Performance of cross-linked enzyme crystals of engineered halohydrin dehalogenase HheG in different chemical reactor systems.

Halohydrin dehalogenase HheG is an industrially interesting biocatalyst for the preparation of different ß-substituted alcohols starting from bulky internal epoxides. We previously demonstrated that the immobilization of different HheG variants in the form of cross-linked enzyme crystals (CLECs) yielded stable and reusable enzyme immobilizes with increased resistance regarding temperature, pH, and the presence of organic solvents. Now, to further establish their preparative applicability, HheG D114C CLECs cross-linked with bis-maleimidoethane have been successfully produced on a larger scale using a stirred crystallization approach, and their application in different chemical reactor types (stirred tank reactor, fluidized bed reactor, and packed bed reactor) was systematically studied and compared for the ring opening of cyclohexene oxide with azide. This revealed the highest obtained space-time yield of 23.9 kgproduct gCLEC -1 h-1 Lreactor volume -1 along with the highest achieved product enantiomeric excess [64%] for application in a packed-bed reactor. Additionally, lyophilization of those CLECs yielded a storage-stable HheG preparation that still retained 67% of initial activity (after lyophilization) after 6 months of storage at room temperature.

Application of electrospun chitosan-based nanofibers as immobilization matrix for biomolecules.

Nanofiber meshes from electrospun chitosan, highly modified with biotin and arylazides, are well-suited for application as enzyme immobilization matrices. To test this, catalytically active biomolecules were immobilized onto photocrosslinked nanofibrous nonwovens consisting mainly of biotinylated fungal chitosan and a small amount (10 w%) of poly ethylene oxide. In this study, we show that over 10 µg eugenol oxidase per milligram dry polymer matrix can be loaded on UV-crosslinked chitosan nanofibers. We further demonstrate that bound enzyme activity can be fully retained for over 7 days of storage at ambient conditions in aqueous buffer. Samples loaded at maximum enzyme carrying capacity were tested in a custom-made plug-flow reactor system with online UV-VIS spectroscopy for activity determination. High wettability and durability of the hydrophilic chitosan support matrix enabled continuous oxidation of model substrate vanillyl alcohol into vanillin with constant turnover at flow rates of up to 0.24 L/h for over 6 h. This proves the above hypothesis and enables further application of the fibers as stacked microfluidic membranes, biosensors, or structural starting points for affinity crosslinked enzyme gels. KEY POINTS: • Biotinylated chitosan-based nanofibers retain enzymes via mild affinity interactions • Immobilized eugenol oxidase shows high activity and resists continuous washing • Nanofiber matrix material tolerated high flow rates in a continuous-flow setup.

Biased Borate Esterification during Nucleoside Phosphorylase-Catalyzed Reactions: Apparent Equilibrium Shifts and Kinetic Implications.

Biocatalytic nucleoside (trans-)glycosylations catalyzed by nucleoside phosphorylases have evolved into a practical and convenient approach to the preparation of modified nucleosides, which are important pharmaceuticals for the treatment of various cancers and viral infections. However, the obtained yields in these reactions are generally determined exclusively by the innate thermodynamic properties of the nucleosides involved, hampering the biocatalytic access to many sought-after target nucleosides. We herein report an additional means for reaction engineering of these systems. We show how apparent equilibrium shifts in phosphorolysis and glycosylation reactions can be effected through entropically driven, biased esterification of nucleosides and ribosyl phosphates with inorganic borate. Our multifaceted analysis further describes the kinetic implications of this in situ reactant esterification for a model phosphorylase.

UV-Spectroscopic Detection of (Pyro-)Phosphate with the PUB Module.

Despite the prevalence of ortho- and pyrophosphate in biochemistry, operationally simple and versatile high-throughput methodologies for their quantification are lacking. We herein introduce PUB, a module for phosphate detection by continuous UV-spectroscopic monitoring of 5-bromouridine phosphorolysis. The PUB module uses cheaply available, bench-stable reagents and can be employed for continuous and discontinuous reaction monitoring in biochemical assays to detect (pyro-)phosphate concentrations spanning almost 4 orders of magnitude, as demonstrated with representative use cases.

Alternative Assay Reagents for UV-Spectroscopic Detection of (Pyro-)Phosphate with the PUB Module.

This report advises against the use of 5-iodoridine or 5-ethynyluridine as alternative assay reagents in the PUB module, primarily due to their lack of an isosbestic point of phosphorolysis under moderately alkaline conditions.

G-type Halohydrin Dehalogenases Catalyze Ring Opening Reactions of Cyclic Epoxides with Diverse Anionic Nucleophiles.

Halohydrin dehalogenases are promiscuous biocatalysts, which enable asymmetric ring opening reactions of epoxides with various anionic nucleophiles. However, despite the increasing interest in such asymmetric transformations, the substrate scope of G-type halohydrin dehalogenases toward cyclic epoxides has remained largely unexplored, even though this subfamily is the only one known to display activity with these sterically demanding substrates. Herein, we report on the exploration of the substrate scope of the two G-type halohydrin dehalogenases HheG and HheG2 and a newly identified, more thermostable member of the family, HheG3, with a variety of sterically demanding cyclic epoxides and anionic nucleophiles. This work shows that, in addition to azide and cyanide, these enzymes facilitate ring-opening reactions with cyanate, thiocyanate, formate, and nitrite, significantly expanding the known repertoire of accessible transformations.

CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering*.

CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure-function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.

Database Mining for Novel Bacterial ß-Etherases, Glutathione-Dependent Lignin-Degrading Enzymes.

Lignin is the most abundant aromatic polymer in nature and a promising renewable source for the provision of aromatic platform chemicals and biofuels. ß-Etherases are enzymes with a promising potential for application in lignin depolymerization due to their selectivity in the cleavage of ß-O-4 aryl ether bonds. However, only a very limited number of these enzymes have been described and characterized so far. Using peptide pattern recognition (PPR) as well as phylogenetic analyses, 96 putatively novel ß-etherases have been identified, some even originating from bacteria outside the order Sphingomonadales A set of 13 diverse enzymes was selected for biochemical characterization, and ß-etherase activity was confirmed for all of them. Some enzymes displayed up to 3-fold higher activity than previously known ß-etherases. Moreover, conserved sequence motifs specific for either LigE- or LigF-type enzymes were deduced from multiple-sequence alignments and the PPR-derived peptides. In combination with structural information available for the ß-etherases LigE and LigF, insight into the potential structural and/or functional role of conserved residues within these sequence motifs is provided. Phylogenetic analyses further suggest the presence of additional bacterial enzymes with potential ß-etherase activity outside the classical LigE- and LigF-type enzymes as well as the recently described heterodimeric ß-etherases.IMPORTANCE The use of biomass as a renewable source and replacement for crude oil for the provision of chemicals and fuels is of major importance for current and future societies. Lignin, the most abundant aromatic polymer in nature, holds promise as a renewable starting material for the generation of required aromatic structures. However, a controlled and selective lignin depolymerization to yield desired aromatic structures is a very challenging task. In this regard, bacterial ß-etherases are especially interesting, as they are able to cleave the most abundant bond type in lignin with high selectivity. With this study, we significantly expanded the toolbox of available ß-etherases for application in lignin depolymerization and discovered more active as well as diverse enzymes than previously known. Moreover, the identification of further ß-etherases by sequence database mining in the future will be facilitated considerably through our deduced etherase-specific sequence motifs.

Microbial ß-etherases and glutathione lyases for lignin valorisation in biorefineries: current state and future perspectives.

Lignin is the major aromatic biopolymer in nature, and it is considered a valuable feedstock for the future supply of aromatics. Hence, its valorisation in biorefineries is of high importance, and various chemical and enzymatic approaches for lignin depolymerisation have been reported. Among the enzymes known to act on lignin, ß-etherases offer the possibility for a selective cleavage of the ß-O-4 aryl ether bonds present in lignin. These enzymes, together with glutathione lyases, catalyse a reductive, glutathione-dependent ether bond cleavage displaying high stereospecificity. ß-Etherases and glutathione lyases both belong to the superfamily of glutathione transferases, and several structures have been solved recently. Additionally, different approaches for their application in lignin valorisation have been reported in the last years. This review gives an overview on the current knowledge on ß-etherases and glutathione lyases, their biochemical and structural features, and critically discusses their potential for application in biorefineries.

Multi-step biocatalytic depolymerization of lignin.

Lignin is a biomass-derived aromatic polymer that has been identified as a potential renewable source of aromatic chemicals and other valuable compounds. The valorization of lignin, however, represents a great challenge due to its high inherent functionalization, what compromises the identification of chemical routes for its selective depolymerization. In this work, an in vitro biocatalytic depolymerization process is presented, that was applied to lignin samples obtained from beech wood through OrganoCat pretreatment, resulting in a mixture of lignin-derived aromatic monomers. The reported biocracking route comprises first a laccase-mediator system to specifically oxidize the Cα hydroxyl group in the ß-O-4 structure of lignin. Subsequently, selective ß-O-4 ether cleavage of the oxidized ß-O-4 linkages is achieved with ß-etherases and a glutathione lyase. The combined enzymatic approach yielded an oily fraction of low-molecular-mass aromatic compounds, comprising coniferylaldehyde and other guaiacyl and syringyl units, as well as some larger (soluble) fractions. Upon further optimization, the reported biocatalytic route may open a valuable approach for lignin processing and valorization under mild reaction conditions.

Recent advances on halohydrin dehalogenases-from enzyme identification to novel biocatalytic applications.

Halohydrin dehalogenases are industrially relevant enzymes that catalyze the reversible dehalogenation of vicinal haloalcohols with formation of the corresponding epoxides. In the reverse reaction, also other negatively charged nucleophiles such as azide, cyanide, or nitrite are accepted besides halides to open the epoxide ring. Thus, novel C-N, C-C, or C-O bonds can be formed by halohydrin dehalogenases, which makes them attractive biocatalysts for the production of various ß-substituted alcohols. Despite the fact that only five individual halohydrin dehalogenase enzyme sequences have been known until recently enabling their heterologous production, a large number of different biocatalytic applications have been reported using these enzymes. The recent characterization of specific sequence motifs has facilitated the identification of novel halohydrin dehalogenase sequences available in public databases and has largely increased the number of recombinantly available enzymes. These will help to extend the biocatalytic repertoire of this enzyme family and to foster novel biotechnological applications and developments in the future. This review gives a general overview on the halohydrin dehalogenase enzyme family and their biochemical properties and further focuses on recent developments in halohydrin dehalogenase biocatalysis and protein engineering.

Biochemical and biocatalytic characterization of 17 novel halohydrin dehalogenases.

Halohydrin dehalogenases are rare but catalytically remarkable enzymes since they are able to form novel C-C, C-O, C-N, or C-S bonds. Very recently, a motif-based sequence database mining approach resulted in the identification of 37 novel halohydrin dehalogenase enzymes, many of them exhibiting only low sequence similarity to previously known halohydrin dehalogenases. In an attempt to explore the biocatalytic potential of these newly identified enzymes, 17 representatives from all six phylogenetic subtypes were heterologously produced in Escherichia coli, purified and characterized to determine their substrate scopes in the dehalogenation and epoxide ring-opening reaction. Several enzymes with broad substrate spectra were identified exhibiting high activities towards a selection of typical substrates. Moreover, four halohydrin dehalogenases were found to be significantly more thermostable than the previously known HheC from Agrobacterium radiobacter AD1. Investigation of the enzymes' stereoselectivity in the dehalogenation of racemic 2-chloro-1-phenylethanol revealed that their stereopreference correlates with the phylogenetic placing of the enzymes in subtypes A through G. Furthermore, the biocatalytic potential of these novel halohydrin dehalogenases was investigated in the preparation of ethyl 4-cyano-3-hydroxybutyrate, a statin side-chain precursor. Though none of the active enzymes selectively formed the required (R)-enantiomer, several halohydrin dehalogenases were identified with significantly higher activity in the conversion compared to HheC, making them promising candidates for this industrially relevant reaction.

Enzyme-substrate complex structures of CYP154C5 shed light on its mode of highly selective steroid hydroxylation.

CYP154C5 from Nocardia farcinica is a bacterial cytochrome P450 monooxygenase active on steroid molecules. The enzyme has recently been shown to exhibit exclusive regioselectivity and stereoselectivity in the conversion of various pregnans and androstans, yielding 16α-hydroxylated steroid products. This makes the enzyme an attractive candidate for industrial application in steroid hormone synthesis. Here, crystal structures of CYP154C5 in complex with four different steroid molecules were solved at resolutions of up to 1.9 Å. These are the first reported P450 structures from the CYP154 family in complex with a substrate. The active site of CYP154C5 forms a flattened hydrophobic channel with two opposing polar regions, perfectly resembling the size and polarity distribution of the steroids and thus resulting in highly specific steroid binding with Kd values in the range 10-100 nM. Key enzyme-substrate interactions were identified that accounted for the exclusive regioselectivity and stereoselectivity of the enzyme. Additionally, comparison of the four CYP154C5-steroid structures revealed distinct structural differences, explaining the observed variations in kinetic data obtained for this P450 with the steroids pregnenolone, dehydroepiandrosterone, progesterone, androstenedione, testosterone and nandrolone. This will facilitate the generation of variants with improved activity or altered selectivity in the future by means of protein engineering.

Expanding the Halohydrin Dehalogenase Enzyme Family: Identification of Novel Enzymes by Database Mining.

Halohydrin dehalogenases are very rare enzymes that are naturally involved in the mineralization of halogenated xenobiotics. Due to their catalytic potential and promiscuity, many biocatalytic reactions have been described that have led to several interesting and industrially important applications. Nevertheless, only a few of these enzymes have been made available through recombinant techniques; hence, it is of general interest to expand the repertoire of these enzymes so as to enable novel biocatalytic applications. After the identification of specific sequence motifs, 37 novel enzyme sequences were readily identified in public sequence databases. All enzymes that could be heterologously expressed also catalyzed typical halohydrin dehalogenase reactions. Phylogenetic inference for enzymes of the halohydrin dehalogenase enzyme family confirmed that all enzymes form a distinct monophyletic clade within the short-chain dehydrogenase/reductase superfamily. In addition, the majority of novel enzymes are substantially different from previously known phylogenetic subtypes. Consequently, four additional phylogenetic subtypes were defined, greatly expanding the halohydrin dehalogenase enzyme family. We show that the enormous wealth of environmental and genome sequences present in public databases can be tapped for in silico identification of very rare but biotechnologically important biocatalysts. Our findings help to readily identify halohydrin dehalogenases in ever-growing sequence databases and, as a consequence, make even more members of this interesting enzyme family available to the scientific and industrial community.

Escherichia coli-based expression system for the heterologous expression and purification of the elicitin ß-cinnamomin from Phytophthora cinnamomi.

Elicitins are sterol carrier proteins from the Oomycete genera Phytophthora and Phytium and elicit a hypersensitive response in many economically important plants, in some cases causing a systemic acquired resistance. Their recombinant expression in bacteria is complicated by the presence of three disulfide bonds in the elicitin structure. In consequence, elicitins have so far only been produced in soluble form by isolation from native Phytophthora or Phytium strains or by recombinant expression in the yeast Pichia pastoris. Here, for the first time, we report the soluble expression of the elicitin ß-cinnamomin from Phytophthora cinnamomi in Escherichia coli by secretion of the protein into the periplasm. ß-Cinnamomin yields have been significantly improved after careful selection of the optimum secretion signal sequence. In total, 17.6 mg ß-cinnamomin per liter cell culture have been obtained in shake flasks with the secretion signal sequence of the maltose-binding protein MalE from E. coli. Furthermore, by making use of a C-terminal His-tag, ß-cinnamomin purification has been significantly simplified with only one step of immobilized metal ion affinity chromatography yielding protein of high purity (>90%). The established protocol has further been successfully applied to the soluble expression of another elicitin.

Selective steroid oxyfunctionalisation by CYP154C5, a bacterial cytochrome P450.

BACKGROUND: Cytochrome P450 monooxygenases--able to regio- and stereoselectively hydroxylate non-activated carbon atoms--are important enzymes for the synthesis of valuable intermediates in the production of steroid hormones in the pharmaceutical industry. However, up to now only a few bacterial enzymes able to hydroxylate steroids have been reported. CYP154C5 from Nocardia farcinica IFM 10152, a bacterial P450 monooxygenase, was previously shown to convert testosterone to 16α-hydroxytestosterone. Since the hydroxylation at 16α-position is of special interest for the pharmaceutical industry, we have studied this enzyme in more detail to investigate its activity and selectivity in bioconversions of further steroids. RESULTS: CYP154C5 was coexpressed in Escherichia coli together with putidaredoxin and putidaredoxin reductase from Pseudomonas putida as redox partners for electron transfer and applied in bioconversions of various pregnanes and androstanes [pregnenolone (1), dehydroepiandrosterone (2), progesterone (3), androstenedione (4), testosterone (5) and nandrolone (6)]. Structure elucidation of the formed products revealed an exclusive regio- and stereoselectivity of CYP154C5, always yielding the corresponding 16α-hydroxylated steroids. Application of whole cells expressing the three components, P450, Pdx and PdR, in steroid biotransformations resulted in significantly higher conversions and total turnover numbers (TTN) compared to reactions using cell-free extracts. Additionally, considerably higher substrate loads (up to 15 mM) were tolerated by the whole-cell system. Furthermore, turnover numbers (TON) were determined for the six different steroids using whole cells. Thus, testosterone was found to be the worst substrate with a TON of only 0.8 µmol substrate consumed min-1 µmol(-1) CYP154C5, while progesterone and pregnenolone were converted the fastest resulting in TON of 3.3 µmol substrate consumed min(-1) µmol(-1) CYP154C5. CONCLUSION: CYP154C5 from N. farcinica constitutes a promising catalyst due to its high regio- and stereoselectivity in the hydroxylation of different steroids as well as its efficient expression in E. coli at high yields. Using this enzyme, 16α-hydroxylated steroids, which are important precursors for the synthesis of high value steroidal drugs in the pharmaceutical industry, can be selectively produced on preparative scale with TTN (µmol substrate consumed µmol(-1) CYP154C5) exceeding 2000.

Chemo-enzymatic synthesis of natural products and their analogs.

Enzymes continue to gain recognition as valuable tools in synthetic chemistry as they enable transformations, which elude conventional organochemical approaches. As such, the progressing expansion of the biocatalytic arsenal has introduced unprecedented opportunities for new synthetic strategies and retrosynthetic disconnections. As a result, enzymes have found a solid foothold in modern natural product synthesis for applications ranging from the generation of early chiral synthons to endgame transformations, convergent synthesis, and cascade reactions for the rapid construction of molecular complexity. As a primer to the state-of-the-art concerning strategic uses of enzymes in natural product synthesis and the underlying concepts, this review highlights selected recent literature examples, which make a strong case for the admission of enzymatic methodologies into the standard repertoire for complex small-molecule synthesis.

Characterization of cytochrome P450 monooxygenase CYP154H1 from the thermophilic soil bacterium Thermobifida fusca.

Cytochrome P450 monooxygenases are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. We have cloned the gene for a new cytochrome P450 monooxygenase, named CYP154H1, from the moderately thermophilic soil bacterium Thermobifida fusca. The enzyme was overexpressed in Escherichia coli at up to 14% of total soluble protein and purified to homogeneity in three steps. CYP154H1 activity was reconstituted using putidaredoxin reductase and putidaredoxin from Pseudomonas putida DSM 50198 as surrogate electron transfer partners. In biocatalytic reactions with different aliphatic and aromatic substrates of varying size, the enzyme converted small aromatic and arylaliphatic compounds like ethylbenzene, styrene, and indole. Furthermore, CYP154H1 also accepted different arylaliphatic sulfides as substrates chemoselectively forming the corresponding sulfoxides and sulfones. The enzyme is moderately thermostable with an apparent melting temperature of 67°C and exhibited still 90% of initial activity after incubation at 50°C.

Two-Phase Biocatalysis in Microfluidic Droplets.

This Perspective discusses the literature related to two-phase biocatalysis in microfluidic droplets. Enzymes used as catalysts in biocatalysis are generally less stable in organic media than in their native aqueous environments; however, chemical and pharmaceutical compounds are often insoluble in water. The use of aqueous/organic two-phase media provides a solution to this problem and has therefore become standard practice for multiple biotransformations. In batch, two-phase biocatalysis is limited by mass transport, a limitation that can be overcome with the use of microfluidic systems. Although, two-phase biocatalysis in laminar flow systems has been extensively studied, microfluidic droplets have been primarily used for enzyme screening. In this Perspective, we summarize the limited published work on two-phase biocatalysis in microfluidic droplets and discuss the limitations, challenges, and future perspectives of this technology.

Asymmetric azidohydroxylation of styrene derivatives mediated by a biomimetic styrene monooxygenase enzymatic cascade.

Enantioenriched azido alcohols are precursors for valuable chiral aziridines and 1,2-amino alcohols, however their chiral substituted analogues are difficult to access. We established a cascade for the asymmetric azidohydroxylation of styrene derivatives leading to chiral substituted 1,2-azido alcohols via enzymatic asymmetric epoxidation, followed by regioselective azidolysis, affording the azido alcohols with up to two contiguous stereogenic centers. A newly isolated two-component flavoprotein styrene monooxygenase StyA proved to be highly selective for epoxidation with a nicotinamide coenzyme biomimetic as a practical reductant. Coupled with azide as a nucleophile for regioselective ring opening, this chemo-enzymatic cascade produced highly enantioenriched aromatic α-azido alcohols with up to >99% conversion. A bi-enzymatic counterpart with halohydrin dehalogenase-catalyzed azidolysis afforded the alternative ß-azido alcohol isomers with up to 94% diastereomeric excess. We anticipate our biocatalytic cascade to be a starting point for more practical production of these chiral compounds with two-component flavoprotein monooxygenases.