expression vector as template. The PCR reaction contained 2 ng of template DNA, 2 μM solution of primer pair (Table S1), 200 μM dNTPs, 2 U/µL of Phusion High-Fidelity DNA polymerase and 10 µL of 5X Phusion HF Buffer, filled up to 50 μL with water. The PCR cycles were initiated at 95 °C for 3 minutes, followed by 30 amplification cycles. Each amplification cycle consisted of denaturation at 95 °C for 1 minute, annealing at temperature of T m no -5 °C for 1 min and extension at 72 °C for 8 minutes. The PCR cycles were finished with a final annealing step at T m pp -5 °C for 1 minute and a final extension step at 72 °C for 30 minutes. Volumes of 10 μL from each PCR reaction were used for analysis by agarose gel electrophoresis. Next, the PCR product was digested with 5 units of DpnI restriction enzyme at 37 °C for 2 h to remove the template DNA. An aliquot of 5 μL from the above digested product was transformed into 100 μL of E. coli XL1-Blue chemically competent cells by heat shock. The transformed cells were plated on a Luria-Bertani (LB) plate containing 50 µg/mL carbenicillin and 10 μg/mL tetracycline and incubated at 37 °C for 16 h. The resulted colonies were tested for the presence of the plasmid using colony PCR and two of the positive colonies from each plate were grown and the plasmid DNA was isolated. To verify the mutations, DNA sequencing was carried out using the sequencing service of Biomi (Gödöllő, Hungary).
In case of mutants PcPAL S542C and PcPAL S707C the optimization of the PCR reactions was required in order to successfully accomplish the site-directed mutagenesis. 4 ng of the template DNA and additional 3% DMSO were added to the PCR reaction, and the final concentration of the primer pair was increased to 3 µM. The temperature of the annealing step in the PCR cycles was risen to 70 °C.
In case of PcPAL double mutant variants (PcPAL L134A/S614C and PcPAL I460V/S614C) the sitedirected mutagenesis was realized under the same conditions as mentioned above, excepting the use of the previously obtained PcPAL L134A and PcPAL I460V variants 1 as template DNA. Table S1. Primers designed for PCR-based site-directed mutagenesis of the Ser residues to Cys in the four selected positions. The primer design and mutagenesis were performed using an earlier described procedure 2 . 2. Activity measurements for the purified enzymes a. Specific activities The activity assays of the purified proteins were performed in Tris buffer, pH 8.8 (100 mM Tris.HCl, 120 mM NaCl) with 2 mM substrate concentration by adding 10 µg of the corresponding purified PcPAL variant in 200 µL final reaction volume, at 30 °C, by monitoring the production of trans-cinnamic acid at 290 nm for 5 minutes using Tecan Infinite Spark 10M UV plate reader.

b. Conversion values in the ammonia elimination and addition reactions
In the reactions with isolated enzymes the same protein quantities were used as in the ammonia elimination and addition reactions with immobilized enzymes presented in Fig. 4 (see Experimental part, general procedures): The ammonia elimination reactions were performed in 1.5 mL Eppendorf tubes, containing 0.049 mg/mL final concentration of the PcPAL variants in 0.5 mL Tris-buffer (20 mM Tris.HCl, 100 mM NaCl, pH 8) at 4 mM D,L-Phe concentration. The reaction mixtures were incubated at 30 °C, at 750 rpm in an Eppendorf ThermoMixer C for different reaction times. For determination of conversion values samples of 50 μL were removed from the reaction mixture, quenched by adding an equal volume of MeOH, vortexed and centrifuged (13300 rpm, 17000 × g, 1 min). The supernatant was filtered through a 0.2 μm modified nylon membrane filter and analyzed by high performance liquid chromatography (HPLC).
The ammonia addition reactions were performed using 1.5 mL Eppendorf tubes, containing the isolated, soluble PcPAL variants. For the biotransformations 0.049 mg/mL final concentration of the purified enzymes was used in 6 M NH 4 OH (pH 10) buffer with 2 mM trans-cinnamic acid concentration. The reaction mixtures were incubated at 30 °C, at 750 rpm in an Eppendorf ThermoMixer C for the specified reaction times. For conversion determinations samples from the reactions were similarly processed as noted above.

Thermal denaturing profiles
The thermal unfolding profiles of the PcPAL variants were determined by real-time protein unfolding experiments performed in a BioRad CFX96 Real-Time Thermal Cycler using the ROX fluorescence filter.

Determination of conversion and enantiomeric excess values by HPLC
a) Reversed-phase high-performance liquid chromatography was used in order to determine the conversions of the PcPAL-catalyzed ammonia elimination and ammonia addition reactions. The analyses were conducted with Agilent (Santa Clara, CA, USA) 1200 and 1100 systems. The samples, retrieved from the biotransformations and prepared accordingly to the descriptions from the experimental part, were injected onto a Gemini NX-C18 column (150×4.5 mm; 5 μm) and eluted with a flow rate of 1.0 mL/min at 25 °C using a gradient of the mobile phase consisting of NH 4 OH buffer (0.1 M, pH 9.0) and MeOH. The conversions were determined using the relative response factor of trans-cinnamic acids compared to L-phenylalanine derivatives (Table S5), that was determined through HPLC analysis of several mixtures of different and known molar ratios of the two reaction partners.   b) The enantiomeric excess values of L-Phe obtained from the ammonia addition reactions were determined employing chiral HPLC. Firstly, the separation of rac-phenylalanine was developed on Crownpak CR-I (+) chiral column (150×3 mm; 5 μm), using as mobile phase a mixture of HClO 4 (pH=1.5) and ACN at 80:20 volume ratio, at 0.4 mL/min flow rate and 25 °C (Figure S4). The absolute configuration of the eluted enantiomers was assessed from their elution order from the chiral CROWNPAK CR-I (+) column according to the manufacturer's instructions and was also confirmed by the obtained retention times for commercial D-and L-Phe standards (Rt D-Phe = 3 min, Rt L-Phe = 5 min).  showing an ee value >99% for the produced L-phenylalanine.  SWCNT NH2 -GDE-wtPcPAL 3.6 18.9 *The ammonia elimination reactions were performed at room temperature, at 750 rpm (vibrational stirring), in 1 mL Tris-buffer (20 mM Tris.HCl, 100 mM NaCl, pH 8.8,) at 4 mM D,L-Phe concentration using 1 mg of biocatalyst (PcPAL site-specifically immobilized through the Cys390 residue and the non-specifically, covalently immobilized wt-PcPAL). The ammonia addition reactions were performed at room temperature, at 750 rpm (vibrational stirring), in 1 mL 6 M NH 4 OH reaction solution containing 2 mM substrate, using 1 mg of biocatalysts (PcPAL site-specifically immobilized through the Cys390 residue and the non-specifically, covalently immobilized wt-PcPAL).

Specific activities of the immobilized enzymes and conversion values in the ammonia addition reaction
Despite our efforts, kinetic measurements for the ammonia addition reactions were not successful, probably due to the high background caused by the elevated ammonia concentration and/or the high UV absorbance of the trans-cinnamic acid substrate. Notable, that in our previous studies 1, 3 we met similar obstacles for the kinetic measurements of the ammonia additions reactions with purified, soluble enzymes, hindering the calculation of specific activities within this important reaction route.
Accordingly the determination of specific activities of the SWCNT NH2 -SS-PALs were limited for the ammonia elimination reaction, where based on their conversion-activity they underperform in comparison with the ammonia additions (see Table S6). Furthermore, we can rely also on the conversion-based activities: as one can see in Table S3 soluble, purified enzymes provided similar conversions after 4 h reaction time, while the immobilized variants (Table S7) clearly possess different activities within the ammonia addition reactions.

Experimental procedure for the determination of specific activities and conversion-activities of the immobilized enzymes:
The ammonia elimination reactions of L-Phe were performed in 2 mL glass bottles (vials), containing 1 mg of the immobilized PcPAL-biocatalysts (SWCNT NH2 -SS-PAL, SWCNT NH2 -GDE-PAL) with optimal 0.13 mg enzyme load in 1 mL Tris-buffer (20 mM Tris.HCl, 100 mM NaCl, pH 8.8) at 2 mM L-Phe concentration. The reaction mixtures were incubated at 30 °C, at 750 rpm in a Heidolph Vibramax 110 incubator. In every 2 minutes an 80 μL sample was removed from the reaction mixture, diluted to 200 μL final volume with Tris-buffer (20 mM Tris.HCl, 100 mM NaCl, pH 8) and the production of trans-cinnamic acid was monitored at 290 nm.
The conversion based activities were similarly determined as described within the main manuscript, (Experimental part, section: ammonia additions under optimal conditions) excepting the use of 1.5 mL Eppendorf tubes as reaction vials. The samples were incubated at 900 rpm in an Eppendorf ThermoMixer C for the specified reaction times. For conversion determinations samples from the reactions were similarly processed as noted above, in section 2.  Figure S9: Recyclability of SWCNT NH2 -SS-PcPAL in the ammonia addition reaction of cinnamic acid using 6 M NH 4 OH (pH=10.0) as ammonia source. The reactions were performed at room temperature, at 1250 rpm, using 1 mg of biocatalyst with the optimal enzyme loading. The samples were taken at specified reaction times. Experimental procedure: 1 mg biocatalyst with the optimal enzyme loading was suspended in 1 mL of 3 M NH 2 CO 2 NH 4 , pH 9.6 buffer. The reaction mixture was incubated at 40 °C, at 750 rpm in a Heidolph Vibramax 110 platform shaker. For conversion determinations samples from the reaction were taken at