An efficient chemoenzymatic methodology was developed for the synthesis of l -3-(2-furyl)alanine using E. coli expressing At PAL as a biocatalyst with an overall isolation yield of 72.3%.

Aromatic d-amino acids (d-AAs) have gained increasing attention as chiral building blocks, with biocatalytic procedures emerging as powerful methods for their asymmetric synthesis. d-Amino acid dehydrogenases (DAADH), developed by protein engineering from meso-diaminopimelate dehydrogenases, step out as highly efficient biocatalysts for the reductive amination-based production of d-AAs. Enzyme immobilization allows the recovery and reuse of biocatalysts, while also enhances their operational stability, which is essential for industrial applications. Since the immobilization of DAADHs have been less explored, we targeted to further progress within the covalent immobilization of DAADH from Ureibacillus thermosphaericus, by exploring its site-specific, covalent immobilization. The individual replacement of several surficial Ser residues to Cys at positions 2, 58, 92, 185, 192 and 317 of UtDAADH, allowed their site-specific immobilization onto the maleimide-functionalized Purolite® ECR8415F methacrylic support. The highest specific activity values, ∼0.078 and ∼0.083 U/mg provided by immobilization through Cys2 and Cys192, respectively, showed 2.1- and 2.2-fold higher values compared to the covalently, but non-specifically immobilized UtDAADH. The recyclability of the immobilized preparations was tested among 10 reductive amination-cycles of phenylpyruvate and based on the retained conversion and specific activities, UtDAADH immobilized through S2C was the best-performing biocatalyst, maintaining 65-70 % conversion and 50 % of the initial activity after the 10th cycle. After the assessment of optimal enzyme/substrate ratio, the immobilized S2C UtDAADH was tested in three consecutive 200 mg-scale reaction, providing the enantiopure d-Phe with complete conversions and excellent > 88 % isolation yields, supporting its synthetic applicability.

Efficient monitoring of the enzymatic PET-hydrolysis is crucial for developing novel plastic-degrading biocatalysts. Herein, we aimed to upgrade in terms of accuracy the analytical methods useful for monitoring enzymatic PET-degradation. For the HPLC-based assessment, the incorporation of an internal standard within the analytic procedure enabled a more accurate quantification of the overall TPA content and the assessment of molar distributions and relative content of each aromatic degradation product. The provided calibration curves cover a broad concentration range, from µM to low mM scale, facilitating assessment of both lower and higher PETase activities, with a limit of detection positioned below the reported PET-degrading activities. The increased reproducibility and accuracy of the improved HPLC method, compared to the previous methods, was supported by lower dispersion of product concentrations and their lower deviation from theoretical values over multiple measurements. The other predominantly employed UV-spectroscopy assay was also improved in terms of employed wavelength and medium extinction coefficient of the three aromatic degradation products, while being cross-validated by the improved HPLC method. Finally, both methods were used for monitoring the product formation within the leaf-branch compost cutinase (LCC)-mediated PET-hydrolysis and provided individual time-productivity profiles for each aromatic degradation product.

The enzymatic synthesis of d-phenylalanines, important chiral building blocks for several pharmaceuticals and fine chemicals, has been widely explored. Their asymmetric synthesis of high atom economy and accessible prochiral starting materials is highly attractive, while the expanding toolbox of protein engineering facilitates access to biocatalysts tailored for these processes. Accordingly, this Review provides an overview of the protein engineering efforts of enzymes involved in the asymmetric synthetic pathways for d-phenylalanines. The engineering efforts on d-amino acid dehydrogenases, d-amino acid transaminases, and phenylalanine ammonia-lyases to produce d-phenylalanines are thoroughly examined, while their application in (chemo)enzymatic cascades is also discussed. For an improved efficiency of the cascades, the protein engineering of l-amino acid deaminases and/or l-amino acid oxidases for an increased transformation of phenylalanines is also addressed.

Characterization of the aromatic ammonia-lyase from Loktanella atrilutea (LaAAL) revealed reduced activity towards canonical AAL substrates: l-Phe, l-Tyr, and l-His, contrasted by its pronounced efficiency towards 3,4-dimethoxy-l-phenylalanine. Assessing the optimal conditions, LaAAL exhibited maximal activity at pH 9.5 in the ammonia elimination reaction route, distinct from the typical pH ranges of most PALs and TALs. Within the exploration of the ammonia source for the opposite, synthetically valuable ammonia addition reaction, the stability of LaAAL exhibited a positive correlation with the ammonia concentration, with the highest stability in 4 M ammonium carbamate of unadjusted pH of ~9.5. While the enzyme activity increased with rising temperatures yet, the highest operational stability and highest stationary conversions of LaAAL were observed at 30 °C. The substrate scope analysis highlighted the catalytic adaptability of LaAAL in the hydroamination of diverse cinnamic acids, especially of meta-substituted and di-/multi-substituted analogues, with structural modelling exposing steric clashes between the substrates' ortho-substituents and catalytic site residues. LaAAL showed a predilection for ammonia elimination, while classifying as a tyrosine ammonia-lyase (TAL) among the natural AAL classes. However, its distinctive attributes, such as genomic context, unique substrate specificity and catalytic fingerprint, suggest a potential natural role beyond those of known AAL classes.

Several procedures were tested for the immobilization of the artificial D -selective amino acid dehydrogenase from Ureibacillus thermosphaericus (UtDAADH) and its co -immobilization with the NADPH-regenerating glucose dehydrogenase (GDH). Based on the conversions of the reductive amination of phenylpyruvate, recyclability, batchto -batch reproducibility, and immobilization costs, DAADH covalently attached onto Purolite (R) ECR8415F or coimmobilized with GDH on polyethylenimine-coated agarose were selected for optimizations. The non -desired substrate/product adsorption occurring in case of the Purolite (R) support, was avoided by increasing the volume of linkers employed for the covalent fixation of the enzyme. The more convenient to prepare immobilization variant, DAADH adsorbed onto the Purolite (R) resin also proved to be applicable. This preparation, despite presenting significant drop of 58.8% of its specific activity over 10 reaction cycles, still provided similar conversions with the covalently immobilized variant. As third effective preparation, the DAADH-GDH co -immobilized system, showed no activity loss over 10 reaction cycles. In this case the additional co -immobilization of the NADP(+) cofactor provided self-sufficient biocatalysts only for limited cycles, after >3 consecutive reactions the conversion dropped with similar to 60%, due to cofactor leakage. The synthetic utility of the immobilized DAADH was demonstrated by the 100 mg -scale reductive amination of phenylpyruvate, obtaining D-phenylalanine with 86% yield.

An efficient preparative-scale synthetic procedure of l-phenylalanine derivatives has been developed using mutant variants of phenylalanine ammonia-lyase from Arabidopsis thaliana (AtPAL). After rigorous reaction engineering, the AtPAL-catalyzed hydroamination reaction of cinnamic acids provided several unnatural amino acids of high synthetic value, such as (S)-m- and (S)-p-methoxyphenylalanine; (S)-o- and (S)-m-methylphenylalanine; and (S)-o- and (S)-p-bromophenylalanine at preparative scale, significantly surpassing the catalytic efficiency in terms of conversions and yields of the previously reported PcPAL-based biotransformations. The AtPAL variants tolerated high substrate and product concentrations, representing an important extension of the PAL-toolbox, while the engineered biocatalytic procedures of improved E-factor and space-time yields fulfill the requirements of sustainable and green chemistry, providing facile access to valuable amino acid building blocks.

In this study we assessed the applicability of the recently reported ancestral l-amino acid oxidase (AncLAAO), for the development of an enzyme-coupled phenylalanine ammonia-lyase (PAL) activity assay. Firstly, the expression and isolation of the AncLAAO-N1 was optimized, followed by activity tests of the obtained octameric N-terminal His-tagged enzyme towards various phenylalanine analogues to assess the compatibility of its substrate scope with that of the well-characterized PALs. AncLAAO-N1 showed high catalytic efficiency towards phenylalanines mono-, di-, or multiple-substituted in the meta- or para-positions, with ortho- substituted substrates being poorly transformed, these results highlighting the significant overlap between its substrate scope and those of PALs. After successful set-up of the AncLAAO-PAL coupled solid phase assay, in a 'proof of concept' approach we demonstrated its applicability for the high-throughput activity screens of PAL-libraries, by screening the saturation mutagenesis-derived I460NNK variant library of PAL from Petroselinum crispum, using p-MeO-phenylalanine as model substrate. Notably, the hits revealed by the coupled assay comprised all the active PAL variants: I460V, I460T, I460S, I460L, previously identified from the tested PAL-library by other assays. Our results validate the applicability of AncLAAO for coupled enzyme systems with phenylalanine ammonia-lyases, including cell-based assays suitable for the high-throughput screening of directed evolution-derived PAL-libraries.

In this study, rational design and saturation mutagenesis efforts for engineering phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) provided tailored PALs active towards challenging, highly valuable di-substituted substrates, such as the l-DOPA precursor 3,4-dimethoxy-l-phenylalanine or the 3-bromo-4-methoxy-phenylalanine. The rational design approach and saturation mutagenesis strategy unveiled identical PcPAL variants of improved activity, highlighting the limited mutational variety of the substrate specificity-modulator residues, L134, F137, I460 of PcPAL. Due to the restricted catalytic efficiency of the best performing L134A/I460V and F137V/I460V PcPAL variants, we imprinted these beneficial mutations to PALs of different origins. The variants of PALs from Arabidopsis thaliana (AtPAL) and Anabaena variabilis (AvPAL) showed higher catalytic efficiency than their PcPAL homologues. Further, the engineered PALs were also compared in terms of catalytic efficiency with a novel aromatic ammonia-lyase from Loktanella atrilutea (LaAAL), close relative of the metagenome-derived aromatic ammonia-lyase AL-11, reported recently to possess atypically high activity towards substrates with electron-donor aromatic substituents. Indeed, LaAAL outperformed the engineered Pc/At/AvPALs in the production of 3,4-dimethoxy-l-phenylalanine; however, in case of 3-bromo-4-methoxy derivatives it showed no activity, with computational results supporting the occurrence of steric hindrance. Transferring the unique array of selectivity modulator residues from LaAAL to the well-characterized PALs did not enhance their activity towards the targeted substrates. Moreover, applying the rational design strategy valid for these well-characterized PALs to LaAAL decreased its activity. These results suggest that distinct tailoring rationale is required for LaAAL/AL-11-like aromatic ammonia-lyases, which might represent a distinct PAL subclass, with natural reaction and substrate scope modified through evolutionary processes. • PAL-activity for challenging substrates generated by protein engineering • Rational/semi-rational protein engineering reveals constrained mutational variability • Engineered PALs are outperformed by novel ALs of distinct catalytic site signature

A new robust, active and selective biocatalyst, prepared by CaL-B entrapment into polyvinyl alcohol–chitosan nanofibers, proved to be efficient for the EKR of nine racemic phenothiazinyl-ethanols, important chiral building blocks.

One of the most promising PET-hydrolysing enzyme, IsPETase, originates from 'plastic-consuming' bacteria Ideonella sakaiensis and operates at moderate temperatures (30-40 °C) in aqueous environment, showing far higher hydrolysing efficiency than several cutinases. Accordingly, the development of proper recombinant expression system providing facile access to the isolated/purified IsPETase is also of high interest. In our aim to produce active recombinant IsPETase, the unsuccessful expression registered by employing several reported expression systems, directed us towards the optimization/ development of a facile/accessible recombinant expression system suitable for IsPETase production. Testing several plasmid constructs, providing different N- or C-terminal affinity tags for the expression of full-length or truncated (residues 28-290) IsPETase and various E. coli expression hosts, revealed several non-reported issues hindering the recombinant expression of IsPETase. After optimization of the construct and expression host, the use of N-terminal His-tag and Rosetta-gami B expression host provided recombinant IsPETase in high purity and titer-yield. The thermal denaturation profile and PET-hydrolysing activity of the obtained IsPETase agree with the reported data, supporting proper folding of the purified protein. The results support that the in vivo folding process of IsPETase might be differently affected among the different E. coli host strains, moreover, underline the importance of the proper selection of the cloning strategy for the successful expression of the IsPETase.

Amine transaminases (TAs) are attractive biocatalysts for the synthesis of chiral aromatic amines representing highly valuable motifs of APIs. The increased industrial need of novel methods to produce chiral amines for APIs resulted in an emerged discovery of new TAs. Joining the current wave of TA related research, this study reports the identification of genes encoding an (S)-selective TA from Pseudomonas psychrotolerans TA (PpS-TA) and an (R)-selective TA from Shinorizobium sp. TA (SrR-TA) by sequence data mining. Functional analysis of the novel TAs revealed their pH profile, thermal stability, optimal buffer system, DMSO tolerance, and operational stability in kinetic resolution (KR) of racemic 1-phenylethane-1-amines. The (S)-selective PpS-TA maintained its operational stability even at high temperature and pH values, enabling conversions from racemates approaching the optimal similar to 50% value of a highly selective KR. Although with lower activity, the (R)-selective SrR-TA remained active at higher DMSO co-solvent concentrations up to 30%, enabling elevated substrate concentrations for aromatic amines of low water-solubility. The kinetic parameters of the novel PpS-TA and SrR-TA in KRs of several racemic 1-arylethane-1-amines (+/-)-2a-d and pyruvate (co-substrate) revealed superior catalytic efficiencies (kcat values) compared to the well-characterized (S)-TA from Chromobacterium violaceum (CvS-TA) indicating the biocatalytic potential of the two newly characterized TAs.

Unnatural substituted amino acids play an important role as chiral building blocks, especially for pharmaceutical industry, where the synthesis of chiral biologically active molecules still represents an open challenge. Recently, modification of the hydrophobic binding pocket of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) resulted in specifically tailored PcPAL variants, contributing to a rational design template for PAL-activity enhancements towards the differently substituted substrate analogues. Within this study we tested the general applicability of this rational design model in case of PALs, of different sources, such as from Arabidopsis thaliana (AtPAL) and Rhodosporidium toruloides (RtPAL). With some exceptions, the results support that the positions of substrate specificity modulating residues are conserved among PALs, thus the mutation with beneficial effect for PAL-activity enhancement can be predicted using the established rational design model. Accordingly, the study supports that tailoring PALs of different origins and different substrate scope, can be performed through a general method. Moreover, the fact that AtPAL variants I461V, L133A and L257V, all outperformed in terms of catalytic efficiency the corresponding, previously reported, highly efficient PcPAL variants, of identical catalytic site, suggests that not only catalytic site differences influence the PAL-activity, thus for the selection of the optimal PAL-biocatalysts for a targeted process, screening of PALs from different origins, should be included.

An efficient, active and stable biocatalyst was prepared by sol–gel CaL-B encapsulation in the presence of a choline–fructose DES, and is able to transform efficiently ten alcohols relevant for various industries.

Nowadays, protein purification by the aid of affinity tags can be carried out with high speed and efficiency. However, in several cases, affinity tags can significantly alter the key properties of enzymes, especially activity and/or thermostability. This study focused on the purification of the non-tagged phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL), as well as on the purification of the TEV (Tobacco Etch Virus) protease, the molecular scissors used to remove the affinity tag from the recombinantly expressed PcPAL. Removal of the 6xHis-tag led to a 1.5-fold increase in the specific activity of PcPAL, while the absence of the affinity tag did not significantly alter the thermostability of the protein. The purity and oligomerization state of the proteins of interest were also analyzed by size exclusion chromatography, both before and after the removal of the affinity tag, confirming the stability of the tetrameric fold of PcPAL

The interest towards ferulic acid decarboxylase (FDC), piqued by the enzyme's unique 1,3-dipolar cycloaddition mechanism and its atypic prFMN cofactor, provided several applications of the FDC mediated decarboxylations, such as the synthesis of styrenes, or its diverse derivatives, including 1,3-butadiene and the enzymatic activation of C-H bonds through the reverse carboligation reactions. While rational design-based protein engineering was successfully employed for tailoring FDC towards diverse substrates of interest, the lack of high-throughput FDC-activity assay hinders its directed evolution-based protein engineering. Herein we report a toolbox, useful for the directed evolution based and/or structure-guided protein engineering of FDC, which was validated representatively on the well described FDC, originary from Saccharomyces cerevisiae (ScFDC). Accordingly, the developed fluorescent plate-assay allows in premiere the FDC-activity screens of a mutant library in a high-throughput manner. Moreover, using the plate-assay for the activity screens of a rationally designed 23-membered ScFDC variant library against a substrate panel comprising of 16, diversely substituted cinnamic acids, revealed several variants of improved activity. The superior catalytic properties of the hits revealed by the plate-assay, were also supported by the conversion values from their analytical scale biotransformations. The computational results further endorsed the experimental findings, showing inactive binding poses of several non-transformed substrate analogues within the active site of the wild-type ScFDC, but favorable ones within the catalytic site of the variants of improved activity. The results highlight several 'hot-spot' residues involved in substrate specificity modulation of FDC, such as I189, I330, F397, I398 or Q192, of which mutations to sterically less demanding residues increased the volume of the active site, thus facilitated proper binding and increased conversions of diverse non-natural substrates. Upon revealing which mutations improve the FDC activity towards specific substrate analogues, we also provide key for the rational substrate-tailoring of FDC.

We report a simple enzymatic procedure for the synthesis of short-chained flavor esters by direct esterification of natural acids with short-chain primary alcohols mediated by lipase B from Candida antarctica entrapped in a tailored sol-gel matrix in the presence of three additives, using vacuum for water removal. Maximal immobilization yields (100%) were obtained for all three biocatalysts, while the enzyme loading and the synthetic activity depend on the used additive (6.7, 7.6, and 7.7 mu g enzyme/mg biocatalyst for beta-cyclodextrin, polyvinyl alcohol, and glycerol, respectively, and 76-110% recovered activity as compared with free lipase). The process was optimized using the reaction of hexan-1-ol with butyric acid as the model with significant conversion improvements (from <40% to >94%) reducing the alcohol/enzyme ratio (from 100:1 to 25:1 weight ratio) for all novel biocatalysts. Other short-chain flavor esters were prepared with excellent yields (>90%) under the previously established optimal conditions. Atom economy (90.53), E-factor (17.22), atom efficiency (88.36), mass intensity (18.58), and reaction mass efficiency (60.07) as relevant sustainability metrics were calculated for the preparative scale model reaction with glycerol as the additive. Based on our results, this green and sustainable new approach can be used for the synthesis of many flavor esters.

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.

An efficient biocatalyst was obtained by covalent immobilization of lipase B from C. antarctica on functionalized SwCNTs for the kinetic resolution of some aryl-ethanols in batch and continuous-flow modes.