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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.
Poly(vinyl alcohol) (PVA) is a water‐soluble synthetic vinyl polymer with remarkable physical properties including thermostability and viscosity. Its biodegradability, however, is low even though a large amount of PVA is released into the environment. Established physical‐chemical degradation methods for PVA have several disadvantages such as high price, low efficiency, and secondary pollution. Biodegradation of PVA by microorganisms is slow and frequently involves pyrroloquinoline quinone (PQQ)‐dependent enzymes, making it expensive due to the costly cofactor and hence unattractive for industrial applications. In this study, we present a modified PVA film with improved properties as well as a PQQ‐independent novel enzymatic cascade for the degradation of modified and unmodified PVA. The cascade consists of four steps catalyzed by three enzymes with in situ cofactor recycling technology making this cascade suitable for industrial applications.
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 %.
An Enzyme Cascade Reaction for the Recovery of Hydroxytyrosol Dervatives from Olive Mill Wastewater
(2022)
Abstract
The valorization of olive mill wastewaters (OMWW), a by‐product of the olive milling, is getting rising attention. Lipophilization of the main phenolic compound 3‐hydroxytyrosol (HT) could facilitate its extraction. An immobilized variant of the promiscuous hydrolase/acyltransferase from Pyrobaculum calidifontis VA1 (PestE) was used to perform acetylation in water using ethyl acetate as acyl donor. PestE was used in a segmented flow setting to allow continuous operation. Additionally, HT precursors were made accessible by pretreatment with almond β‐glucosidase and the hydrolytic activity of PestE_I208A_L209F_N288A.
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.
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.
Abstract
Halide methyltransferases (HMTs) enable the enzymatic synthesis of S‐adenosyl‐l‐methionine (SAM) from S‐adenosyl‐l‐homocysteine (SAH) and methyl iodide. Characterisation of a range of naturally occurring HMTs and subsequent protein engineering led to HMT variants capable of synthesising ethyl, propyl, and allyl analogues of SAM. Notably, HMTs do not depend on chemical synthesis of methionine analogues, as required by methionine adenosyltransferases (MATs). However, at the moment MATs have a much broader substrate scope than the HMTs. Herein we provide an overview of the discovery and engineering of promiscuous HMTs and how these strategies will pave the way towards a toolbox of HMT variants for versatile chemo‐ and regioselective biocatalytic alkylations.
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.
Abstract
Erucic (22:1, cisΔ13) and gondoic acids (20:1, cisΔ11) are building blocks obtained from renewable sources for the oleochemical industry. Different biocatalytic strategies for the enrichment of these compounds with high recovery yields were developed in our group. Geotrichum candidum lipases (GCL) strongly discriminate against fatty acids longer than 18 carbon atoms. Thus, GCL‐I and ‐II were investigated using hydrolysis or ethanolysis reactions with Crambe and Camelina oils. Hydrolysis was also studied using fatty acid ethyl esters (FAEE) derived from the corresponding oil. Both isoforms were highly selective; however, interesting differences were observed. Although it has been reported that GCL‐I displays a higher preference toward 18 cisΔ9, which is present in the studied oils at high levels, GCL‐II showed higher enrichment values during hydrolysis independent of the substrate used. Hence, enrichments of 87% (Crambe oil) and 82% (Crambe FAEE) for erucic acid and 50% (Camelina oil) and 45% (Camelina FAEE) for gondoic acid, with recovery values between 89% and 99%, were achieved. On the contrary, the best enzyme for ethanolysis was GCL‐I (82% and 41% for erucic and gondoic acid, respectively). In this case, although GCL‐II also displayed good enrichment and recovery levels (77% and 28%, respectively), they were lower compared to the former reactions. In both ethanolysis reactions, the FAEE fraction contained between 92% and 97% of 18 unsaturated fatty acids.
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
Enzyme activity data for biocatalytic applications are currently often not annotated with standardized conditions and terms. This makes it extremely hard to retrieve, compare, and reuse enzymatic data. With advances in the fields of artificial intelligence (AI) and machine learning (ML), the automated usability of data in the form of machine‐readable annotations will play a crucial role for their success. It is becoming increasingly easy to retrieve complex data sets and extract relevant information; however, standardized data readability is a current limitation. In this contribution, we outline an iterative approach to develop standardized terms and create semantic relations (ontologies) to achieve this highly desirable goal of improving the discoverability, accessibility, interoperability, and reuse of digital resources in the field of biocatalysis.
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.
Fatty aldehydes (FALs) can be derived from fatty acids (FAs) and related compounds and are frequently used as flavors and fragrances. Although chemical methods have been conventionally used, their selective biotechnological production aiming at more efficient and eco-friendly synthetic routes is in demand. α-Dioxygenases (α-DOXs) are heme-dependent oxidative enzymes biologically involved in the initial step of plant FA α-oxidation during which molecular oxygen is incorporated into the Cα-position of a FA (Cn) to generate the intermediate FA hydroperoxide, which is subsequently converted into the shortened corresponding FAL (Cn-1). α-DOXs are promising biocatalysts for the flavor and fragrance industries, they do not require NAD(P)H as cofactors or redox partner proteins, and they have a broad substrate scope. Here, we highlight recent advances in the biocatalytic utilization of α-DOXs with emphasis on newly discovered cyanobacterial α-DOXs as well as analytical methods to measure α-DOX activity in vitro and in vivo.
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
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
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.
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.
Marine Bacteroidetes that degrade polysaccharides contribute to carbon cycling in the ocean. Organic matter, including glycans from terrestrial plants, might enter the oceans through rivers. Whether marine bacteria degrade structurally related glycans from diverse sources including terrestrial plants and marine algae was previously unknown. We show that the marine bacterium Flavimarina sp. Hel_I_48 encodes two polysaccharide utilization loci (PULs) which degrade xylans from terrestrial plants and marine algae. Biochemical experiments revealed activity and specificity of the encoded xylanases and associated enzymes of these PULs. Proteomics indicated that these genomic regions respond to glucuronoxylans and arabinoxylans. Substrate specificities of key enzymes suggest dedicated metabolic pathways for xylan utilization. Some of the xylanases were active on different xylans with the conserved β-1,4-linked xylose main chain. Enzyme activity was consistent with growth curves showing Flavimarina sp. Hel_I_48 uses structurally different xylans. The observed abundance of related xylan-degrading enzyme repertoires in genomes of other marine Bacteroidetes indicates similar activities are common in the ocean. The here presented data show that certain marine bacteria are genetically and biochemically variable enough to access parts of structurally diverse xylans from terrestrial plants as well as from marine algal sources.
Long-chain aliphatic amines such as (S,Z)-hepta- dec-9-en-7-amine and 9-aminoheptadecane were synthesized from ricinoleic acid and oleic acid, respectively, by whole-cell cascade reactions using the combination of an alcohol dehydrogenase (ADH) from Micrococcus luteus, an engi- neered amine transaminase from Vibrio fluvialis (Vf-ATA), and a photoactivated decarboxylase from Chlorella variabilis NC64A (Cv-FAP) in a one-pot process. In addition, long chain aliphatic esters such as 10-(heptanoyloxy)dec-8-ene and octyl- nonanoate were prepared from ricinoleic acid and oleic acid, respectively, by using the combination of the ADH, a Baeyer– Villiger monooxygenase variant from Pseudomonas putida KT2440, and the Cv-FAP. The target compounds were produced at rates of up to 37 U g1 dry cells with conversions up to 90 %. Therefore, this study contributes to the preparation of industrially relevant long-chain aliphatic chiral amines and esters from renewable fatty acid resources.
Formaldehyde is a toxic metabolite that is formed in large quantities during bacterial utilization of the methoxy sugar 6-O-methyl-d-galactose, an abundant monosaccharide in the red algal polysaccharide porphyran. Marine bacteria capable of metabolizing porphyran must therefore possess suitable detoxification systems for formaldehyde. We demonstrate here that detoxification of formaldehyde in the marine Flavobacterium Zobellia galactanivorans proceeds via the ribulose monophosphate pathway. Simultaneously, we show that the genes encoding the key enzymes of this pathway are important for maintaining high formaldehyde resistance. Additionally, these genes are upregulated in the presence of porphyran, allowing us to connect porphyran degradation to the detoxification of formed formaldehyde.