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Fast screening of enzyme variants is crucial for tailoring biocatalysts for the asymmetric synthesis of non-natural chiral chemicals, such as amines. However, most existing screening methods either are limited by the throughput or require specialized equipment. Herein, we report a simple, high-throughput, low-equipment dependent, and generally applicable growth selection system for engineering amine-forming or converting enzymes and apply it to improve biocatalysts belonging to three different enzyme classes. This results in (i) an amine transaminase variant with 110-fold increased specific activity for the asymmetric synthesis of the chiral amine intermediate of Linagliptin; (ii) a 270-fold improved monoamine oxidase to prepare the chiral amine intermediate of Cinacalcet by deracemization; and (iii) an ammonia lyase variant with a 26-fold increased activity in the asymmetric synthesis of a non-natural amino acid. Our growth selection system is adaptable to different enzyme classes, varying levels of enzyme activities, and thus a flexible tool for various stages of an engineering campaign.
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.
Acute pancreatitis (AP) is a major, globally increasing gastrointestinal disease and a biliary origin is the most common cause. However, the effects of bile acids (BAs), given systemically, on the pancreas and on disease severity remains elusive. In this study, we have investigated the roles of different circulating BAs in animal models for AP to elucidate their impact on disease severity and the underlying pathomechanisms. BAs were incubated on isolated acini and AP was induced through repetitive injections of caerulein or L-arginine; pancreatic duct ligation (PDL); or combined biliopancreatic duct ligation (BPDL). Disease severity was assessed using biochemical and histological parameters. Serum cholecystokinin (CCK) concentrations were determined via enzyme immunoassay. The binding of the CCK1 receptor was measured using fluorescence-labeled CCK. In isolated acini, hydrophobic BAs mitigated the damaging effects of CCK. The same BAs further enhanced pancreatitis in L-arginine- and PDL-based pancreatitis, whereas they ameliorated pancreatic damage in the caerulein and BPDL models. Mechanistically, the binding affinity of the CCK1 receptor was significantly reduced by hydrophobic BAs. The hydrophobicity of BAs and the involvement of CCK seem to be relevant in the course of AP. Systemic BAs may affect the severity of AP by interfering with the CCK1 receptor.
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.
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
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.
Entdeckung und Design promiskuitiver Acyltransferase‐Aktivität in Carboxylesterasen der Familie VIII
(2021)
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.
Polybutylene adipate terephthalate (PBAT) is a biodegradable alternative to polyethylene and can be broadly used in various applications. These polymers can be degraded by hydrolases of terrestrial and aquatic origin. In a previous study, we identified tandem PETase-like hydrolases (Ples) from the marine microbial consortium I1 that were highly expressed when a PBAT blend was supplied as the only carbon source. In this study, the tandem Ples, Ple628 and Ple629, were recombinantly expressed and characterized. Both enzymes are mesophilic and active on a wide range of oligomers. The activities of the Ples differed greatly when model substrates, PBAT-modified polymers or PET nanoparticles were supplied. Ple629 was always more active than Ple628. Crystal structures of Ple628 and Ple629 revealed a structural similarity to other PETases and can be classified as member of the PETases IIa subclass, α/β hydrolase superfamily. Our results show that the predicted functions of Ple628 and Ple629 agree with the bioinformatic predictions, and these enzymes play a significant role in the plastic degradation by the consortium.
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.
Enzymes, the driving biocatalysts in living organisms, are typically not suited for large-scale industrial use. In the last decade, enzyme engineering has evolved into the key technology to design tailor-made enzymes for chemical and pharmaceutical applications. We highlight current trends in enzyme engineering and biocatalysis based on outstanding examples from the pharmaceutical industry.