Structural Analysis of a Genetically Encoded FRET Biosensor by SAXS and MD Simulations.

Inspired by the modular architecture of natural signaling proteins, ligand binding proteins are equipped with two fluorescent proteins (FPs) in order to obtain Förster resonance energy transfer (FRET)-based biosensors. Here, we investigated a glucose sensor where the donor and acceptor FPs were attached to a glucose binding protein using a variety of different linker sequences. For three resulting sensor constructs the corresponding glucose induced conformational changes were measured by small angle X-ray scattering (SAXS) and compared to recently published single molecule FRET results (Höfig et al., ACS Sensors, 2018). For one construct which exhibits a high change in energy transfer and a large change of the radius of gyration upon ligand binding, we performed coarse-grained molecular dynamics simulations for the ligand-free and the ligand-bound state. Our analysis indicates that a carefully designed attachment of the donor FP is crucial for the proper transfer of the glucose induced conformational change of the glucose binding protein into a well pronounced FRET signal change as measured in this sensor construct. Since the other FP (acceptor) does not experience such a glucose induced alteration, it becomes apparent that only one of the FPs needs to have a well-adjusted attachment to the glucose binding protein.

A cascade reaction for the synthesis of d-fagomine precursor revisited: Kinetic insight and understanding of the system.

The synthesis of aldol adduct (3S,4R)-6-[(benzyloxycarbonyl)amino]-5,6-dideoxyhex-2-ulose, a precursor of the interesting dietary supplement, iminosugar d-fagomine, was studied in a cascade reaction with three enzymes starting from Cbz-N-3-aminopropanol. This system was studied previously using a statistical optimization method which enabled a 79 % yield of the aldol adduct with a 10 % yield of the undesired amino acid by-product. Here, a kinetic model of the cascade, including enzyme operational stability decay rate and the undesired overoxidation of the intermediate product, was developed. The validated model was instrumental in the optimization of the cascade reaction in the batch reactor. Simulations were carried out to determine the variables with the most significant impact on substrate conversion and product yield. As a result, process conditions were found that provided the aldol adduct in 92 % yield with only 0.7 % yield of the amino acid in a one-pot one-step reaction. Additionally, compared to previous work, this improved process outcome was achieved at lower concentrations of two enzymes used in the reaction. With this study the advantages are demonstrated of a modelling approach in developing complex biocatalytical processes. Mathematical models enable better understanding of the interactions of variables in the investigated system, reduce cost, experimental efforts in the lab and time necessary to obtain results since the simulations are carried out in silico.

Construction and comprehensive characterization of an EcLDCc-CatIB set-varying linkers and aggregation inducing tags.

BACKGROUND: In recent years, the production of inclusion bodies that retained substantial catalytic activity was demonstrated. These catalytically active inclusion bodies (CatIBs) were formed by genetic fusion of an aggregation inducing tag to a gene of interest via short linker polypeptides and overproduction of the resulting gene fusion in Escherichia coli. The resulting CatIBs are known for their high stability, easy and cost efficient production, and recyclability and thus provide an interesting alternative to conventionally immobilized enzymes. RESULTS: Here, we present the construction and characterization of a CatIB set of the lysine decarboxylase from Escherichia coli (EcLDCc), constructed via Golden Gate Assembly. A total of ten EcLDCc variants consisting of combinations of two linker and five aggregation inducing tag sequences were generated. A flexible Serine/Glycine (SG)- as well as a rigid Proline/Threonine (PT)-Linker were tested in combination with the artificial peptides (18AWT, L6KD and GFIL8) or the coiled-coil domains (TDoT and 3HAMP) as aggregation inducing tags. The linkers were fused to the C-terminus of the EcLDCc to form a linkage between the enzyme and the aggregation inducing tags. Comprehensive morphology and enzymatic activity analyses were performed for the ten EcLDCc-CatIB variants and a wild type EcLDCc control to identify the CatIB variant with the highest activity for the decarboxylation of L-lysine to 1,5-diaminopentane. Interestingly, all of the CatIB variants possessed at least some activity, whilst most of the combinations with the rigid PT-Linker showed the highest conversion rates. EcLDCc-PT-L6KD was identified as the best of all variants allowing a volumetric productivity of 457 g L- 1 d- 1 and a specific volumetric productivity of 256 g L- 1 d- 1 gCatIB-1. Noteworthy, wild type EcLDCc, without specific aggregation inducing tags, also partially formed CatIBs, which, however showed lower activity compared to most of the newly constructed CatIB variants (volumetric productivity: 219 g L- 1 d- 1, specific volumetric activity: 106 g L- 1 d- 1 gCatIB- 1). Furthermore, we demonstrate that microscopic analysis can serve as a tool to find CatIB producing strains and thus allow for prescreening at an early stage to save time and resources. CONCLUSIONS: Our results clearly show that the choice of linker and aggregation inducing tag has a strong influence on the morphology and the enzymatic activity of the CatIBs. Strikingly, the linker had the most pronounced influence on these characteristics.

Synthesis of α-hydroxy ketones and vicinal diols with the Bacillus licheniformis DSM 13T butane-2,3-diol dehydrogenase.

The enantioselective synthesis of α-hydroxy ketones and vicinal diols is an intriguing field because of the broad applicability of these molecules. Although, butandiol dehydrogenases are known to play a key role in the production of 2,3-butandiol, their potential as biocatalysts is still not well studied. Here, we investigate the biocatalytic properties of the meso-butanediol dehydrogenase from Bacillus licheniformis DSM 13T (BlBDH). The encoding gene was cloned with an N-terminal StrepII-tag and recombinantly overexpressed in E. coli. BlBDH is highly active towards several non-physiological diketones and α-hydroxyketones with varying aliphatic chain lengths or even containing phenyl moieties. By adjusting the reaction parameters in biotransformations the formation of either the α-hydroxyketone intermediate or the diol can be controlled.

Synthesis of α-hydroxy ketones and vicinal (R,R)-diols by Bacillus clausii DSM 8716T butanediol dehydrogenase.

α-hydroxy ketones (HK) and 1,2-diols are important building blocks for fine chemical synthesis. Here, we describe the R-selective 2,3-butanediol dehydrogenase from B. clausii DSM 8716T (BcBDH) that belongs to the metal-dependent medium chain dehydrogenases/reductases family (MDR) and catalyzes the selective asymmetric reduction of prochiral 1,2-diketones to the corresponding HK and, in some cases, the reduction of the same to the corresponding 1,2-diols. Aliphatic diketones, like 2,3-pentanedione, 2,3-hexanedione, 5-methyl-2,3-hexanedione, 3,4-hexanedione and 2,3-heptanedione are well transformed. In addition, surprisingly alkyl phenyl dicarbonyls, like 2-hydroxy-1-phenylpropan-1-one and phenylglyoxal are accepted, whereas their derivatives with two phenyl groups are not substrates. Supplementation of Mn2+ (1 mM) increases BcBDH's activity in biotransformations. Furthermore, the biocatalytic reduction of 5-methyl-2,3-hexanedione to mainly 5-methyl-3-hydroxy-2-hexanone with only small amounts of 5-methyl-2-hydroxy-3-hexanone within an enzyme membrane reactor is demonstrated.

A FRET-based biosensor for the quantification of glucose in culture supernatants of mL scale microbial cultivations.

BACKGROUND: In most microbial cultivations D-glucose is the main carbon and energy source. However, quantification of D-glucose especially in small scale is still challenging. Therefore, we developed a FRET-based glucose biosensor, which can be applied in microbioreactor-based cultivations. This sensor consists of a glucose binding protein sandwiched between two fluorescent proteins, constituting a FRET pair. Upon D-glucose binding the sensor undergoes a conformational change which is translated into a FRET-ratio change. RESULTS: The selected sensor shows an apparent Kd below 1.5 mM D-glucose and a very high sensitivity of up to 70% FRET-ratio change between the unbound and the glucose-saturated state. The soluble sensor was successfully applied online to monitor the glucose concentration in an Escherichia coli culture. Additionally, this sensor was utilized in an at-line process for a Corynebacterium glutamicum culture as an example for a process with cell-specific background (e.g. autofluorescence) and medium-induced quenching. Immobilization of the sensor via HaloTag® enabled purification and covalent immobilization in one step and increased the stability during application, significantly. CONCLUSION: A FRET-based glucose sensor was used to quantify D-glucose consumption in microtiter plate based cultivations. To the best of our knowledge, this is the first method reported for online quantification of D-glucose in microtiter plate based cultivations. In comparison to D-glucose analysis via an enzymatic assay and HPLC, the sensor performed equally well, but enabled much faster measurements, which allowed to speed up microbial strain development significantly.

An Enzymatic 2-Step Cofactor and Co-Product Recycling Cascade towards a Chiral 1,2-Diol. Part I: Cascade Design.

Alcohol dehydrogenases are of high interest for stereoselective syntheses of chiral building blocks such as 1,2-diols. As this class of enzymes requires nicotinamide cofactors, their application in biotechnological synthesis reactions is economically only feasible with appropriate cofactor regeneration. Therefore, a co-substrate is oxidized to the respective co-product that accumulates in equal concentration to the desired target product. Co-product removal during the course of the reaction shifts the reaction towards formation of the target product and minimizes undesired side effects. Here we describe an atom efficient enzymatic cofactor regeneration system where the co-product of the ADH is recycled as a substrate in another reaction set. A 2-step enzymatic cascade consisting of a thiamine diphosphate (ThDP)-dependent carboligase and an alcohol dehydrogenase is presented here as a model reaction. In the first step benzaldehyde and acetaldehyde react to a chiral 2-hydroxy ketone, which is subsequently reduced by to a 1,2-diol. By choice of an appropriate co-substrate (here: benzyl alcohol) for the cofactor regeneration in the alcohol dehydrogenases (ADH)-catalyzed step, the co-product (here: benzaldehyde) can be used as a substrate for the carboligation step. Even without any addition of benzaldehyde in the first reaction step, this cascade design yielded 1,2-diol concentrations of >100 mM with optical purities (ee, de) of up to 99%. Moreover, this approach overcomes the low benzaldehyde solubility in aqueous systems and optimizes the atom economy of the reaction by reduced waste production. The example presented here for the 2-step recycling cascade of (1R,2R)-1-phenylpropane-1,2-diol can be applied for any set of enzymes, where the co-products of one process step serve as substrates for a coupled reaction.

Navigating within thiamine diphosphate-dependent decarboxylases: Sequences, structures, functional positions, and binding sites.

Thiamine diphosphate-dependent decarboxylases catalyze both cleavage and formation of CC bonds in various reactions, which have been assigned to different homologous sequence families. This work compares 53 ThDP-dependent decarboxylases with known crystal structures. Both sequence and structural information were analyzed synergistically and data were analyzed for global and local properties by means of statistical approaches (principle component analysis and principal coordinate analysis) enabling complexity reduction. The different results obtained both locally and globally, that is, individual positions compared with the overall protein sequence or structure, revealed challenges in the assignment of separated homologous families. The methods applied herein support the comparison of enzyme families and the identification of functionally relevant positions. The findings for the family of ThDP-dependent decarboxylases underline that global sequence identity alone is not sufficient to distinguish enzyme function. Instead, local sequence similarity, defined by comparisons of structurally equivalent positions, allows for a better navigation within several groups of homologous enzymes. The differentiation between homologous sequences is further enhanced by taking structural information into account, such as BioGPS analysis of the active site properties or pairwise structural superimpositions. The methods applied herein are expected to be transferrable to other enzyme families, to facilitate family assignments for homologous protein sequences.

Impact of Molecular Crowding on Translational Mobility and Conformational Properties of Biological Macromolecules.

Effects of molecular crowding on structural and dynamical properties of biological macromolecules do depend on the concentration of crowding agents but also on the molecular mass and the structural compactness of the crowder molecules. By employing fluorescence correlation spectroscopy (FCS), we investigated the translational mobility of several biological macromolecules ranging from 17 kDa to 2.7 MDa. Polyethylene glycol and Ficoll polymers of different molecular masses were used in buffer solutions to mimic a crowded environment. The reduction in translational mobility of the biological tracer molecules was analyzed as a function of crowder volume fractions and was generally more pronounced in PEG as compared to Ficoll solutions. For several crowding conditions, we observed a molecular sieving effect, in which the diffusion coefficient of larger tracer molecules is reduced to a larger extent than predicted by the Stokes-Einstein relation. By employing a FRET-based biosensor, we also showed that a multiprotein complex is significantly compacted in the presence of macromolecular crowders. Importantly, with respect to sensor in vivo applications, ligand concentration determining sensors would need a crowding specific calibration in order to deliver correct cytosolic ligand concentration.

Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species.

Bacterial periplasmic binding proteins (PBPs) undergo a pronounced ligand-induced conformational change which can be employed to monitor ligand concentrations. The most common strategy to take advantage of this conformational change for a biosensor design is to use a Förster resonance energy transfer (FRET) signal. This can be achieved by attaching either two fluorescent proteins (FPs) or two organic fluorescent dyes of different colors to the PBPs in order to obtain an optical readout signal which is closely related to the ligand concentration. In this study we compare a FP-equipped and a dye-labeled version of the glucose/galactose binding protein MglB at the single-molecule level. The comparison demonstrates that changes in the FRET signal upon glucose binding are more pronounced for the FP-equipped sensor construct as compared to the dye-labeled analog. Moreover, the FP-equipped sensor showed a strong increase of the FRET signal under crowding conditions whereas the dye-labeled sensor was not influenced by crowding. The choice of a labeling scheme should therefore be made depending on the application of a FRET-based sensor.

Structural and Mutagenesis Studies of the Thiamine-Dependent, Ketone-Accepting YerE from Pseudomonas protegens.

A wide range of thiamine diphosphate (ThDP)-dependent enzymes catalyze the benzoin-type carboligation of pyruvate with aldehydes. A few ThDP-dependent enzymes, such as YerE from Yersinia pseudotuberculosis (YpYerE), are known to accept ketones as acceptor substrates. Catalysis by YpYerE gives access to chiral tertiary alcohols, a group of products difficult to obtain in an enantioenriched form by other means. Hence, knowledge of the three-dimensional structure of the enzyme is crucial to identify structure-activity relationships. However, YpYerE has yet to be crystallized, despite several attempts. Herein, we show that a homologue of YpYerE, namely, PpYerE from Pseudomonas protegens (59 % amino acid identity), displays similar catalytic activity: benzaldehyde and its derivatives as well as ketones are converted into chiral 2-hydroxy ketones by using pyruvate as a donor. To enable comparison of aldehyde- and ketone-accepting enzymes and to guide site-directed mutagenesis studies, PpYerE was crystallized and its structure was determined to a resolution of 1.55 Å.

Genetically Encoded Förster Resonance Energy Transfer-Based Biosensors Studied on the Single-Molecule Level.

Genetically encoded Förster resonance energy transfer (FRET)-based biosensors for the quantification of ligand molecules change the magnitude of FRET between two fluorescent proteins upon binding a target metabolite. When highly sensitive sensors are being designed, extensive sensor optimization is essential. However, it is often difficult to verify the ideas of modifications made to a sensor during the sensor optimization process because of the limited information content of ensemble FRET measurements. In contrast, single-molecule detection provides detailed information and higher accuracy. Here, we investigated a set of glucose and crowding sensors on the single-molecule level. We report the first comprehensive single-molecule study of FRET-based biosensors with reasonable counting statistics and identify characteristics in the single-molecule FRET histograms that constitute fingerprints of sensor performance. Hence, our single-molecule approach extends the toolbox of methods aiming to understand and optimize the design of FRET-based biosensors.

A Synthetic Reaction Cascade Implemented by Colocalization of Two Proteins within Catalytically Active Inclusion Bodies.

In nature, enzymatic reaction cascades, i.e., realized in metabolic networks, operate with unprecedented efficacy, with the reactions often being spatially and temporally orchestrated. The principle of "learning from nature" has in recent years inspired the setup of synthetic reaction cascades combining biocatalytic reaction steps to artificial cascades. Hereby, the spatial organization of multiple enzymes, e.g., by coimmobilization, remains a challenging task, as currently no generic principles are available that work for every enzyme. We here present a tunable, genetically programmed coimmobilization strategy that relies on the fusion of a coiled-coil domain as aggregation inducing-tag, resulting in the formation of catalytically active inclusion body coimmobilizates (Co-CatIBs). Coexpression and coimmobilization was proven using two fluorescent proteins, and the strategy was subsequently extended to two enzymes, which enabled the realization of an integrated enzymatic two-step cascade for the production of (1 R,2 R)-1-phenylpropane-1,2-diol (PPD), a precursor of the calicum channel blocker diltiazem. In particular, the easy production and preparation of Co-CatIBs, readily yielding a biologically produced enzyme immobilizate renders the here presented strategy an interesting alternative to existing cascade immobilization techniques.

Catalytically active inclusion bodies of L-lysine decarboxylase from E. coli for 1,5-diaminopentane production.

Sustainable and eco-efficient alternatives for the production of platform chemicals, fuels and chemical building blocks require the development of stable, reusable and recyclable biocatalysts. Here we present a novel concept for the biocatalytic production of 1,5-diaminopentane (DAP, trivial name: cadaverine) using catalytically active inclusion bodies (CatIBs) of the constitutive L-lysine decarboxylase from E. coli (EcLDCc-CatIBs) to process L-lysine-containing culture supernatants from Corynebacterium glutamicum. EcLDCc-CatIBs can easily be produced in E. coli followed by a simple purification protocol yielding up to 43% dry CatIBs per dry cell weight. The stability and recyclability of EcLDCc-CatIBs was demonstrated in (repetitive) batch experiments starting from L-lysine concentrations of 0.1 M and 1 M. EcLDC-CatIBs exhibited great stability under reaction conditions with an estimated half-life of about 54 h. High conversions to DAP of 87-100% were obtained in 30-60 ml batch reactions using approx. 180-300 mg EcLDCc-CatIBs, respectively. This resulted in DAP titres of up to 88.4 g l-1 and space-time yields of up to 660 gDAP l-1 d-1 per gram dry EcLDCc-CatIBs. The new process for DAP production can therefore compete with the currently best fermentative process as described in the literature.

Advantages of Hydrogel-Based 3D-Printed Enzyme Reactors and Their Limitations for Biocatalysis.

The development of process steps catalyzed by immobilized enzymes usually encompasses the screening of enzyme variants, as well as the optimization of immobilization protocols and process parameters. Direct immobilization of biocatalysts by physical entrapment into hydrogels can be applied to reduce the effort required for immobilization, as the enzyme-specific optimization of the immobilization procedure is omitted. Physical entrapment is applicable for purified enzymes as well as crude cell extracts. Therefore, it can be used to quickly assess and compare activities of immobilized enzymes. For the application in flow reactors, we developed 3D-printed hydrogel lattices for enzyme entrapment as well as matching housings, also manufactured by 3D-printing. Testing the resulting enzyme reactors for three different enzymes, namely alcohol dehydrogenase from Lactobacillus brevis, benzoylformate decarboxylase from Pseudomonas putida and ß-galactosidase from Aspergillus oryzae, and four different enzymatic reactions showed the broad applicability of the approach but also its limitations. The activity of the immobilized biocatalysts was measured in batch experiments and compared to the kinetics of the respective free enzymes in solution. This comparison yields an effectiveness factor, which is a key figure to describe the extent the immobilized catalyst is effectively utilized. For the examined systems the effectiveness factor ranged between 6 and 14% and decreased with increasing absolute activity of the entrapped enzymes due to mass transfer limitations. To test the suitability of the hydrogel lattices for continuous operation, they were inserted into 3D-printed reactor housings and operated at constant flow. Stable product formation could be monitored over a period of 72 h for all four enzymatic systems, including two reactions with redox cofactor regeneration. Comparing calculated and experimental conversion in the continuous setup, higher values of the effectiveness factor in batch experiments also hint at good performance in continuous flow. This can be used to optimize complex biocatalytic reactions on a small scale.

(R,R)-Butane-2,3-diol dehydrogenase from Bacillus clausii DSM 8716T: Cloning and expression of the bdhA-gene, and initial characterization of enzyme.

The gene encoding a putative (R,R)-butane-2,3-diol dehydrogenase (bdhA) from Bacillus clausii DSM 8716T was isolated, sequenced and expressed in Escherichia coli. The amino acid sequence of the encoded protein is only distantly related to previously studied enzymes (identity 33-43%) and exhibited some uncharted peculiarities. An N-terminally StrepII-tagged enzyme variant was purified and initially characterized. The isolated enzyme catalyzed the (R)-specific oxidation of (R,R)- and meso-butane-2,3-diol to (R)- and (S)-acetoin with specific activities of 12U/mg and 23U/mg, respectively. Likewise, racemic acetoin was reduced with a specific activity of up to 115U/mg yielding a mixture of (R,R)- and meso-butane-2,3-diol, while the enzyme reduced butane-2,3-dione (Vmax 74U/mg) solely to (R,R)-butane-2,3-diol via (R)-acetoin. For these reactions only activity with the co-substrates NADH/NAD+ was observed. The enzyme accepted a selection of vicinal diketones, α-hydroxy ketones and vicinal diols as alternative substrates. Although the physiological function of the enzyme in B. clausii remains elusive, the data presented herein clearly demonstrates that the encoded enzyme is a genuine (R,R)-butane-2,3-diol dehydrogenase with potential for applications in biocatalysis and sensor development.

Phenylalanine ammonia lyase from Arabidopsis thaliana (AtPAL2): A potent MIO-enzyme for the synthesis of non-canonical aromatic alpha-amino acids.: Part II: Application in different reactor concepts for the production of (S)-2-chloro-phenylalanine.

Phenylalanine ammonia lyase (PAL) from Arabidopsis thaliana (AtPAL2) is in general a very good catalyst for the amination of fluoro- and chloro-cinnamic acid derivatives yielding halogenated (S)-phenylalanine derivatives with ≥85% conversion and excellent ee values >99%. We have studied the application of this enzyme as whole cell biocatalyst and immobilized on the cellulose carrier Avicel® for the production of the hypertension drug precursor (S)-2-chloro-phenylalanine using batch, fed-batch, as well as continuous membrane reactor and plug-flow reactor. For immobilization, a C-terminal fusion of the enzyme with a carbohydrate binding module (CBM) was produced, which selectively binds to Avicel® directly from crude cell extracts, thus enabling a fast and cheap immobilization, stabilization and recycling of the enzyme. 1g Avicel was loaded with 10mg enzyme. Best results were obtained with whole cells using the continuous membrane reactor (47gproduct/gDryCellWeight) and using the immobilized enzyme in a repetitive fed-batch (274gproduct/gimmobilized enzyme) or in a continuous plug-flow reactor (288gproduct/gimmobilize enzyme). Therewith the productivity of AtPAL2 outperforms the established fed-batch process at DSM using PAL from Rhodotorula glutinis in E. coli as whole cell biocatalyst with a productivity of 0.14gproduct/gWetCellWeight (ca. 0.7gproduct/gDryCellWeight) (de Lange et al., 2011; doi:10.1002/cctc.201000435).

Catalytically-active inclusion bodies-Carrier-free protein immobilizates for application in biotechnology and biomedicine.

Bacterial inclusion bodies (IBs) consist of unfolded protein aggregates and represent inactive waste products often accumulating during heterologous overexpression of recombinant genes in Escherichia coli. This general misconception has been challenged in recent years by the discovery that IBs, apart from misfolded polypeptides, can also contain substantial amounts of active and thus correctly or native-like folded protein. The corresponding catalytically-active inclusion bodies (CatIBs) can be regarded as a biologically-active sub-micrometer sized biomaterial or naturally-produced carrier-free protein immobilizate. Fusion of polypeptide (protein) tags can induce CatIB formation paving the way towards the wider application of CatIBs in synthetic chemistry, biocatalysis and biomedicine. In the present review we summarize the history of CatIBs, present the molecular-biological tools that are available to induce CatIB formation, and highlight potential lines of application. In the second part findings regarding the formation, architecture, and structure of (Cat)IBs are summarized. Finally, an overview is presented about the available bioinformatic tools that potentially allow for the prediction of aggregation and thus (Cat)IB formation. This review aims at demonstrating the potential of CatIBs for biotechnology and hopefully contributes to a wider acceptance of this promising, yet not widely utilized, protein preparation.

Phenylalanine ammonia lyase from Arabidopsis thaliana (AtPAL2): A potent MIO-enzyme for the synthesis of non-canonical aromatic alpha-amino acids: Part I: Comparative characterization to the enzymes from Petroselinum crispum (PcPAL1) and Rhodosporidium toruloides (RtPAL).

Phenylalanine ammonia lyase (PAL) from Arabidopsis thaliana (AtPAL2) was comparatively characterized to the well-studied enzyme from parsley (PcPAL1) and Rhodosporidium toruloides (RtPAL) with respect to kinetic parameters for the deamination and the amination reaction, pH- and temperature optima and the substrate range of the amination reaction. Whereas both plant enzymes are specific for phenylalanine, the bifunctional enzyme from Rhodosporidium toruloides shows KM-values for L-Phe and L-Tyr in the same order of magnitude and, compared to both plant enzymes, a 10-15-fold higher activity. At 30°C all enzymes were sufficiently stable with half-lives of 3.4days (PcPAL1), 4.6days (AtPAL2) and 9.7days (RtPAL/TAL). Very good results for the amination of various trans-cinnamic acid derivatives were obtained using E. coli cells as whole cell biocatalysts in ammonium carbonate buffer. Investigation of the substrate ranges gave interesting results for the newly tested enzymes from A. thaliana and R. toruloides. Only the latter accepts besides 4-hydroxy-CA also 3-methoxy-4-hydroxy-CA as a substrate, which is an interesting intermediate for the formation of pharmaceutically relevant L-Dopa. AtPAL2 is a very good catalyst for the formation of (S)-3-F-Phe, (S)-4-F-Phe and (S)-2-Cl-Phe. Such non-canonical amino acids are valuable building blocks for the formation of various drug molecules.

HaloTag™: Evaluation of a covalent one-step immobilization for biocatalysis.

Easy, fast and gentle immobilization for the efficient reuse of important biocatalysts is highly demanded. We used the commercially available HaloTag™ technology (Promega), so far relatively unknown in the context of biocatalysis, to immobilize the benzaldehyde lyase from P. fluorescence (PfBAL). Immobilization mediated by this fusion tag proceeds rapidly within minutes from crude extracts yielding covalently attached enzymes in high purity, making expensive and laborious previous chromatographic purification steps obsolete, which strongly reduces the costs for biocatalyst immobilization. Further, we introduce a novel design of HaloTag fusions and demonstrate the positive effect of the tag on soluble expression and activity of PfBAL. The immobilized biocatalyst was stable at 4°C for months and was successfully reused in several repetitive batches for the carboligation of aggressive aldehydes.