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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 %.
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.
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.
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
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
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
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.
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.