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Baeyer-Villiger monooxygenases (BVMOs) are important flavin-dependent enzymes which perform oxygen insertion reactions leading to valuable products. As reported in many studies, BVMOs are usually unstable during application, preventing a wider usage in biocatalysis. Here, we discovered a novel NADPH-dependent BVMO which originates from Halopolyspora algeriensis using sequence similarity networks (SSNs). The enzyme is stable at temperatures between 10 °C to 30 °C up to five days after the purification, and yields the normal ester product. In this study, the substrate scope was investigated for a broad range of aliphatic ketones and the enzyme was biochemically characterized to identify optimum reaction conditions. The best substrate (86 % conversion) was 2-dodecanone using purified enzyme. This novel BVMO could potentially be applied as part of an enzymatic cascade or in bioprocesses which utilize aliphatic alkanes as feedstock.
Combining solid acid catalysts with enzyme reactions in aqueous environments is challenging because either very acidic conditions inactivate the enzymes, or the solid acid catalyst is neutralized. In this study, Amberlyst-15 encapsulated in polydimethylsiloxane (Amb-15@PDMS) is used to deprotect the lignin depolymerization product G−C2 dioxolane phenol in a buffered system at pH 6.0. This reaction is directly coupled with the biocatalytic reduction of the released homovanillin to homovanillyl alcohol by recombinant horse liver alcohol dehydrogenase, which is subsequently acylated by the promiscuous acyltransferase/hydrolase PestE_I208A_L209F_N288A in a one-pot system. The deprotection catalyzed with Amb-15@PDMS attains up to 97 % conversion. Overall, this cascade enables conversions of up to 57 %.
Amine transaminases (ATAs) are pyridoxal-5′-phosphate (PLP)-dependent enzymes that catalyze the transfer of an amino group from an amino donor to an aldehyde and/or ketone. In the past decade, the enzymatic reductive amination of prochiral ketones catalyzed by ATAs has attracted the attention of researchers, and more traditional chemical routes were replaced by enzymatic ones in industrial manufacturing. In the present work, the influence of the presence of an α,β-unsaturated system in a methylketone model substrate was investigated, using a set of five wild-type ATAs, the (R)-selective from Aspergillus terreus (Atr-TA) and Mycobacterium vanbaalenii (Mva-TA), the (S)-selective from Chromobacterium violaceum (Cvi-TA), Ruegeria pomeroyi (Rpo-TA), V. fluvialis (Vfl-TA) and an engineered variant of V. fluvialis (ATA-256 from Codexis). The high conversion rate (80 to 99%) and optical purity (78 to 99% ee) of both (R)- and (S)-ATAs for the substrate 1-phenyl-3-butanone, using isopropylamine (IPA) as an amino donor, were observed. However, the double bond in the α,β-position of 4-phenylbut-3-en-2-one dramatically reduced wild-type ATA reactivity, leading to conversions of <10% (without affecting the enantioselectivity). In contrast, the commercially engineered V. fluvialis variant, ATA-256, still enabled an 87% conversion, yielding a corresponding amine with >99% ee. Computational docking simulations showed the differences in orientation and intermolecular interactions in the active sites, providing insights to rationalize the observed experimental results.
Abstract
Olive mill wastewater (OMWW) is produced annually during olive oil extraction and contains most of the health‐promoting 3‐hydroxytyrosol of the olive fruit. To facilitate its recovery, enzymatic transesterification of hydroxytyrosol (HT) was directly performed in an aqueous system in the presence of ethyl acetate, yielding a 3‐hydroxytyrosol acetate rich extract. For this, the promiscuous acyltransferase from Pyrobaculum calidifontis VA1 (PestE) was engineered by rational design. The best mutant for the acetylation of hydroxytyrosol (PestE_I208A_L209F_N288A) was immobilized on EziG2 beads, resulting in hydroxytyrosol conversions between 82 and 89 % in one hour, for at least ten reaction cycles in a buffered hydroxytyrosol solution. Due to inhibition by other phenols in OMWW the conversions of hydroxytyrosol from this source were between 51 and 62 %. In a preparative scale reaction, 13.8 mg (57 %) of 3‐hydroxytyrosol acetate was extracted from 60 mL OMWW.
First Aid Kits are collections of the most important medical equipment required for quick medical assistance. Similarly, enzyme kits can provide a proficient, ready‐ and easy‐to‐use collection of biocatalysts that can be applied with high reproducibility. In this article, we illustrate how kits of oxyfunctionalisation enzymes could operate as synthetic ‘First Aid’ for chemists working on complex natural product total synthesis in an early‐ or late‐stage fashion, as well as in lead diversification in drug discovery processes. We reason that enzyme kits could catalyse the integration of biocatalysis into (synthetic) organic chemistry and describe how we envision their future application.
p-Coumaric acid (p-CA) is a key precursor for the biosynthesis of flavonoids. Tyrosine ammonia lyases (TALs) specifically catalyze the synthesis of p-CA from l-tyrosine, which is a convenient enzymatic pathway. To explore novel and highly active TALs, a phylogenetic tree-building approach was conducted including 875 putative TALs and 46 putative phenylalanine/tyrosine ammonia lyases (PTALs). Among them, 5 TALs and 3 PTALs were successfully characterized and found to exhibit the proposed enzymatic activity. The TAL from Chryseobacterium luteum sp. nov (TALclu) has the highest affinity (Km=0.019 mm) and conversion efficiency (kcat/Km=1631 s−1 ⋅ mm−1) towards l-tyrosine. The reaction conditions for two purified enzymes and their E. coli recombinant cells were optimized and p-CA yields of 2.03 g/L after 8 hours by TALclu and 2.35 g/L after 24 h by TAL from Rivularia sp. PCC 7116 (TALrpc) in whole cells were achieved. These TALs are thus candidates for the construction of whole-cell systems to produce the flavonoid precursor p-CA.
Abstract
The aldehyde tag is appropriate to selectively label proteins, prepare antibody‐drug conjugates or to immobilize enzymes or antibodies for biotechnological and medical applications. The cysteine within the consensus sequence CxPxR of the aldehyde tag is specifically oxidized by the formylglycine‐generating enzyme (FGE) to the non‐canonical and electrophilic amino acid Cα‐formylglycine (FGly). Subsequent reductive amination is a common method for site‐directed immobilization, which usually results in poor immobilization efficiency due to the reaction conditions. Here, we introduce a new solid support like agarose modified with an aryl substituted pyrazolone (Knoevenagel reagent) that was obtained in a facile and efficient 2‐step synthesis. The modified agarose allowed the site‐selective and efficient immobilization of aldehyde‐containing small molecules, peptides and proteins – in particular enzymes – at physiological pH (6.2–8.2) without any additive or catalyst needed. In comparison to reductive amination, higher loadings and activities were achieved in various buffers at different concentrations and temperatures.
Abstract
First Aid Kits are collections of the most important medical equipment required for quick medical assistance. Similarly, enzyme kits can provide a proficient, ready‐ and easy‐to‐use collection of biocatalysts that can be applied with high reproducibility. In this article, we illustrate how kits of oxyfunctionalisation enzymes could operate as synthetic ‘First Aid’ for chemists working on complex natural product total synthesis in an early‐ or late‐stage fashion, as well as in lead diversification in drug discovery processes. We reason that enzyme kits could catalyse the integration of biocatalysis into (synthetic) organic chemistry and describe how we envision their future application.
Abstract
Chiral and enantiopure amines can be produced by enantioselective transaminases via kinetic resolution of amine racemates. This transamination reaction requires stoichiometric amounts of co‐substrate. A dual‐enzyme recycling system overcomes this limitation: l‐amino acid oxidases (LAAO) recycle the accumulating co‐product of (S)‐selective transaminases in the kinetic resolution of racemic amines to produce pure (R)‐amines. However, availability of suitable LAAOs is limited. Here we use the heterologously produced, highly active fungal hcLAAO4 with broad substrate spectrum. H2O2 as byproduct of hcLAAO4 is detoxified by a catalase. The final system allows using sub‐stoichiometric amounts of 1 mol% of the transaminase co‐substrate as well as the initial application of l‐amino acids instead of α‐keto acids. With an optimized protocol, the synthetic potential of this kinetic resolution cascade was proven at the preparative scale (>90 mg) by the synthesis of highly enantiomerically pure (R)‐methylbenzylamine (>99 %ee) at complete conversion (50 %).
Abstract
Promiscuous acyltransferase activity is the ability of certain hydrolases to preferentially catalyze acyl transfer over hydrolysis, even in bulk water. However, poor enantioselectivity, low transfer efficiency, significant product hydrolysis, and limited substrate scope represent considerable drawbacks for their application. By activity‐based screening of several hydrolases, we identified the family VIII carboxylesterase, EstCE1, as an unprecedentedly efficient acyltransferase. EstCE1 catalyzes the irreversible amidation and carbamoylation of amines in water, which enabled the synthesis of the drug moclobemide from methyl 4‐chlorobenzoate and 4‐(2‐aminoethyl)morpholine (ca. 20 % conversion). We solved the crystal structure of EstCE1 and detailed structure–function analysis revealed a three‐amino acid motif important for promiscuous acyltransferase activity. Introducing this motif into an esterase without acetyltransferase activity transformed a “hydrolase” into an “acyltransferase”.
Abstract
Certain hydrolases preferentially catalyze acyl transfer over hydrolysis in an aqueous environment. However, the molecular and structural reasons for this phenomenon are still unclear. Herein, we provide evidence that acyltransferase activity in esterases highly correlates with the hydrophobicity of the substrate‐binding pocket. A hydrophobicity scoring system developed in this work allows accurate prediction of promiscuous acyltransferase activity solely from the amino acid sequence of the cap domain. This concept was experimentally verified by systematic investigation of several homologous esterases, leading to the discovery of five novel promiscuous acyltransferases. We also developed a simple yet versatile colorimetric assay for rapid characterization of novel acyltransferases. This study demonstrates that promiscuous acyltransferase activity is not as rare as previously thought and provides access to a vast number of novel acyltransferases with diverse substrate specificity and potential applications.
Abstract
Biocatalysis has found numerous applications in various fields as an alternative to chemical catalysis. The use of enzymes in organic synthesis, especially to make chiral compounds for pharmaceuticals as well for the flavors and fragrance industry, are the most prominent examples. In addition, biocatalysts are used on a large scale to make specialty and even bulk chemicals. This review intends to give illustrative examples in this field with a special focus on scalable chemical production using enzymes. It also discusses the opportunities and limitations of enzymatic syntheses using distinct examples and provides an outlook on emerging enzyme classes.
Abstract
Environmentally‐friendly processes for the manufacturing of valuable industrial compounds like ω‐hydroxy fatty acids (ω‐OHFAs) are highly desirable. Herein, we present such an approach by establishing a two‐step enzymatic cascade reaction for the production of 2,15,16‐trihydroxy hexadecanoic acid (THA). Starting with the easily accessible natural compound ustilagic acid (UA) that is secreted by the corn smut fungus Ustilago maydis, the recombinantly expressed esterase BS2 from Bacillus subtilis and the commercial β‐glucosidase from almonds were applied yielding 86 % product. Both hydrolases do not require expensive cofactors, making the process economically attractive. Additionally, no harmful solvents are required, so that the product THA can be labelled natural to be used in food and cosmetic products.
Abstract
A device for the transaminase‐catalysed synthesis combined with continuous recovery of chiral amines was designed. The system enabled the separation of the reaction components in three liquid phases: a reaction phase, an organic solvent phase (where the poorly water soluble ketone substrate was supplied), and an aqueous extraction phase for continuous product recovery. The transaminase‐mediated asymmetric synthesis of (S)‐1‐methyl‐3‐phenylpropylamine was employed as model reaction. Factors influencing the performance of the system, such as reactor geometry, working volumes and operating parameters, were investigated. Specifically, reaction yield and product recovery were enhanced by i) reducing the thickness of the reaction phase, while continuously stirring and ii) reducing the volume of the extraction phase. Under the optimal condition tested, 85 % of the product formed was extracted and a product concentration value of 9 g/L was reached. However, co‐extraction of the unreacted amine donor (17 %) was observed. Advantages and drawbacks of this process compared to existing technologies, as well as possible optimization strategies are discussed.
Abstract
Methylation of free hydroxyl groups is an important modification for flavonoids. It not only greatly increases absorption and oral bioavailability of flavonoids, but also brings new biological activities. Flavonoid methylation is usually achieved by a specific group of plant O‐methyltransferases (OMTs) which typically exhibit high substrate specificity. Here we investigated the effect of several residues in the binding pocket of the Clarkia breweri isoeugenol OMT on the substrate scope and regioselectivity against flavonoids. The mutation T133M, identified as reported in our previous publication, increased the activity of the enzyme against several flavonoids, namely eriodictyol, naringenin, luteolin, quercetin and even the isoflavonoid genistein, while a reduced set of amino acids at positions 322 and 326 affected both, the activity and the regioselectivity of the methyltranferase. On the basis of this work, methylated flavonoids that are rare in nature were produced in high purity.
In this work, the regioselectivity of different Baeyer-Villiger monooxygenases (BVMOs) for the conversion of selected substrates was reversed or improved by protein engineering. These studies highlight the importance of substrate positioning for the regioselectivity and that the position of the substrate can be efficiently influenced by introducing proper mutations. It was shown that the beneficial mutations for all BVMOs were partly in corresponding positions. Additionally, the sulfoxidation activity and the stability of BVMOs were targeted and improved by applying protein engineering.
Chiral amines represent high-value fine chemicals serving as key intermediate products in pharmaceutical, chemical and agrochemical industries. In the past decades, application of amine transaminases (ATAs) for stereoselective amination of prochiral ketones emerged to an environmentally benign and economically attractive alternative to transition metal-catalyzed asymmetric synthesis to afford optically pure amines at industrial scale. However, the restricted substrate scope of wild-type transaminases prohibited the conversion of particularly sterically demanding substrates, making protein engineering indispensable. The following thesis covers elaboration of a novel assay for transaminases (Article I) and identification and development of transaminase variants in order to achieve biocatalytic preparation of a set of pharmaceutically relevant model amines, ideally in optically pure form for both stereoisomers, preferentially using asymmetric synthesis and most preferably using isopropylamine as cost-efficient amine donor co-substrate (Article II-IV). The aforementioned target amines and the corresponding precursor ketones (see Scheme 4.1) were conceived and provided by the company F. Hoffmann-La Roche to attain suitable biocatalysts for a variety of potential intermediates for active pharmaceutical ingredients. Protein engineering of the transaminase scaffolds investigated in this thesis comprised: Initial screening for suitable starting enzyme scaffolds, structure-guided rational design of these scaffolds to enable bulky planar substrate acceptance, elaboration of a sequence motif, verification of the motif and preparative-scale asymmetric synthesis reactions (Article II). For non-planar and structurally different target substrates, namely spatially bulky or bi-cyclic bridged substrates, the transaminase variants were specifically refined and a different evolutionary route had to be pursued (Article III and Article IV). These results (Article II) represent not only the first successful endeavor to engineer a PLP-fold type I amine transaminase (commonly denoted as (S)-selective) for the conversion of highly sterically demanding substrates, but also generally expanded the scope of available fold type I amine transaminases by enzymes having a novel and exceptionally broad substrate spectrum. Aside from structure-guided rational protein engineering, as well non-rational methods, such as site-specific saturation mutagenesis or directed evolution, were applied for protein-engineering. In order to do so for all of the target compounds, a novel high-throughput solid phase activity assay for transaminases that was actually developed during the master thesis, was refined and published (Article I). In the context of this thesis, the same assay principle was as well adapted for quantification of specific activities in liquid phase (Article III). A comparison of different methodologies for developing agar plate assays and a detailed step by step protocol of our transaminase assay are illustrated in a book chapter.
Die Ziele der vorliegenden Arbeit ergaben sich aus zwei Arbeitsschwerpunkten - dem Nachweis einer neuartigen prokaryotischen Phenoloxidase bei dem Bakterienisolat Azotobacter chroococcum SBUG 1484 und der Durchführung Phenoloxidase-katalysierter Biotransformationsreaktionen zur Derivatisierung von ortho- bzw. para-dihydroxylierten Verbindungen. Der zunächst unbekannte, eine neue Phenoloxidase bildende, Bakterienstamm sollte mittels morphologischer und physiologischer Tests sowie 16S-rDNA-Analysen einer Art zugeordnet werden. Da die Expression der Phenoloxidase nur unter bestimmten Bedingungen auftrat sollten die in Abhängigkeit von verschiedenen Kultivierungsparametern zahlreich auftretenden Zelldifferenzierungsprozesse des Stammes untersucht und eine standardisierte Kultivierungsmethode zur Erzielung hoher Phenoloxidase-Aktivitäten entwickelt werden. Die Untersuchung wesentlicher Eigenschaften der neubeschriebenen Phenoloxidase war für eine Zuordnung in die Gruppe der Multikupfer-Oxidasen und eine Prüfung der Eignung des Enzyms für biotechnologische Anwendungen eine unbedingte Voraussetzung. In Phenoloxidase-katalysierten Reaktionen sollte die Aminierung von einfach alkylsubstituierten Brenzkatechinen und Hydrochinonen sowie mehrfach-substituierten ein- bzw. zweikernigen dihydroxylierten Aromaten mit aliphatischen sowie alicyclischen Amindonoren untersucht werden. Im Mittelpunkt der Betrachtungen standen dabei die Aufklärung von Reaktionsmechanismen bei homo- und heteromolekularen Kopplungsreaktionen sowie die Prüfung des Einflusses verschiedener Reaktionsparameter (u.a. Hydroxylierungspositionen der Enzymsubstrate, Substituenten, Eduktkonzentrationen, Katalysatoren, pH-Werte der Reaktionssysteme, Lösungsmittel) auf die Nebenreaktionen und Ausbeuten der anvisierten Zielverbindungen (sekundäre Amine). Eine strukturchemische Analyse der Syntheseprodukte war dazu unerlässlich.
Tertiary alcohols have become interesting targets for organic synthesis themselves or as building blocks for valuable pharmaceutical compounds. However, the synthesis of optically pure tertiary alcohols is still a challenge both chemical and enzymatic means. Enzymes containing the GGG(A)X motif in the active site region have been known to show activity towards these sterically demanding substrates. Several tertiary alcohols have been resolved with high enantioselectivity by using this biocatalytic synthetic route. This thesis aims at providing a better understanding of enantiorecognition of GGG(A)X motif hydrolases in the enzymatic synthesis of enantiomerically enriched tertiary alcohols. Kinetic resolution of a wide range of tertiary alcohols using hydrolases provided insights on factors that can influence enantioselectivity of GGG(A)X motif enzymes. Additionally, a newly proposed chemoenzymatic method to synthesize protected alpha,alpha-dialkyl-alpha-hydroxycarboxylic acids has broadened the application of these enzymes to synthesize optically pure tertiary alcohols. Newly found biocatalysts through functional screening, database mining and rational protein design approaches provided a better enzyme platform for optically pure tertiary alcohol resolution.