Selective metal ion affinity binding as a simple and renewable enzyme immobilization was investigated using the same affinity function on various supports. Phenylalanine ammonia-lyase from parsley (PcPAL) and an amine transaminase from Vibrio fluvialis (VfTA) with His-tag were used as model enzymes. Metal ion chelating groups on the surface of six enzyme carriers were created from the surface-alkylamino moieties by treatment with ethylenediaminetetraacetic dianhydride and subsequent complexation with cobalt(II) ions. Three porous polymer beads, two silica-based supports, and a silica-coated magnetic nanoparticle (MNP) were investigated as carriers. The most effective PcPAL biocatalyst forms were tested in kinetic resolution and ammonia addition reactions with substrates containing phenyl and thiophen-2-yl rings. In the selective ammonia addition reaction onto the (hetero)arylacrylates needing a harsh medium of a 6 M ammonia solution, the biocatalysts exhibited excellent stability and led to l-amino acids in high yield and excellent enantiomeric excess. The recharging of the MNP supports was investigated by five subsequent cycles of reactions-after elution with 5% diethylenetriamine and reloading with fresh PcPAL or VfTA-retaining over 80% of the relative activities until the third cycle. The repurposing of the supports was also investigated by changing one enzyme to the other on the MNP-immobilized metal ion affinity chromatography support.

Tyrosine hydroxylase (hTH) catalyzes the conversion of l-tyrosine to l-DOPA, the rate-limiting step in dopamine biosynthesis. As impaired hTH function is implicated in dopamine-related neurodegenerative disorders, conventional therapies relying on l-DOPA supplementation often cause adverse side effects. In this study, we investigate the immobilization of recombinant hTH onto silica nanoparticles (SNPs) as a molecularly engineered platform for enzyme replacement therapy (ERT) via intranasal administration. Recombinant GST-tagged hTH was expressed in E. coli with high yield and purity, while SNPs were synthesized with controlled size distribution and surface functionalization to enable efficient ionic binding. Immobilization efficiencies exceeding 70% were achieved. Dynamic light scattering and ζ-potential measurements confirmed enhanced colloidal stability and a reduction in protein aggregation upon immobilization. Enzymatic assays based on dopachrome formation demonstrated that catalytic activity was preserved post-immobilization. Collectively, these findings highlight SNP-based immobilization as a promising liquid-phase nanobiotechnological approach, offering a stable and non-invasive therapeutic platform for dopamine-related pathologies.

Chiral secondary amine dicarboxylic acid derivatives are key building blocks of several peptidomimetic angiotensin-converting enzyme (ACE2) inhibitors. Reductive amination of alpha-keto acids with alpha-amino esters can yield orthogonally functionalized derivatives facilitating further chemical modifications. Biocatalysis offers a sustainable and selective way to implement this synthetic strategy. Here we report the selection of a ketimine reductase enzyme (RnKIRED) for this transformation from a pool of three separate enzyme classes (opine dehydrogenases, imino acid reductases, and imine reductases). We describe the extended substrate scope of this enzyme among alpha-keto acids and alpha-amino esters and demonstrate the synthetic applicability of our approach in sixteen preparative scale reactions. In addition, we report the unique stereoselective behavior of RnKIRED that allows controlling the configuration on the newly formed stereocenter. Coupling small amino esters to large keto acids yields (S) configuration, while coupling large amino esters with small keto acids results in (R) configuration. The molecular background for this unique stereospecificity is also provided.

Utilizing the electrospinning technique to entrap enzymes within nanofibers (providing nanofibrous enzyme catalysts, NEC) offers promising avenues for the scientific and industrial sectors as well. While numerous instances of poly(vinyl alcohol) (PVA)-based nanofibrous biocatalysts have been documented, a comprehensive investigation to characterize the effect of PVA on catalytic activity is notably lacking. This study systematically investigates the effect of PVA properties, such as molecular weight (24-205 kDa), degree of hydrolysis (DoH, 88% and 98%), and polymer concentration (6-16 wt%), on the structure of PVA nanofibers and on the biocatalytic properties of a lipase from Burkholderia cepacia (BcL) entrapped into PVA nanofibers, ensuring 100% yield of immobilization. The PVA-enzyme interactions were studied using viscometry, scanning electron microscopy, Raman mapping, differential scanning calorimetry, and computational docking simulations. Stereoselective hydrolysis and transesterification were used to evaluate enzyme activity. Results showed that the molecular weight and DoH of PVA have a significant effect on the biocatalytic activity of nanofibrous enzyme catalysts. Remarkably, activity yields (mostly exceeded 100%, with maximal yields achieved using fully hydrolyzed PVA) with a molecular weight of 61 kDa provided a 10-fold increase in the specific enzymatic activity of BcL immobilized in nanofibers (90.6 U x g-1) compared to its native form (9.0 U x g-1).

The binding ability of human serum albumin (HSA) on active pharmaceutical ingredients (APIs) is one of the most important parameters in the early stages of drug discovery. In this study, an immobilized HSA-based tool was developed for the rapid and easy in vitro screening of API binding. The work explored the serious incompleteness in the identification of HSA used for in vitro screening published in the last five years. To mitigate this problem, a comprehensive analysis and immobilization studies were performed on the most used HSA types. Serious differences in the colloidal stability of HSAs and their API binding ability on a selected set of APIs were observed. HSAs were immobilized on magnetic nanoparticles with glutardialdehyde (GDA) or cyclohexyl-diglycidyl ether (CDGE) linkers, which have never been used for HSA immobilization before. The HSA-MNP-CDGE complexes achieved a higher immobilization yield and preserved API binding ability; however, the esterase-like enzymatic activity of HSA reduced significantly.

Fumonisins are sphingolipid-like mycotoxins that cause serious damage by contaminating food and feed. The tricarballylic acid (TCA) units of fumonisin B-1 (FB1; accounting for 70 % of fumonisin contamination) can be removed by fumonisin B-1 esterase (FE, EC 3.1.1.87) providing a biotechnological FB1 detoxification possibility. Here, we report the regioselective cleavage of the TCA ester at C6 in the first step of FB1 hydrolysis and kinetic characterization for two FEs. The low K-M values (4.76-44.3 mu M) are comparable to concentrations of environmental contaminations, and the high catalytic efficiencies are promising for practical applications. The X-ray structure of one of the FEs enabled the understanding of the FB1 hydrolysis at molecular level and revealed an arginine pocket key for substrate binding, and the catalytic role of the glutamate preceding the catalytic serine. Computations showed that this FE is likely capable of detoxifying any fumonisin indicating its potential applicability in food and feed products.

A systematic investigation of the catalyst‐free Strecker reaction is conducted in aqueous buffer, offering an efficient and green alternative that leads to α‐aminonitriles without the need of chromatography. Optimization reveals that low pH and high buffer concentration significantly enhance conversion, with yields up to 97%. Broad substrate scope is demonstrated with various aldehydes, ketones, and amines, leading to key intermediates for natural and unnatural amino acids. Potassium cyanide and acetone cyanohydrin are established, latter as a safer and effective cyanide source, and reactions are conducted also in buffer–methyl tert‐butyl ether mixed solvent further improving the methodology. It is hypothesized that, based on the similarity to cyanohydrin formation, the hydroxynitrile lyases (HNLs) might transform imines to aminonitriles. Adding AtHNL and HbHNL further accelerates the reaction suggesting an undiscovered reactivity of these enzymes.

Enzymatic processes play an increasing role in synthetic organic chemistry which requires the access to a broad and diverse set of enzymes. Metagenome mining is a valuable and efficient way to discover novel enzymes with unique properties for biotechnological applications. Here, we report the discovery and biocatalytic characterization of six novel metagenomic opine dehydrogenases from a hot spring environment (mODHs) (EC 1.5.1.X). These enzymes catalyze the asymmetric reductive amination between an amino acid and a keto acid resulting in opines which have defined biochemical roles and represent promising building blocks for pharmaceutical applications. The newly identified enzymes exhibit unique substrate specificity and higher thermostability compared to known examples. The feature that they preferably utilize negatively charged polar amino acids is so far unprecedented for opine dehydrogenases. We have identified two spatially correlated positions in their active sites that govern this substrate specificity and demonstrated a switch of substrate preference by site-directed mutagenesis. While they still suffer from a relatively narrow substrate scope, their enhanced thermostability and the orthogonality of their substrate preference make them a valuable addition to the toolbox of enzymes for reductive aminations. Importantly, enzymatic reductive aminations with highly polar amines are very rare in the literature. Thus, the preparative-scale enzymatic production, purification, and characterization of three highly functionalized chiral secondary amines lend a special significance to our work in filling this gap. Key points • Six new opine dehydrogenases have been discovered from a hot spring metagenome • The newly identified enzymes display a unique substrate scope • Substrate specificity is governed by two correlated active-site residues

Human tyrosine hydroxylase (hTH) has key role in the production of catecholamine neurotransmitters. The structure, function and regulation of hTH has been extensively researched area and the possibility of enzyme replacement therapy (ERT) involving hTH through nanocarriers has been raised as well. However, our understanding on how hTH may interact with nanocarriers is still lacking. In this work, we attempted to investigate the immobilization of hTH on magnetic nanoparticles (MNPs) with various surface linkers in quantitative and mechanistic detail. Our results showed that the activity of hTH was retained after immobilization via secondary and covalent interactions as well. The colloidal stability of hTH could be also enhanced proved by Dynamic light scattering and Zeta potential analysis and a homogenous enzyme layer could be achieved, which was investigated by Raman mapping. The covalent attachment of hTH on MNPs via aldehyde or epoxy linkers provide irreversible immobilization and 38.1 % and 16.5 % recovery (ER). The hTH-MNPs catalyst had 25 % ER in average in simulated nasal electrolyte solution (SNES). This outcome highlights the relevance of immobilization applying MNPs as a potential formulation tool of sensitive therapeutic enzymes offering new opportunities for ERT related to neurodegenerative disorders.

Opines and opine-type chemicals are valuable natural products with diverse biochemical roles, and potential synthetic building blocks of bioactive compounds. Their synthesis involves reductive amination of ketoacids with amino acids. This transformation has high synthetic potential in producing enantiopure secondary amines. Nature has evolved opine dehydrogenases for this chemistry. To date, only one enzyme has been used as biocatalyst, however, analysis of the available sequence space suggests more enzymes to be exploited in synthetic organic chemistry. This review summarizes the current knowledge of this underexplored enzyme class, highlights key molecular, structural, and catalytic features with the aim to provide a comprehensive general description of opine dehydrogenases, thereby supporting future enzyme discovery and protein engineering studies.

Biotechnology and BioengineeringVolume 120, Issue 10 p. 2771-2775 ISSUE INFORMATIONFree Access Biotechnology and Bioengineering: Volume 120, Number 10, October 2023 First published: 14 September 2023 https://doi.org/10.1002/bit.28143AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Volume120, Issue10October 2023Pages 2771-2775 RelatedInformation

The increasing application of recombinant enzymes demands not only effective and sustainable fermentation, but also highly efficient downstream processing and further stabilization of the enzymes by immobilization. In this study, a novel approach for the isolation and immobilization of His‐tagged transaminase from Chromobacterium violaceum ( Cv TA) has been developed. A recombinant of Cv TA was simultaneously isolated and immobilized by binding on silica nanoparticles (SNPs) with metal affinity linkers and additionally within poly(lactic acid) (PLA) nanofibers. The linker length and the nature of the metal ion significantly affected the enzyme binding efficiency and biocatalytic activity of Cv TA‐SNPs. The formation of PLA nanofibers by electrospinning enabled rapid embedding of Cv TA‐SNPs biocatalysts and ensured enhanced stability and activity. The developed advanced immobilization method reduces the time required for enzyme isolation, purification and immobilization by more than fourfold compared to a classical stepwise technique.

Aspartate ammonia-lyases (AALs) catalyze the non-oxidative elimination of ammonia from l-aspartate to give fumarate and ammonia. In this work the AAL coding gene from Pseudomonas fluorescens R124 was identified, isolated, and cloned into the pET-15b expression vector and expressed in E. coli. The purified enzyme (PfAAL) showed optimal activity at pH 8.8, Michaelis-Menten kinetics in the ammonia elimination from l-aspartate, and no strong dependence on divalent metal ions for its activity. The purified PfAAL was covalently immobilized on epoxy-functionalized magnetic nanoparticles (MNP), and effective kinetics of the immobilized PfAAL-MNP was compared to the native solution form. Glycerol addition significantly enhanced the storability of PfAAL-MNP. Inhibiting effect of the growing viscosity (modulated by addition of glycerol or glucose) on the enzymatic activity was observed for the native and immobilized form of PfAAL, as previously described for other free enzymes. The storage stability and recyclability of PfAAL-MNP is promising for further biocatalytic applications.

Immobilized metal ion affinity chromatography principles were applied for selective immobilization of recombinant polyhistidine tag fused phenylalanine ammonia-lyase from parsley (PcPAL) on porous polymeric support with aminoalkyl moieties modified with an EDTA dianhydride (EDTADa)-derived chelator and charged with transition metal ions. Out of the five investigated metal ions - Fe3+, Co2+, Ni2+, Cu2+, Zn2+ - the best biocatalytic activity of PcPAL was achieved when the enzyme was immobilized on the Co2+ ion-charged support (31.8 ± 1.2 U/g). To explore the features this PcPAL obtained by selective immobilization, the thermostability and reusability of this PAL biocatalyst were investigated. To maximize the activity of the immobilized PcPAL the surface functionalization of the aminoalkylated polymeric carrier was fine-tuned with using glycidol as a thinning group beside EDTADa. The maximal activity yield (YA=103 %) was earned when the EDTADa and glycidol were used in 1 to 24 ratio. The reversibility of the immobilization method allowed the development of a support regeneration protocol which enables easy reuse of the functionalized support in case of enzyme inactivation.

This study describes six yeast strains for stereoselective ketone reductions. The reaction conditions for the yeast strains (Pichia carsonii, Lodderomyces elongisporus, Candida norvegica, Candida guillermondi, Debaromyces fabryi and Candida parapsilosis) were optimized in a design of experiments for three ketones of different properties. The pH tolerance, temperature stability and productivity of the bioreductions with lyophilized cells of the yeast strains were characterized. In several cases, the optimized bioreductions resulted in enantiopure alcohols (ee > 99 %) with conversions ranging from moderate to excellent.

In this study, we investigated the influence of different modes of magnetic mixing on effective enzyme activity of aspartate ammonia-lyase from Pseudomonas fluorescens immobilized onto epoxy-functionalized magnetic nanoparticles by covalent binding (AAL-MNP). The effective specific enzyme activity of AAL-MNPs in traditional shake vial method was compared to the specific activity of the MNP-based biocatalyst in two devices designed for magnetic agitation. The first device agitated the AAL-MNPs by moving two permanent magnets at two opposite sides of a vial in x-axis direction (being perpendicular to the y-axis of the vial); the second device unsettled the MNP biocatalyst by rotating the two permanent magnets around the y-axis of the vial. In a traditional shake vial, the substrate and biocatalyst move in the same direction with the same pattern. In magnetic agitation modes, the MNPs responded differently to the external magnetic field of two permanent magnets. In the axial agitation mode, MNPs formed a moving cloud inside the vial, whereas in the rotating agitation mode, they formed a ring. Especially, the rotating agitation of the MNPs generated small fluid flow inside the vial enabling the mixing of the reaction mixture, leading to enhanced effective activity of AAL-MNPs compared to shake vial agitation.

Immobilization of the recombinant, plant-derived Petroselinum crispum phenylalanine ammonia lyase (PcPAL) in electrospun matrices have the potential to create promising, easy-to-use biocatalysts. Polylactic acid (PLA) a biologically inert, commercial biopolymer, was chosen as the material of the carrier system. PLA could be electrospun properly only from water-immiscible organic solvents, which limits its application as a carrier of sensitive biological objects. The emulsion electrospinning is a proper solution to overcome this issue using non-ionic emulsifiers with different hydrophilic-lipophilic balance (HLB) values. The stabilized emulsion could protect the sensitive PcPAL dissolved in the aqueous buffer phase and improve fiber formation, plus help to keep the biocatalytic activity of enzymes. In this study, the first approach is described to produce PLA nanofibers containing PcPAL enzymes by emulsion electrospinning and to use the resulted biocatalyst in the ammonia elimination reaction from l-phenylalanine.

The enzyme family harboring the post-translationally formed 5-methylene-3,5-dihydro-4H-imidazol-4-one (MIO) catalytic residue comprises both aromatic amino acid ammonia-lyases (ALs) and 2,3-aminomu...

A novel phenylalanine ammonia-lyase of the psychrophilic yeast Pseudozyma antarctica (PzaPAL) was identified by screening microbial genomes against known PAL sequences. PzaPAL has a significantly different substrate binding pocket with an extended loop (26 aa long) connected to the aromatic ring binding region of the active site as compared to the known PALs from eukaryotes. The general properties of recombinant PzaPAL expressed in E. coli were characterized including kinetic features of this novel PAL with L-phenylalanine (S) -1a and further racemic substituted phenylalanines rac-1b-g,k. In most cases, PzaPAL revealed significantly higher turnover numbers than the PAL from Petroselinum crispum (PcPAL). Finally, the biocatalytic performance of PzaPAL and PcPAL was compared in the kinetic resolutions of racemic phenylalanine derivatives (rac-1a-s) by enzymatic ammonia elimination and also in the enantiotope selective ammonia addition reactions to cinnamic acid derivatives (2a-s). The enantiotope selectivity of PzaPAL with o-, m-, p-fluoro-, o-, p-chloro-and o-, m-bromo-substituted cinnamic acids proved to be higher than that of PcPAL.