Saturation Mutagenesis for Phenylalanine Ammonia Lyases of Enhanced Catalytic Properties

biomolecules

Article Saturation Mutagenesis for Phenylalanine Ammonia Lyases of Enhanced Catalytic Properties

Raluca Bianca Tomoiagă, Souad Diana Tork, Ilka Horváth, Alina Filip, Levente Csaba Nagy and László Csaba Bencze *

Biocatalysis and Biotransformation Research Center, Faculty of Chemistry and Chemical Engineering,

Babes, -Bolyai University, Arany János street 11, RO-400028 Cluj-Napoca, Romania; [email protected] (R.B.T.); [email protected] (S.D.T.); [email protected] (I.H.); aﬁ[email protected] (A.F.); [email protected] (L.C.N.) * Correspondence: [email protected]

Received: 11 April 2020; Accepted: 23 May 2020; Published: 30 May 2020

Abstract: Phenylalanine ammonia-lyases (PALs) are attractive biocatalysts for the stereoselective synthesis of non-natural phenylalanines. The rational design of PALs with extended substrate scope, highlighted the substrate specificity-modulator role of residue I460 of Petroselinum crispum PAL. Herein, saturation mutagenesis at key residue I460 was performed in order to identify PcPAL variants of enhanced activity or to validate the superior catalytic properties of the rationally explored I460V PcPAL compared with the other possible mutant variants. After optimizations, the saturation mutagenesis employing the NNK-degeneracy generated a high-quality transformant library. For high-throughput enzyme-activity screens of the mutant library, a PAL-activity assay was developed, allowing the identification of hits showing activity in the reaction of non-natural substrate, p-MeO-phenylalanine. Among the hits, besides the known I460V PcPAL, several mutants were identified, and their increased catalytic efficiency was confirmed by biotransformations using whole-cells or purified PAL-biocatalysts. Variants I460T and I460S were superior to I460V-PcPAL in terms of catalytic efficiency within the reaction of p-MeO-Phe. Moreover, I460T PcPAL maintained the high specificity constant of the wild-type enzyme for the natural substrate, l-Phe. Molecular docking supported the favorable substrate orientation of p-MeO-cinnamic acid within the active site of I460T variant, similarly as shown earlier for I460V PcPAL (PDB ID: 6RGS).

Keywords: biocatalysis; phenylalanine ammonia-lyases; saturation mutagenesis; protein engineering

1. Introduction Phenylalanine ammonia-lyases (PALs, EC 4.3.1.24, and PAL/TALs with combined phenylalanine and tyrosine ammonia-lyase activities, EC 4.3.1.25) play essential roles in the non-oxidative ammonia elimination of phenylalanine to cinnamic acid, an important precursor for the biosynthesis of phenylpropanoids. From a synthetic point of view, PALs, under high ammonia concentration, can perform the reverse ammonia addition reaction onto α,β-unsaturated arylacrylates, to produce l-α-amino acids in high enantiomeric purity. Accordingly, PALs from Rhodotorula glutinis (RgPAL) [1,2], Petroselinum crispum (PcPAL) [3], and Anabaena variabilis (AvPAL) [4] due to their broad substrate tolerance emerged as eﬃcient biocatalysts for the production of phenylalanine analogs [5–10]. Recent protein engineering increased the catalytic activity [11–14] and/or extended the substrate scope of PALs [15–17]. Despite these advances, the broader application of PALs for the large-scale synthesis of various speciﬁc target molecules still remains a challenge due to the substrate/product inhibition [14,18] and/or enzyme stability [16,19,20] related issues, especially in the synthetically attractive ammonia addition reaction, that requires high (4–6 M) ammonia concentrations.

Biomolecules 2020, 10, 838; doi:10.3390/biom10060838 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, 838 2 of 13

Directed evolution driven protein engineering is one of the most powerful tools to extend the substrate scope of enzymes, or to increase their catalytic activity, but also to alleviate the substrate/product inhibition or protein stability related dilemma [21–24]. The bottleneck of the directed evolution is the screening effort, necessary for the proper selection of hits from the largely sized mutant libraries (> 103–104 clones), that requires high-throughput enzyme activity assays applicable at whole cell/cell lysate level. Among the PAL-activity assays, the high-performance liquid chromatography (HPLC) based procedure [14,16] allows the determination of conversion and enantiomeric excess values but can only be employed for the screening of limited sized libraries derived from rational design mutagenesis or single combinatorial active-site testing (CAST) [25]. The UV-spectroscopy based PAL-activity assay monitors the production or consumption of the cinnamic acid derivatives [26,27] and possesses low sensitivity in the case of using cellular biocatalysts. Moreover, the reported fluorescence PAL-activity assays, despite their sensitivity, are limited for activity tests using the non-natural substrate o-NH2-phenylalanine [28] or require high instrumentation facilities, such as inductively coupled plasma–mass spectrometry (ICP-MS) [29], thus their general applicability is suppressed. Another important issue is the quality of the transformant library, obtained through different mutagenesis methods, high-quality libraries, with a higher frequency of hits, providing the advantage of a reduced screening effort. In this context, iterative saturation mutagenesis (ISM) [30,31] has emerged as an effective tool, where multiple protein sites composed of one or more amino acid residues are chosen for randomization through saturation mutagenesis, while the obtained hits are used as templates to perform randomization at the other sites in iterative cycles until the desired degree of catalyst improvement is reached. Combinatorial active-site saturation test (CAST) employs the methodology of ISM for residues within the catalytic site, creating smaller, high-quality libraries [25,32,33]. For phenylalanine ammonia-lyases (PALs), limited directed evolution and/or saturation mutagenesis studies have been performed, focusing on inducing d-selectivity [34] or increased activity towards p-substituted bulky [12,13] or electron donor substrates [17]. Modification of the catalytic site of PcPAL through smaller amino acid substitutions of the active site residues I460 or/and F137, extended its substrate scope towards bulky phenylalanine analogs [16]. Furthermore, mapping the hydrophobic region of the active site of PcPAL [14] revealed the importance of residue I460 for the enzyme activity modulation towards substrates bearing bulky substituents mostly in m- or p-positions on the aromatic ring. Based on the above, the synthetic utility of I460V variant was also demonstrated by the production of several m-, or p-substituted phenylalanine analogs of high synthetic value, employing biotransformations catalyzed efficiently by mutant I460V, but with low efficiency by wild-type PcPAL [18]. In this context, this study focused on the randomization by site-saturation mutagenesis of the key activity modulator residue I460 of PcPAL in order to identify novel mutant variants with increased activity towards selected substrates of synthetic interest. For obtaining high-quality libraries, with increased frequency of hits within the transformant library, the mutagenesis procedure was optimized, while the PAL-activity assay was adapted to allow the high-throughput activity screens of cell lysates providing candidates of which superior activity was validated.

2. Materials and Methods

2.1. Saturation Mutagenesis Procedure: Site-saturation mutagenesis experiments were carried out using as template the pET19b vector harboring the gene encoding PAL from Petroselinum crispum [35]. The primers containing the NNK degenerate codons (Tables S1 and S2) were purchased from Invitrogen (Karlsruhe, Germany). Biomolecules 2020, 10, 838 3 of 13

2.1.1. Procedure Using Megaprimers: PCR reactions were performed in a total volume of 50 µL, using 100 ng template DNA, 0.1 µM NNK degenerate (forward) and ﬂanking (reverse) primer, 0.1 µM dNTPs, 1 U Phusion High Fidelity polymerase. The PCR protocol consisted of initial denaturation for 3 min at 95 ◦C followed by 10 cycles of 30 s at 95 ◦C, 1 min at 64 ◦C annealing temperature, established by initial gradient PCR experiments and extension for 1 min at 72 ◦C. The second stage PCR consisted of 25 cycles of 30 s at 95 ◦C and extension for 8 min at 72 ◦C; followed by a ﬁnal extension of 16 min at 72 ◦C.

2.1.2. Procedure Using Partially Overlapping Primers: The PCR reactions for the overlapping method used 2 ng template, 2 µM of each primer, 0.1 µM dNTPs, 1 U Phusion High Fidelity polymerase in a total volume of 50 µL. The PCR protocol consisted of: initial denaturation for 3 min at 95 ◦C followed by 30 cycles of 1 min at 95 ◦C, annealing 1 min at Tm no-5 ◦C and extension 15 min at 72 ◦C, followed by final denaturation at 95 ◦C for 1 min, annealing at Tm pp-5 ◦C for 1 min and extension for 15 min at 72 ◦C. The PCR products from both procedures were digested with DpnI (overnight, 37 ◦C) and purified through ethanol precipitation. 5 µL of the purified PCR product was electroporated into 50 µL E. coli BL21 GOLD (DE3) electrocompetent cells, followed by incubation in SOC medium for 1 h, at 37 ◦C. The mixture was plated on Luria Bertani (LB) agar plates supplemented with carbenicillin (50 µg/mL) and incubated overnight at 37 ◦C, obtaining the corresponding transformant libraries.

2.2. Activity Screens of Transformant Libraries The colonies of the transformant libraries (113 colonies from each library) were picked and transferred to 100 µL carbenicillin supplemented (50 µg/mL) LB-medium distributed in the wells of a 96-deep-well polypropylene plate, followed by overnight incubation at 37 ◦C, 200 rpm. 8 µL of each preculture were used to inoculate 800 µL of LB-medium distributed within a 96-deep-well plate, followed by cell growth and induction at OD600 0.5–0.7 using 0.5 mM IPTG. The colonies were grown to an OD600 of ~2 (corresponding to a wet cell concentration of ~12 mg/mL) by incubation at 25 ◦C, 200 rpm for 20–24 h. The cultures were pelleted by centrifugation, 20 min, 4000 rpm (1751 g) at 4 C, × ◦ and the cell pellets were deposited at 80 C prior to their use. − ◦ For activity screens, each cell pellet was resuspended in 400 µL lysis buffer (20 mM PBS pH = 7.00, NaCl 50 mM, 0.07 mM lysozyme, 0.36 mM PMBS), followed by incubation for 40 min, 800 rpm at 30 C, obtaining by centrifugation (20 min, 4000 rpm or 1751 g, 4 C) the clarified lysates as ◦ × ◦ supernatant. The cell lysates of the PAL variants of the transformant libraries were assayed using racemic p-methoxyphenylalanine as substrate monitoring the production of p-methoxy trans-cinnamic acid at 290 nm. Thus, 50 µL of cell lysate was added to the solution of p-methoxyphenylalanine (2 mM final concentration) in 50 mM Tris.HCl, 100 mM NaCl pH = 8.8, yielding a final reaction volume of 200 µL. The enzyme activity was assessed by monitoring the absorbance at 290 nm over 15 min, at 30 ◦C, using a Tecan Infinite Spark 10M plate reader and UV-transparent polyacrylate 96-well plate (Costar #3635, Corning, Waltham, MA, USA) The DNA of the hits with similar or increased enzyme activity, compared to I460V-PcPAL, was extracted and sequenced (BIOMI, Gödöll˝o,Hungary) to identify the mutations.

2.3. Protein Expression, Isolation, and Thermal Unfolding Assessments Wild-type PcPAL and variants I460S, I460T, and I460V were expressed, isolated, and purified using our previously reported method [14,35]. The protein homogeneity/oligomerization state was determined through size-exclusion chromatography using Superdex 10/300 (GE Healthcare Bio-Sciences, Uppsala, Sweden), while the purity of the enzymes was assessed by SDS-PAGE. The thermal unfolding of the PcPAL mutants was determined by nanoscale differential scanning fluorimetry measurements, using Prometheus NT.48 nanoDSF instrument (NanoTemper Technologies, München, Germany). Biomolecules 2020, 10, 838 4 of 13

2.4. Enzyme Kinetics The kinetic measurements were performed by monitoring the production of trans-cinnamic acid and/or p-methoxy trans-cinnamic acid at 290 nm, using substrate concentrations of 50–5000 µM and purified PcPAL variants at a fixed enzyme concentration of 0.1543 µM. The vmax,KM, and kcat values were obtained from the non-linear regression fitting of the Michaelis–Menten curves.

2.5. HPLC Analysis for Conversion and Enantioselectivity Determinations The reactions with whole cell biocatalysts were performed in 20 mM Tris.HCl, 100 mM NaCl pH = 8.8 using 2 mM substrate concentration and induced whole cells of a cell density of OD600 ~2 (corresponding to a wet cell concentration of ~12 mg/mL) in a final volume of 200 µL. In case of reactions using purified enzymes as biocatalysts, similar conditions were employed using 50 µg purified PcPAL variant. The reaction mixtures were incubated at 800 rpm, 30 ◦C, taking samples after 2, 4, 6, and 16 h reaction times. Samples were quenched by adding an equal volume of MeOH and centrifugated at 13,400 rpm (12,000 g) for 10 min, filtered through a 0.22 µm nylon membrane filter and analyzed by × HPLC. In order to determine the conversions values, a Gemini NX-C18 column (150 4.5 mm; 5 µm) × was chosen, using as mobile phase: A: NH4OH buffer (0.1 M, pH 9.0)/ B: MeOH, with a flow rate of 1.0 mL/min. The enantiomeric excess was determined by chiral HPLC separation, using Crownpak CR-I (+) chiral column (150 3 mm; 5 µm) and HClO (pH = 1.5)/acetonitrile as mobile phase at a flow × 4 rate of 0.4 mL/min. All measurements were performed at 25 ◦C.

2.6. Analyzing Thermal Unfolding of PcPAL Mutants through nanoDSF Measurements Nanoscale differential scanning fluorimetry (nanoDSF) measurements were performed using Prometheus NT.48 nanoDSF instrument, which monitors the shift of intrinsic tryptophan fluorescence of proteins upon unfolding by detecting the fluorescence at emission wavelengths of 330 and 350 nm. For determination of the protein melting point (Tm), the maximum of the first derivative of the fluorescence ratios F350/F330 was used. PcPAL variants were diluted with 50 mM Tris.HCl, 100 mM NaCl pH 8.8 buffer to a final concentration of 1 mg/mL. 10 µL of each sample was loaded into UV capillaries (NanoTemper Technologies, München, Germany) and protein unfolding was detected during heating in a linear thermal ramp of 1 ◦C/min between 20 and 95 ◦C, with an excitation power of 20%. Data analysis was performed using NT Melting Control software and melting temperature (Tm) was determined by fitting the experimental data using a polynomial function, in which the maximum slope was indicated by the peak of its first derivative (F350/F330). All measurements were performed in triplicate.

2.7. Molecular Docking The enzyme active site was specified from the coordinates of the co-crystallized ligand (E)-3-(4-methoxyphenyl)acrylic acid within the crystal structure of PcPAL I460V (PDB ID: 6RGS). A docking grid with a size of 16 Å 16 Å 16 Å was used, and the grid center was obtained × × from the coordinates of the co-crystallized ligand from 6RGS. Ligand docking was performed using Autodock Vina [36], with the degree of exhaustiveness set to 200. Residues V460, T460, and I460 were explicitly specified as flexible side-chains in mutant I460V, I460T, and wild-type PcPAL, respectively. Protonation states of the protein scaffold were predicted at pH 8.8 and 10 by the PROPKA method using PDB2PQR [37]. The ground state geometries of substrates p-MeO-Phe and p-MeO-cinnamic acid were obtained at the DFT level of theory, employing the B3LYP functional and the 6–311++G(d,p) basis set, using the Gaussian 09 package. Harmonic vibrational frequencies have been checked for the optimized structures to confirm that the true energy minimum has been found. Biomolecules 2020, 10, x FOR PEER REVIEW 5 of 13

In the initial randomization library for I460, the commonly employed NNK degeneracy, defining Biomolecules32 codons2020, 10encoding, 838 all 20 canonical amino acids and one Stop codon, was applied. For the generation5 of 13 of the mutant libraries, two different mutagenesis strategies were employed, one based on partially overlapping primers [38] and the Megaprimers method [39]. 3. ResultsThe and procedure Discussion using partially overlapping primers is known for its increased efficiency in comparison with the QuikChange procedure and employs primer pairs containing non-overlapping 3.1. Site-Saturation Mutagenesis sequences at their 3′ end and mutagenic, primer-primer complementary (overlapping) sequences at Inthe the 5′ end initial [30,31]. randomization The melting librarypoint of forthe I460,non-overl the commonlyapping region employed (Tm no) positions NNK degeneracy, 5 to 10 °C higher defining 32 codonsthan those encoding of primer all 20-primer canonical complementary amino acids region and one (Tm Stoppp), in codon, order was to avoid applied. the competing For the generation self- pairing. For a high-quality DNA library, two primer pairs (Table S1), one favoring the annealing of of the mutant libraries, two different mutagenesis strategies were employed, one based on partially the codons rich in bases G, C, and one favoring those based predominantly on A or T, were employed. overlapping primers [38] and the Megaprimers method [39]. Thus, the products of the two PCR reactions, performed at different annealing temperatures, were Thecombined, procedure digested using with partiallyDpnI and transformed overlapping into primers E. coli BL21 is known (DE3) p forLys competent its increased cells ethroughfficiency in comparisonheat shock with and the electroporation. QuikChange procedure and employs primer pairs containing non-overlapping sequencesIn at the their case 30 ofend the and megaprimer mutagenic, procedure primer-primer [25,32,33], complementary a forward mutagenic (overlapping) primer carrying sequences the at the 50 endNNK [30, 31 degeneracy]. The melting and apoint reverse of the non non-overlapping-mutagenic primer region were (T usedm no in) positions a two-stage 5 to PCR 10 ◦ C reaction. higher than thoseReverse of primer-primer primers for megaprimers complementary of the region different, (Tm small, pp), in medium, order to and avoid large the lengths competing were designed self-pairing. For a(Figure high-quality 1a, Table DNA S1). library, two primer pairs (Table S1), one favoring the annealing of the codons rich in basesWhile G, by C, andthe use one of favoring large megaprimer those based (Figure predominantly 1b lane 4), no on plasmid A or T, wereamplification employed. could Thus, be the observed, the medium-sized megaprimer procedure (Figure 1b lane 5) provided low plasmid products of the two PCR reactions, performed at different annealing temperatures, were combined, concentration within the second PCR step compared to the megaprimer concentration resulted from digested with DpnI and transformed into E. coli BL21 (DE3) pLys competent cells through heat shock the first PCR step. Comparing the concentration of the desired plasmid product with that of the and electroporation.megaprimer product, it was found that the use of small-sized megaprimers (Figure 1b lane 3) Incompeted the case with of thethe megaprimerpartially overlapping procedure primer [25 procedure,32,33], a ( forwardFigure 1b mutagenic lanes 1 and primer2). Accordingly, carrying the NNKfurther degeneracy optimization and a reverse steps of non-mutagenic the PCR protocol, primer related were to the used template in a two-stage concentration PCR and reaction. extension Reverse primerstem forperature megaprimers were performed, of the di significantlyfferent, small, increasing medium, the and quality large of lengthsthe PCR were product designed of the small (Figure- 1a, Tablemegaprimer S1). protocol (Figure 1b lane 6).

(a) (b)

FigureFigure 1. (a ) 1. Saturation (a) Saturation mutagenesis mutagenesis using using the Megaprimers the Megaprimers procedure, procedure, representing representing the genethe geneencoding PcPALencoding (green), thePcPAL pET19b-vector (green), the pET19b backbone-vector (black backbone), and the (black megaprimers), and the megaprimers of the different, of the small, different, medium, small, medium, large lengths (blue). In the first PCR stage, the forward mutagenic primer and the large lengths (blue). In the first PCR stage, the forward mutagenic primer and the reverse, antiprimer reverse, antiprimer anneal to the template resulting the amplified sequences of the corresponding, anneal to the template resulting the amplified sequences of the corresponding, large, medium, and large, medium, and small-sized megaprimers. In the second PCR stage through annealing of the small-sized megaprimers. In the second PCR stage through annealing of the forward primer and the forward primer and the obtained megaprimers, the mutated plasmid products are obtained. (b) obtainedAgarose megaprimers, gel analysis theof the mutated PCR products plasmid obtaine productsd through are obtained. the different (b) mutagenesis Agarose gel procedures: analysis of the PCR productslanes 1, 2 using obtained the two through sets of thepartially different overlapping mutagenesis primers, procedures: lane 3 smalllanes-megaprimer1, 2 using protocol, the two lane sets of partially overlapping primers, lane 3 small-megaprimer protocol, lane 4 large-megaprimer protocol, lane 5 medium-megaprimer protocol, lane 6 optimized small-megaprimer protocol, L 10 kb DNA ladder.

While by the use of large megaprimer (Figure1b lane 4), no plasmid amplification could be observed, the medium-sized megaprimer procedure (Figure1b lane 5) provided low plasmid concentration within the second PCR step compared to the megaprimer concentration resulted from the first PCR step. Comparing the concentration of the desired plasmid product with that of the megaprimer product, it was found that the use of small-sized megaprimers (Figure1b lane 3) competed with the Biomolecules 2020, 10, 838 6 of 13 partially overlapping primer procedure (Figure1b lanes 1 and 2). Accordingly, further optimization steps of the PCR protocol, related to the template concentration and extension temperature were Biomolecules 2020, 10, x FOR PEER REVIEW 6 of 13 performed, significantly increasing the quality of the PCR product of the small-megaprimer protocol (Figure1b4 lane large 6).-megaprimer protocol, lane 5 medium-megaprimer protocol, lane 6 optimized small- megaprimer protocol, L 10 kb DNA ladder. 3.2. Quality of Transformant Libraries 3.2. Quality of Transformant Libraries Directed evolution procedures, such as (iterative) saturation mutagenesis, require a high-efficiency transformationDirected of evolution the mutagenesis procedures, product, such as providing (iterative) a susaturationfficient number mutagenesis, of colonies require for a high the- 95% coverageefficiency of residue transformation randomization, of the mutagenesis 96 colonies product, in case providing of a single-site a sufficient NNK number degeneracy. of colonies In order for to generatethe 95% the coverage requested of residue high colony randomization, number both96 colonies heat shock in case and of a electroporation single-site NNK transformationdegeneracy. In of order to generate the requested high colony number both heat shock and electroporation the PCR products obtained through the partially overlapping primers strategy and by the optimized transformation of the PCR products obtained through the partially overlapping primers strategy and small-sized megaprimer strategy were tested using BL21 (DE3) pLys, Rosetta (DE3) pLys, or BL21 (DE3) by the optimized small-sized megaprimer strategy were tested using BL21 (DE3) pLys, Rosetta (DE3) GoldpLys, strains or E.BL21 coli (DE3)competent Gold strains cells. While E.coli bycompetent heat-shock, cells. low While transformation by heat-shock, effi lowciency transformation (<10 colonies) wasefficiency obtained (<10 for colonies) both PCR was products obtained forfor both each PCR host products strains, for the each electroporation host strains, the protocol electroporation provided <31 coloniesprotocol provided in case of <31E. coloniescoli BL21 in (DE3) case of pLys, E.coliRossetta BL21 (DE3) (DE3) pLys, pLys Rossetta and > (DE3)140 colonies pLys and in >140 case of BL21colonies (DE3) Goldin case host. of BL21 Further, (DE3) Gold to evaluate host. Further, the diversity to evaluate and the quality diversity of librariesand quality obtained of libraries by the partiallyobtained overlapping by the primers partially and overlapping the small-sized primers megaprimer and the small mutagenesis-sized megaprimer methods, we mutagenesis compared the distributionmethods, of we the compared encountered the basesdistribution at the degeneracyof the encountered site for bases the plasmids at the degeneracy isolated from site the for pooled the agarplasmids plate of the isolated two transformant from the pooled libraries agar plate with of the the values two transforexpectedmant of NNK-degeneracy libraries with the values(Figure 2, Figureexpected S1). While of NNK the-degeneracy transformant (Figure library 2, Figure generated S1). While by the the small-megaprimer transformant library mutational generated by strategy the resembledsmall-megaprimer the theoretical mutational base distribution, strategy resembled in case of the partially theoretical overlapping base distribution, primer in strategy-derived case of the partially overlapping primer strategy-derived library, within the first and second base of the NNK- library, within the first and second base of the NNK-degeneracy, cytosine (C) was underrepresented or degeneracy, cytosine (C) was underrepresented or completely missing, with a high dominance of completely missing, with a high dominance of thymine (T) within the first degenerated nucleotide. thymine (T) within the first degenerated nucleotide. Accordingly, from the two transformant Accordingly,libraries, from only the the two higher transformant-quality one libraries, generated only by the the higher-quality small megaprimer one generated-strategy limits by the the small megaprimer-strategyoccurrence of amino limits acid the bias occurrence and ensures of amino that allacid the bias desired and ensures mutations that are all thepresent desired within mutations the are presenttransformant within library. the transformant The differences library. in the distribution The differences percentage in thes for distribution the different percentages bases, obtained for the differentfor the bases, small obtained megaprimer for the-transformant small megaprimer-transformant library, with respect to the library, theoretical with one, respect is similar to the to theoretical those one,observed is similar in to previous those observed saturation in previousmutagenesis saturation studies [40] mutagenesis and approaches studies the [40 limits] and of approaches saturation the limitsmutagenesis of saturation procedu mutagenesisres [41]. procedures [41].

(a) Expected base distribution.

(b) Base distribution—small

base

rd megaprimer nd

3 method 2

FigureFigure 2. Nucleotide-base 2. Nucleotide-base distribution distribution within within randomized randomized position position 460 of 460Pc PAL. of Pc (PAL.a) Theoretical, (a) Theoretical, expected distributionexpected for distribution NNK-degeneracy, for NNK-degeneracy, with the four with bases the (A,four T, bases G, C) (A, equally T, G, C) represented equally represented in the first in two nucleotides,the first whiletwo nucleotides, G, T having while the sameG, T having ratio within the same the ratio third within position); the third (b) experimental position); (b) base-distribution experimental base-distribution from the small megaprimer-transformant library; (c) experimental base-distribution from the transformant library obtained by the partially overlapping primers mutational strategy. For Biomolecules 2020, 10, x FOR PEER REVIEW 7 of 13

both (a) and (c) cases, the base-distribution was assessed by sequencing the DNA plasmid isolated from the cellular mixture of all colonies pooled from the LB-agar plate of the corresponding transformant library. The G, T, C, A encodes for the corresponding nucleotide bases thymine (T), adenosine (A), cytosine (C), and guanine (G).

3.3. Screening of Transformant Libraries

3.3.1. Assay Development Since saturation mutagenesis-based enzyme engineering requires a reliable high-throughput Biomoleculesactivity2020 , assay10, 838 able to screen the large transformant libraries, the compatibility of the UV7 of 13 spectroscopy-based PAL-activity assay [26,42] with the use of whole cell PAL-biocatalysts was tested. Unfortunately, the low sensitivity, derived from the high UV-background of cell suspensions from the small megaprimer-transformant library; (c) experimental base-distribution from the hindered the detection of mutant variants with still low, but increased activity compared to the wild- transformanttype enzyme, library that obtained would serve by the as partially potential overlapping hits and startingprimers mutational points for strategy. additional For saturation both (a) andmutagenesis (c) cases, therounds. base-distribution Therefore, the was UV assessed-assay—based by sequencing on monitoring the DNA the pr plasmidoduction isolated or consumption from the cellularof cinnamic mixture acid of derivatives all colonies— pooledwas appropriately from the LB-agar adapted plate for of thereliable corresponding enzyme activity transformant measurements library. Theusing G, T,cell C, lysates A encodes as biocatalysts for the corresponding (Figure 3a,b). nucleotide For cell bases lysis, thymine various (T),known adenosine lysis-inducing (A), cytosine chemical (C), andagents, guanine such (G). as Tween-20, Triton-X-100, lysozyme, polymyxin B sulphate, glucose, and glycerol solutions were employed comparing their efficiency by activity measurement of cell lysates within 3.3. Screeningthe natural ofTransformant PAL-reaction.Libraries Cell lysates obtained by the use of Triton X-100 or polymyxin B sulfate (PMBS) or their combined 3.3.1. Assayuse with Development lysozyme showed significantly higher specific activity compared to those obtained by SinceTween saturation-20, glucose, mutagenesis-based or glycerol-based enzyme solutions engineering (Figure 3a). requires Next, a reliablethe optimization high-throughput of the most activity assay ableefficient to screen cell lysis the procedures large transformant was performed. libraries, A combination the compatibility of the three of the efficient UV spectroscopy-based cell-lysis agents, Triton-X, PMBS, and lysozyme, significantly increased the viscosity of the cell lysate, hindering the PAL-activity assay [26,42] with the use of whole cell PAL-biocatalysts was tested. Unfortunately, the appropriate purification of cell lysate by centrifugation, providing as a consequence a high standard low sensitivity,deviation within derived the fromactivity the assay. high The UV-background additional use of of benzonase cell suspensions provided a hindered four-component the detection cell of mutantlysis cocktail variants with with increased still low, efficiency but increased (Figure 3a, activitylane 8). However, compared in high to the-throughputwild-type experiments,enzyme,that wouldthis serve method as potentialwas improper hits due and to startingthe high price points of benzonase. for additional Therefore, saturation the two- mutagenesiscomponent PMBS rounds.- Therefore,lysozyme the UV-assay—basedlysis buffer was selected, on monitoring and its efficiency the production was further orimproved consumption by optimizing of cinnamic the pH, acid derivatives—wasincubation conditions appropriately (temperature, adapted rpm) for (Figure reliable 3 enzymeb), thus finally activity themeasurements cell lysis performed using for cell 40 min lysates as biocatalystsat 800 rpm, (Figure 30 °C 3witha,b). lysis For buffer cell lysis, containing various 0.36 known mM of lysis-inducing PMBS and 0.07 chemicalmM lysozyme, agents, pH such 7.0, as Tween-20,provided Triton-X-100, homogenous, lysozyme, clear cell polymyxin lysate with B low sulphate, UV-background, glucose, and applicable glycerol for solutions the high were-throughput employed comparingPAL-activity their efficiency screens (Figure by activity 3b, lane measurement 4, Table S3). of cell lysates within the natural PAL-reaction.

(a) (b)

Figure 3. Assay optimizations monitoring the relative activity of the different cell lysates of I460V-PcPAL, in the ammonia elimination reaction of p-MeO-Phe. The following lysis buffers have been employed: (a) 1. Tween 20 buffer (50 mM NaH PO 2H O, 300 mM NaCl, 10 mM imidazole, 0.07 mM lysozyme, 2 4· 2 400 U/mL DNAse I. 1% Tween20 pH 8), 2. GTE buffer (50 mM glucose, 10 mM EDTA, 25 mM Tris.HCl, pH 8), 3. GTE/Lys. buffer (50 mM glucose, 10 mM EDTA, 0.07 mM lysozyme, 25 mM Tris.HCl, pH 8), 4. 5% glycerol buffer (5 % glycerol, 100 mM NaCl, 1 mM DTT, 50 mM Tris.HCl, pH 7,5), 5. 5% glycerol/Lys. buffer (5 % glycerol, 100 mM NaCl, 1 mM DTT, 0.07 mM lysozyme, 50 mM Tris.HCl, pH 7,5), 6. PMBS/Lys. buffer (0.36 mM PMBS, 0.07 mM lysozyme, 50 mM NaCl, 20 mM Tris.HCl, pH 8.8), 7. Triton X-100 buffer (2% Triton X-100, 50 mM NaCl, 20 mM Tris.HCl, pH 8.8), 8. Triton X-100/PMBS/Lys./Benz. buffer (2% Triton X-100, 0.36 mM PMBS, 0.07 mM lysozyme, 25 U/mL benzonase, 50 mM NaCl, 20 mM Tris.HCl, pH 8.8). (b) Optimizations using the two component PMBS/Lys. buffer and the four-component Triton X-100/PMBS/Lys./Benz. buffer systems with different pHs and/or lysis conditions. Abbreviations: Lys., PMBS, T. X-100 and Benz. denote for lysozyme, polymyxin B sulphate, Triton X-100 and benzonase, correspondingly. Biomolecules 2020, 10, 838 8 of 13

Cell lysates obtained by the use of Triton X-100 or polymyxin B sulfate (PMBS) or their combined use with lysozyme showed signiﬁcantly higher speciﬁc activity compared to those obtained by

Tween-20,Biomolecules glucose, 2020, 10 or, x glycerol-basedFOR PEER REVIEW solutions (Figure3a). Next, the optimization of the most8 of e ffi13 cient cell lysis procedures was performed. A combination of the three efficient cell-lysis agents, Triton-X, PMBS, andFigure lysozyme, 3. Assay significantly optimizations monitoring increased the relative viscosity activity of the of the cell different lysate, cell hindering lysates of theI460V appropriate- purificationPcPAL, of cellin the lysate ammonia by centrifugation,elimination reaction providing of p-MeO-Phe. as a The consequence following lysis a highbuffers standard have been deviation within theemployed: activity assay.(a) 1. Tween The additional20 buffer (50 usemM ofNaH benzonase2PO4·2H2O, provided300 mM NaCl, a four-component 10 mM imidazole, 0.07 cell mM lysis cocktail lysozyme, 400 U/mL DNAse I. 1% Tween20 pH 8), 2. GTE buffer (50 mM glucose, 10 mM EDTA, 25 with increased efficiency (Figure3a, lane 8). However, in high-throughput experiments, this method mM Tris.HCl, pH 8), 3. GTE/Lys. buffer (50 mM glucose, 10 mM EDTA, 0.07 mM lysozyme, 25 mM was improperTris.HCl, due pH to 8), the 4. high5% glycerol price buffer of benzonase. (5 % glycerol, Therefore, 100 mM NaCl, the two-component 1 mM DTT, 50 mM PMBS-lysozyme Tris.HCl, pH lysis buffer was7,5), selected, 5. 5% glycerol/Lys. and its effi bufferciency (5 was % glycerol, further 100 improved mM NaCl, by 1 mM optimizing DTT, 0.07 the mM pH, lysozyme, incubation 50 mM conditions (temperature,Tris.HCl, rpm) pH (Figure 7,5), 6. 3PMBS/Lys.b), thus finallybuffer (0.36 the mM cell PMBS, lysis performed 0.07 mM lysozyme, for 40 min50 mM at NaCl, 800 rpm, 20 mM 30 ◦C with lysis bufferTris.HCl, containing pH 8.8), 0.36 7. Triton mM X- of100 PMBS buffer (2% and Triton 0.07 X mM-100, 50 lysozyme, mM NaCl, pH20 mM 7.0, Tris.HCl, provided pH 8.8), homogenous, 8. clear cellTriton lysate X with-100/PMBS/Lys./Benz. low UV-background, buffer (2% applicableTriton X-100, for0.36 themM high-throughputPMBS, 0.07 mM lysozyme, PAL-activity 25 U/mL screens benzonase, 50 mM NaCl, 20 mM Tris.HCl, pH 8.8). (b) Optimizations using the two component (Figure3b, lane 4, Table S3). PMBS/Lys. buffer and the four-component Triton X-100/PMBS/Lys./Benz. buffer systems with different pHs and/or lysis conditions. Abbreviations: Lys., PMBS, T. X-100 and Benz. denote for 3.3.2. Activity Screens of Transformant Libraries lysozyme, polymyxin B sulphate, Triton X-100 and benzonase, correspondingly. As previously shown [14,16], mutation I460V confers activity to PcPAL towards several phenylalanine3.3.2. Activity analogs, Screens such of Transformant as p-methoxyphenylalanine, Libraries poorly or not accepted as substrates by the wild-type enzyme.As previously In order shown to identify [14,16], other mutation possible I460V mutants confers with activity significantly to PcPAL increased towards activity,several the saturationphenylalanine mutagenesis analogs, libraries such as obtainedp-methoxyphenylalanine, from randomization poorly or at not residue accepted 460 as of substratesPcPAL were by the tested in a high-throughputwild-type enzyme. In format order to for identify the ammoniaother possible elimination mutants with from significantlyp-MeO-Phe increased using activity, the optimized the activitysaturation assay. mutagenesis libraries obtained from randomization at residue 460 of PcPAL were tested in a high-throughput format for the ammonia elimination from p-MeO-Phe using the optimized The activity of >100 colonies/saturation library, providing >95% coverage for the used NNK activity assay. randomization,The activity was screened, of >100 colonies/saturation of which 10 hits library, displayed providing similar >95% or higher coverage enzyme for the activity used NNK than the cell lysaterandomization, of the highly was screened, active I460V of whichPcPAL, 10 hits used displayed as a positive similar control.or higher Among enzyme theactivity hits, than besides the the I460Vcell mutation lysate of identified the highly in active 4 selected I460V Pc colonies,PAL, used 5 novel as a positive active mutants,control. Among I460C-, the I460L-, hits, besides I460W-, the I460T-, I460S-I460VPcPAL mutation were also identified revealed. in 4 selected colonies, 5 novel active mutants, I460C-, I460L-, I460W-, I460T- To, I460S confirm-PcPAL the were increased also revealed. enzyme activity of the identified mutants, the conversion values of the ammoniaTo elimination confirm the fromincreased racemic enzymep-MeO-Phe activity of usingthe identified the corresponding mutants, the conversionPcPAL variants values of as the whole cell-biocatalystsammonia elimination were determined from racemic by HPLC p-MeO (Figure-Phe using4). While the corresponding variants I460C, PcPAL I460L, variants and I460W as whole showed low conversionscell-biocatalysts after were 16 determinedh reaction time, by HPLC both (Figure variants 4). I460T While and variants I460S I460C, showed I460L, high and conversions, I460W showed low conversions after 16 h reaction time, both variants I460T and I460S showed high exceeding those obtained by the positive control I460V (Figure4, Figures S3–S5) and approaching the conversions, exceeding those obtained by the positive control I460V (Figure 4, Figures S3-S5) and maximal,approaching 50% conversion the maximal, values 50% ofconversion a kinetic values resolution of a kinetic process. resolution process.

FigureFigure 4. Ammonia 4. Ammonia elimination elimination from from racemic racemic p--MeO-PheMeO-Phe using using the the induced induced whole whole-cells-cells of the of the correspondingcorrespondingPcPAL PcPAL variants variants showing showing increased increased activity activity within within the high-throughput the high-throughput activity activity screens. screens. Biomolecules 2020, 10, x FOR PEER REVIEW 9 of 13

3.4. Functional and Structural Characterization of Improved PcPAL Variants Further, mutants I460S, I460T of enhanced activity and I460V, as reference were isolated and the ammoniaBiomolecules 2020eliminations, 10, 838 from p-MeO-Phe were performed using the purified enzymes as biocatalysts.9 of 13 While both I460V and I460T showed conversions approaching the maximal 50% conversion of the kinetic resolution process after only 2 h reaction time, I460S provided significantly lower, 17% 3.4. Functional and Structural Characterization of Improved PcPAL Variants conversion, approaching the maximal value only after 16 h (Figure S10). The significant activity discrepancyFurther, between mutants the I460S, free I460T and ofintracellular enhanced activityenzyme andsuggested I460V, asstability reference issues were for isolated variant and I460S, the whichammonia was eliminations supported by from the pthermal-MeO-Phe unfolding were performed profiles analysis using the of purifiedall active enzymes mutants. as As biocatalysts. depicted in FigureWhile 5a, theboth melting I460V and temperature I460T showed (Tm) conversions of variant I460S approaching (55 ± 0.2 the °C) maximal was significantly 50% conversion lower of comparedthe kinetic to resolution the wt-PcPAL process (75.1±0.2 after only°C) or 2 to h reactionthe variant time, I460T I460S (70 provided± 0.2 °C), supporting significantly a decrease lower, 17% of theconversion, protein stability approaching induced the by maximal the mutation. value only A similar after 16effect h (Figure was also S10). reported The significant for the rationally activity designeddiscrepancy I460A between PcPAL the mutant free and [16] intracellular with decreased enzyme stability suggested and consequently, stability issues low for enzyme variant activity I460S, ofwhich the purified was supported enzyme. by the thermal unfolding profiles analysis of all active mutants. As depicted in FigureIn case5a, of the reactions melting performed temperature with (T purifiedm) of variant enzymes, I460S besi (55des the0.2 conversions,C) was significantly the enantiomeric lower ± ◦ excesscompared (ee) toof the wt-PcunreactedPAL (75.1D-p-MeO0.2-PheC) was or to also the monitored variant I460T by (70chiral0.2 HPLC.C), supportingBoth I460S and a decrease I460T- ± ◦ ± ◦ catalyzedof the protein reactions stability resulted induced in optically by the mutation. pure (ee >99%) A similar D-p-MeO effect-Phe was (Figure also reported S11, Figure for the S12), rationally at 50% conversions,designed I460A equalPcPAL to the mutant theoretical [16] with maximum decreased of stabilitya kinetic and resolution consequently, process, low which enzyme supports activity the of completethe purified enantioselectivity enzyme. of variant I460S and I460T within the model reaction.

(a) (b)

Figure 5. ((a) The thermal unfoldingunfolding temperaturetemperature (T (Tmm)) of of I460S I460SPc PcPALPAL and and I460T I460TPc PcPAL,PAL, determined determined by bynanoDSF nanoDSF (Prometheus (Prometheus NT.48). NT.48). Fluorescence Fluorescence intensity intensity ratios ratios F350 F350/F330/F330 and and their their first first derivative derivative are arerepresented represented as a function as a function of the applied of the applied linear thermal linear ramp thermal (b) Michaelis–Menten ramp (b) Michaelis curves–Menten obtained curves by obtainedinitial reaction by initial velocity reaction measurements velocity usingmeasurementp-MeO-Phes using as substrate p-MeO-Phe at di asfferent substrate concentration at different and concentrationpurified PcPAL and variants purified as catalysts.PcPAL variants as catalysts.

Further,In case of the reactions kinetic performed parameters with of purifiedPcPAL variants enzymes, I460T, besides I460V the conversions, were determined the enantiomeric within the ammoniaexcess (ee) elimination of the unreacted of p-MeOd-Phep-MeO-Phe (Figure 5b, was Table also 1) monitored and of the bynatural chiral substrate HPLC. L Both-phenylalanine I460S and (I460T-catalyzedL-Phe) (Figure S14 reactions–S16, Table resulted 1). Both in optically I460V- pureand (eeI460T>99%)-PcPALd-p -MeO-Pheshowed similar (Figures catalytic S11 and efficiency S12), at within50% conversions, the ammonia equal elimination to the theoretical from the maximum non-natural of a kinetic substrate resolution p-MeO process,-Phe, while which the supports wild-type the PccompletePAL poorly enantioselectivity catalyzed this of rea variantction. Despite I460S and of I460Tthe ~10 within fold lower the model kcat values reaction. shown by the mutants withinFurther, the ammonia the kinetic elimination parameters from ofp-MeOPcPAL-Phe variants than that I460T, of the I460V wild- weretype-Pc determinedPAL in the withinnatural, the L- Pheammonia deamination elimination reaction of p-MeO-Phe (Table 1), (Figure these mutants5b, Table 1 could) and ofbe the considered natural substrate potent biocatalystsl-phenylalanine, since PALs(l-Phe) with (Figures similar S14–S16, catalytic Table efficiencies1). Both were I460V- employed and I460T- forPc efficientPAL showed preparative similar biotransformations catalytic efficiency ofwithin several the phenylalanine ammonia elimination analogs [14,16,43]. from the non-naturalInterestingly, substrate variant I460Tp-MeO-Phe, exhibits whilesignificantly the wild-type higher affinityPcPAL poorlytowards catalyzed the natural this substrate, reaction. Despitein comparison of the ~10 to reference fold lower mutant kcat values I460V, shown thus also by the preserving mutants thewithin specificity the ammonia order shown elimination by the fromwild-typep-MeO-Phe PcPAL. than that of the wild-type-PcPAL in the natural, l-Phe deamination reaction (Table 1), these mutants could be considered potent biocatalysts, since PALs with similar catalytic efficiencies were employed for efficient preparative biotransformations of several phenylalanine analogs [14,16,43]. Interestingly, variant I460T exhibits significantly higher affinity towards the natural substrate, in comparison to reference mutant I460V, thus also preserving the specificity order shown by the wild-type PcPAL. Biomolecules 2020, 10, 838 10 of 13

Biomolecules 2020, 10, x FOR PEER REVIEW 10 of 13 Table 1. Enzyme kinetic parameters for the ammonia eliminations from p-methoxyphenylalanine and Table 1. Enzyme kinetic parameters for the ammonia eliminations from p-methoxyphenylalanine and natural substrate l-Phe catalyzed by wild-type PcPAL and variants I460. natural substrate L-Phe catalyzed by wild-type PcPAL and variants I460. Compound 4-MeO-Phe l-Phe Compound 4-MeO-Phe L-Phe PcPALPcPAL wtwt-- I460TI460T I460V I460V wtwt- I460T I460TI460V I460V KMK(µM M)(μM) 17411741 ± 191.2191.2 210.2210.2 ± 27.927.9 256.2 256.2 ± 26.826.8 110.9 110.9± 12.3 12.3127.1 ± 127.1 20.7 474.620.7 ± 39.8 474.6 39.8 1 ± ± ± ± ± ± kcatk(scat− (s)-1) 0.0120.012 0.129 0.129 0.139 0.139 0.698 0.698 0.805 0.8050.617 0.617 vmaxvmax(µ (μM/min)M/min) 0.450.45 ± 0.030.03 4.8 4.8 ± 0.10.1 5.22 5.22 ± 0.1 0.126.2 ± 26.2 0.6 0.635.4 ± 35.41.1 1.15.2 ± 0.1 5.2 0.1 3 ± ± ± ± ± ± k / K *10 -3 cat kcatM/ KM *10− 0.007 0.617 0.543 6.297 6.334 1.300 1 µ 1 0.007 0.617 0.543 6.297 6.334 1.300 (s− (sx -1Mxμ−M)-1)

3.5.3.5. Molecular Molecular Docking Docking BasedBased on on the the recently recently reported reported crystal crystal structure structure of variant of variant I460V I460V of Pc ofPALPcPAL in complex in complex with with p- methoxyp-methoxy--transtrans-cinnamic-cinnamic acid acid (PDB (PDB ID: 6RGS) ID: 6RGS) [14], [we14], performed we performed molecular molecular docking docking of the ofL-p the- methoxyphenylalaninel-p-methoxyphenylalanine and the and corresponding the corresponding cinnamic cinnamic acid acid in case in case of of variant variant I460T, I460T, reference reference mutantmutant I460V I460V and and wildwild-type-type Pc PcPALPAL (Figure (Figure 6).6). The The provided provided orientation orientation of of p-pMeO-MeO-cinnamic-cinnamic acid, acid, similarsimilar to to one one observed within within the the crystal crystal structure structure (Figure (Figure S20), S20), supported supported the validity the validity of the dockingof the dockingprocedure, procedure, which supports which supports almost identical, almost favorable identical, substrate favorable orientations substrate orientationswithin the catalytic within site the of catalyticboth I460V site of and both I460T I460V variants. and I460T Moreover, variants. the Moreover, docking results the docking conﬁrmed results the confirmed inactivity ofthe the inactivitywild-type ofPc thePAL. wild In-type this case,PcPAL. no activeIn this conformation case, no active for conformationp-MeO-Phe or for of itsp-MeO cinnamic-Phe acidor of counterpart its cinnamic could acid be counterpartobserved, duecould to be the observed, steric repulsion due to withthe steric residue repulsion I460 (Figure with residue S21). I460 (Figure S21).

(a) (b)

FigureFigure 6. 6.ViewView of ofthe the catalytic catalytic site, site, including including the the electrophilic electrophilic 4- 4-methylideneimidazol-5-onemethylideneimidazol-5-one (MIO) (MIO) prostheticprosthetic group; group; overlay overlay of of the the (a ()a p)-pMeO-MeO cinnamic cinnamic acid acid and and (b ()b p)-pMeO-MeO-Phe-Phe orientations orientations within within the the catalyticcatalytic site site of of I460V I460V and and I460T. I460T. 4. Conclusions 4. Conclusions This study focuses on the saturation mutagenesis of activity modulator residue I460 of PcPAL in This study focuses on the saturation mutagenesis of activity modulator residue I460 of PcPAL order to explore and identify novel PAL-variants with increased activity towards non-natural substrates. in order to explore and identify novel PAL-variants with increased activity towards non-natural Using the NNK-degeneracy, providing access to all possible single-site mutants, the optimized substrates. Using the NNK-degeneracy, providing access to all possible single-site mutants, the mutagenesis experiments provided high-quality mutant libraries, with nucleotide-base distributions optimized mutagenesis experiments provided high-quality mutant libraries, with nucleotide-base approaching the theoretical values. For the activity screens of mutant libraries, a high-throughput distributions approaching the theoretical values. For the activity screens of mutant libraries, a high- PAL-activity assay was adapted for use with cell-lysate biocatalysts. During the activity screen for throughput PAL-activity assay was adapted for use with cell-lysate biocatalysts. During the activity the ammonia elimination reaction of the non-natural substrate, p-methoxyphenylalanine, several screen for the ammonia elimination reaction of the non-natural substrate, p-methoxyphenylalanine, hits have been identiﬁed, showing similar or higher activity than the cell lysate of the rationally several hits have been identified, showing similar or higher activity than the cell lysate of the developed, highly active I460V PcPAL. Interestingly, among the hits the mutation I460V occurred rationally developed, highly active I460V PcPAL. Interestingly, among the hits the mutation I460V with the highest frequency, supporting the eﬃciency of the reported rational design approach [14,16]. occurred with the highest frequency, supporting the efficiency of the reported rational design approach [14,16]. Further biotransformations using the whole-cell and/or purified PAL-biocatalysts of the corresponding mutants confirmed the activity of the novel variants towards p-MeO-Phe, Biomolecules 2020, 10, 838 11 of 13

Further biotransformations using the whole-cell and/or purified PAL-biocatalysts of the corresponding mutants confirmed the activity of the novel variants towards p-MeO-Phe, poorly transformed by the wild-type enzyme. While within the whole-cell biotransformations, variants I460T and I460S presented an improved catalytic efficiency compared to the rationally explored I460V PcPAL, mutant I460S presented stability issues and low catalytic efficiency after purification. Variant I460T, in the model reaction with p-MeO-Phe, showed similar kinetic profile with the reference variant I460V, moreover, in contrast to I460V, maintained the catalytic efficiency of the wild-type enzyme within the natural reaction. Molecular docking based on the recently reported crystal structure of I460V PcPAL (6RGS) complexed with the product of the investigated model reaction, p-MeO-cinnamic acid, supported similar substrate orientations within the active site of variants I460V and I460T, while no active orientation of the substrate could be observed in the case of the wild-type PcPAL. The developed/optimized procedures provide tools for efficient saturation mutagenesis based directed evolution of PALs, while the identified variants, due to the highly conserved nature of residue I460V among phenylalanine-ammonia lyases provide further perspective for PAL mediated biotransformations of industrially relevant phenylalanine analogs.

Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/10/6/838/s1: Supporting Information describing experimental details/results for saturation mutagenesis, assay development, HPLC reaction monitoring, enzyme kinetics, enzyme purification and molecular docking. Author Contributions: L.C.B. conceived the project, while R.B.T. designed and performed the saturation mutagenesis experiments. R.B.T., together with I.H. was also responsible for the adaptation of the PAL-activity assay under the supervision of L.C.B. and S.D.T. The whole-cell biotransformations and their monitoring were performed by R.B.T. and S.D.T., while A.F. was responsible for enzyme purifications. L.C.N. contributed to molecular docking. L.C.B. supervised all data and wrote the paper together with all authors. All authors reviewed and agreed to the published version of the manuscript. Funding: This work was financed by the Swiss National Science Foundation (SNSF)—project PROMYS, grant nr. IZ11Z0_166543. R.B.T. thanks the STAR-Institute of the Babes, -Bolyai University for the provided student-research fellowship. Acknowledgments: We gratefully acknowledge the infrastructural support of the Biocatalysis and

Biotransformation Research Center and the helpful comments from C. Paizs, M.I. Tos, a and F.D. Irimie. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

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