Search

Hydroxynitrile lyase from the white rabbit's foot fern Davallia tyermannii (DtHNL) catalyzes the enantioselective synthesis of α-cyanohydrins, which are key building blocks for pharmaceutical and agrochemical industries. An efficient and competitive process necessitates the availability and robustness of the biocatalyst. Herein, the recombinant production of DtHNL1 in Komagataella phaffii, yielding approximately 900 000 U L -1 , is described. DtHNL1 constitutes approximately 80 % of the total protein content. The crude enzyme was immobilized. Crosslinked enzyme aggregates (CLEAs) resulted in significant enhancement of the biocatalyst stability under acidic conditions (activity retained after 168 h at pH 2.4). The DtHNL1-CLEA was employed for (R)-mandelonitrile synthesis (99 % conversion, 98 % enantiomeric excess) in a biphasic system, and evaluated for the synthesis of (R)-hydroxypivaldehyde cyanohydrin under reaction conditions that immediately inactivated non-immobilized DtHNL1. The results show the DtHNL1-CLEA to be a stable biocatalyst for the synthesis of enantiomerically pure cyanohydrins under acidic conditions., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Increasing demand for chemicals from renewable resources calls for the development of new biotechnological methods for the reduction of oxidized bio-based compounds. Enzymatic carboxylate reduction is highly selective, both in terms of chemo- and product selectivity, but not many carboxylate reductase enzymes (CARs) have been identified on the sequence level to date. Thus far, their phylogeny is unexplored and very little is known about their structure-function-relationship. CARs minimally contain an adenylation domain, a phosphopantetheinylation domain and a reductase domain. We have recently identified new enzymes of fungal origin, using similarity searches against genomic sequences from organisms in which aldehydes were detected upon incubation with carboxylic acids. Analysis of sequences with known CAR functionality and CAR enzymes recently identified in our laboratory suggests that the three-domain architecture mentioned above is modular. The construction of a distance tree with a subsequent 1000-replicate bootstrap analysis showed that the CAR sequences included in our study fall into four distinct subgroups (one of bacterial origin and three of fungal origin, respectively), each with a bootstrap value of 100%. The multiple sequence alignment of all experimentally confirmed CAR protein sequences revealed fingerprint sequences of residues which are likely to be involved in substrate and co-substrate binding and one of the three catalytic substeps, respectively. The fingerprint sequences broaden our understanding of the amino acids that might be essential for the reduction of organic acids to the corresponding aldehydes in CAR proteins., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Thiamine diphosphate (ThDP) dependent carboligases catalyze the formation of carbon‐carbon bonds. These enzymes are prominent biocatalysts for the production of valuable α‐hydroxy carbonyl compounds, which serve as key building blocks in the synthesis of various pharmaceuticals and fine chemicals. Carboligases act in selective manner to afford regio‐ and stereochemically defined products from simple starting materials. This review explores the catalytic prowess of carboligases for synthetic purposes and is aimed at providing a selection tool of enzymes for particular sets of substrates or desired products. The comprehensive overview encompasses structural insights, relationships of the currently known sequence space and practical applications with a focus on recent literature, showcasing carboligases as potent tools for sustainable and efficient synthesis. [ABSTRACT FROM AUTHOR]

Many aldehydes are volatile compounds with distinct and characteristic olfactory properties. The aldehydic functional group is reactive and, as such, an invaluable chemical multi‐tool to make all sorts of products. Owing to the reactivity, the selective synthesis of aldehydic is a challenging task. Nature has evolved a number of enzymatic reactions to produce aldehydes, and this review provides an overview of aldehyde‐forming reactions in biological systems and beyond. Whereas some of these biotransformations are still in their infancy in terms of synthetic applicability, others are developed to an extent that allows their implementation as industrial biocatalysts. [ABSTRACT FROM AUTHOR]

Carboxylate reductases (CARs) are valuable catalysts for the selective one‐step reduction of carboxylic acids to their corresponding aldehydes. In recent years, numerous new CARs have been made available, studied and applied in the context of biocatalytic syntheses. The preparation of aldehydes as end products for the flavor and fragrance sector and the integration of CARs in cascade reactions with aldehydes as the key intermediates represent the two major fields of applications. This review gives a comprehensive overview of the current toolbox of recombinant CARs and their numerous carboxylate substrates. Non‐natural functions of CARs are highlighted and recent insight into the structure‐function relationship of CARs are summarized. [ABSTRACT FROM AUTHOR]

Among nature's arsenal of oxidative enzymes, cytochrome P450s (CYPs) catalyze the most challenging reactions, the hydroxylations of non‐activated C−H bonds. Human CYPs are studied in drug development due to their physiological role at the forefront of metabolic detoxification, but their challenging handling makes them unsuitable for application. CYPs have a great potential for biocatalysis, but often lack appropriate features such as high and soluble expression, self‐sufficient internal electron transport, high stability, and an engineerable substrate scope. We have probed these characteristics for a recently described CYP that originates from the thermophilic fungus Thermothelomyces thermophila (CYP505A30), a homolog of the well‐known P450‐BM3 from Bacillus megaterium. CYP505A30 is a natural monooxygenase‐reductase fusion, is well expressed, and moderately tolerant towards temperature and solvent exposure. Although overall comparable, we found the stability of the enzyme's domains to be inverse to P450‐BM3, with a more stable reductase compared to the heme domain. After analysis of a homology model, we created mutants of the enzyme based on literature data for P450‐BM3. We then probed the enzyme variants in bioconversions using a panel of active pharmaceutical ingredients, and activities were detected for a number of structurally diverse compounds. Ibuprofen was biooxidized in a preparative scale whole cell bioconversion to 1‐, 2‐ and 3‐hydroxyibuprofen. [ABSTRACT FROM AUTHOR]

Abstract: Human enzymes have been widely studied in various disciplines. The number of reactions taking place in the human body is vast, and so is the number of potential catalysts for synthesis. Herein, we focus on the application of human enzymes that catalyze chemical reactions in course of the metabolism of drugs and xenobiotics. Some of these reactions have been explored on the preparative scale. The major field of application of human enzymes is currently drug development, where they are applied for the synthesis of drug metabolites. [ABSTRACT FROM AUTHOR]

Carboxylate reductases (CARs, E.C. 1.2.1.30) generate aldehydes from their corresponding carboxylic acid with high selectivity. Little is known about the structure of CARs and their catalytically important amino acid residues. The identification of key residues for carboxylate reduction provides a starting point to gain deeper understanding of enzymatic carboxylate reduction. A multiple sequence alignment of CARs with confirmed activity recently identified in our lab and fromthe literature revealed a fingerprint of conserved amino acids. We studied the function of conserved residues by multiple sequence alignments and mutational replacements of these residues. In this study, single-site alanine variants of Neurospora crassa CAR were investigated to determine the contribution of conserved residues to the function, expressability or stability of the enzyme. The effect of amino acid replacements was investigated by analyzing enzymatic activity of the variants in vivo and in vitro. Supported by molecular modeling, we interpreted that five of these residues are essential for catalytic activity, or substrate and co-substrate binding. We identified amino acid residues having significant impact on CAR activity. Replacement of His 237, Glu 433, Ser 595, Tyr 844, and Lys 848 by Ala abolish CAR activity, indicating their key role in acid reduction. These results may assist in the functional annotation of CAR coding genes in genomic databases. While some other conserved residues decreased activity or had no significant impact, four residues increased the specific activity of NcCAR variants when replaced by alanine. Finally, we showed that NcCAR wild-type and mutants efficiently reduce aliphatic acids. [ABSTRACT FROM AUTHOR]

3-Hydroxytyrosol (3-HT) is a phenolic antioxidant that has a number of beneficial effects on human health and is a valuable building block in the synthesis of various pharmaceuticals. Herein, we report a new method for the production of 3-HT through reduction of 3,4-dihydroxyphenylacetic acid. The reduction was performed in whole Escherichia coli BL21 (DE3) cells overexpressing carboxylic acid reductase from Nocardia and phosphopantetheinyl transferase from E. coli. An endogenous E. coli aldehyde reducing activity turned out to be highly efficient for further reduction of the aldehyde intermediate to the desired alcohol. The influence of different buffer components, cofactors, and cofactor recycling systems was investigated. A very economic combination of glucose, citrate, and air proved sufficient for recycling of the essential cofactors ATP and NAD(P)H. Selected crucial parameters were then further optimized within a 'design of experiments' approach. Finally, first preparative-scale bioreductions resulted in pure 3-HT. [ABSTRACT FROM AUTHOR]

Recombinant human aldehyde oxidase (AO) was expressed in Escherichia coli. Different cell disruption methods and conditions of cell culture in shake flasks and bioreactors and of biotransformation on an analytical scale were tested to optimize the synthesis of oxidized AO drug metabolites. The volumetric productivity was increased 24-fold by optimizing the cell culture conditions. The highest yield was achieved in a 25 L stirred tank bioreactor under non-oxygen-limited conditions and high lactose feed rate. Suspensions of highly concentrated and well-aerated whole cells at neutral pH and relatively low temperatures led to the best conversion. The solvent for the substrate and the buffering agent for the biotransformation had an important effect. In a biotransformation with AO, 210 mg of famciclovir was converted to diacetyl penciclovir a yield of 82 %. The optimized protocol represents a viable method for the preparative synthesis of oxidized AO metabolites of drugs. [ABSTRACT FROM AUTHOR]

Nitrile reductase QueF catalyzes the reduction of 2-amino-5-cyanopyrrolo[2,3- d]pyrimidin-4-one (preQ0) to 2-amino-5-aminomethylpyrrolo[2,3- d]pyrimidin-4-one (preQ1) in the biosynthetic pathway of the hypermodified nucleoside queuosine. It is the only enzyme known to catalyze a reduction of a nitrile to its corresponding primary amine and could therefore expand the toolbox of biocatalytic reactions of nitriles. To evaluate this new oxidoreductase for application in biocatalytic reactions, investigation of its substrate scope is prerequisite. We report here an investigation of the active site binding properties and the substrate scope of nitrile reductase QueF from Escherichia coli. Screenings with simple nitrile structures revealed high substrate specificity. Consequently, binding interactions of the substrate to the active site were identified based on a new homology model of E. coli QueF and modeled complex structures of the natural and non-natural substrates. Various structural analogues of the natural substrate preQ0 were synthesized and screened with wild-type QueF from E. coli and several active site mutants. Two amino acid residues Cys190 and Asp197 were shown to play an essential role in the catalytic mechanism. Three non-natural substrates were identified and compared to the natural substrate regarding their specific activities by using wild-type and mutant nitrile reductase. [ABSTRACT FROM AUTHOR]

2-Hydroxy-3-pentanone and 3-hydroxy-2-pentanone are flavor molecules present in various foods, such as cheese, wine, durian, and honey, where they impart buttery, hay-like, and caramel-sweet aromas. However, their utilization as flavoring agents is constrained by a lack of developed synthesis methods. In this study, we present their synthesis from simple starting compounds available in natural quality, catalyzed by previously characterized ThDP-dependent carboligases. Additionally, we demonstrate that newly discovered homologues of pyruvate dehydrogenase from E. coli (Ec PDH E1), namely La PDH from Leclercia adecarboxylata , Cn PDH from Cupriavidus necator , and Tc PDH from Tanacetum cinerariifolium , exhibit promising potential for α-hydroxy pentanone synthesis in form of whole-cell biocatalysts. Enzyme stability at varying pH levels, kinetic parameters, and reaction intensification were investigated. Cn PDH, for example, exhibits superior stability across different pH levels compared to Ec PDH E1. Both α-hydroxy pentanones can be produced with Cn PDH in satisfactory yields (74% and 59%, respectively). • Synthesis of aroma constituents of wine, cheese and durian by ThDP-dependent carboligases from simple starting compounds. • Heterologous production and functional characterization of homologues of pyruvate dehydrogenase from E. coli (Ec PDH E1). • Cupriavidus necator PDH is more stable across different pH levels compared to Ec PDH E1. • Product distribution of carboligations can be tuned by choice of enzyme and also by choice of donor and acceptor substrate. [ABSTRACT FROM AUTHOR]

Nitrile hydratases (NHase) catalyze the hydration of nitriles to the corresponding amides. We report on the heterologous expression of various nitrile hydratases. Some of these enzymes have been investigated by others and us before, but sixteen target proteins represent novel sequences. Of 21 target sequences, 4 iron and 16 cobalt containing proteins were functionally expressed from Escherichia coli BL21 (DE3) Gold. Cell free extracts were used for activity profiling and basic characterization of the NHases using the typical NHase substrate methacrylonitrile. Co-type NHases are more tolerant to high pH than Fe-type NHases. A screening for activity on three structurally diverse nitriles was carried out. Two novel Co-dependent NHases from Afipia broomeae and Roseobacter sp. and a new Fe-type NHase from Gordonia hydrophobica were very well expressed and hydrated methacrylonitrile, pyrazine-carbonitrile, and 3-amino-3-(p-toluoyl)propanenitrile. The Co-dependent NHases from Caballeronia jiangsuensis and Microvirga lotononidis, as well as two Fe-dependent NHases from Pseudomonades, were—in addition—able to produce the amide from cinnamonitrile. Summarizing, seven so far uncharacterized NHases are described to be promising biocatalysts. [ABSTRACT FROM AUTHOR]

Mycobacterium marinum CAR (Mm CAR) is one of the most widely used CARs as the key enzyme for the synthesis of aldehydes, alcohols and further products from the respective carboxylic acids. Herein, we describe the first functionally secreted 131 kDa CAR and its isolated A-domain using Komagataella phaffii and a methanol-free constitutive expression strategy. Precipitated and lyophilized Mm CAR (500 µg) was isolated from the culture supernatant and showed no decrease in activity for piperonylic acid (80% conversion), even when stored for up to 3 weeks at 4°C. Lyophilized Mm CAR precipitate gave 48% yield of E/Z -nonanal-4-nitrobenzoyloxime from the reduction of nonanoic acid and in-situ derivatization with O -4-nitrobenzoyl-hydroxylamine. Furthermore, K. phaffii could successfully secrete the Mm CAR adenylation domain. Its activity was confirmed by the amidation of benzoic acid with n -hexylamine. Neither enzyme variant was glycosylated by the yeast. In summary, functional CAR can be secreted by K. phaffii and used for cell free conversion of carboxylic acids to various products. [Display omitted] • Active full-length Mm CAR and Mm CAR A-domain were secreted by Komagataella phaffii for the first time. • Mm CAR A-domain was active for amidation of benzoic acid with n-hexylamine. • Precipitated, lyophilized full-length Mm CAR can be stored without activity loss at 4°C for at least 3 weeks. [ABSTRACT FROM AUTHOR]

Nitrile reductase QueF catalyzes the reduction of a nitrile to its corresponding amine. In their Full Paper on page 7007 ff., N. Klempier et al. report on the investigation of active‐site binding and the substrate scope of E. coli QueF by modeling, enzyme, and substrate engineering. The active‐site residues and structural features of the substrates essential for catalysis were identified in spectrophotometric and HPLC‐MS based screenings of active‐site mutants and natural substrate analogues. [ABSTRACT FROM AUTHOR]

Carboxylic acid reductases (CARs) are well-known for their eminent selective one-step synthesis of carboxylic acids to aldehydes. To date, however, few CARs have been identified and characterized, especially from fungal sources. In this study, the CAR from the white rot fungus Pycnoporus cinnabarinus (Pc CAR2) was expressed in Escherichia coli. Pc CAR2′s biochemical properties were explored in vitro after purification, revealing a melting temperature of 53 °C, while the reaction temperature optimum was at 35 °C. In the tested buffers, the enzyme showed a pH optimum of 6.0 and notably, a similar activity up to pH 7.5. Pc CAR2 was immobilized to explore its potential as a recyclable biocatalyst. Pc CAR2 showed no critical loss of activity after six cycles, with an average conversion to benzaldehyde of more than 85% per cycle. Immobilization yield and efficiency were 82% and 76%, respectively, on Ni-sepharose. Overall, our findings contribute to the characterization of a thermotolerant fungal CAR, and established a more sustainable use of the valuable biocatalyst. [Display omitted] • The carboxylic acid reductase (CAR) from Pycnoporus cinnabarinus Pc CAR2 is a pH and thermotolerant enzyme. • Targeted immobilization of Pc CAR2 maintains essential conformational flexibility of the enzyme and enables recycling. • All known CARs from subtype IV are from Basidiomycetes. [ABSTRACT FROM AUTHOR]

• The Pycnoporus cinnabarinus genome encodes for a repertoire of carboxylic acid reductase (CAR) enzymes. • Type IV CARs can be classified into more than one subclass (or subtype). • A new functionally-distinct member of the CAR enzyme family was identified. Carboxylic acid reductases (CARs) are attracting burgeoning attention as biocatalysts for organic synthesis of aldehydes and their follow-up products from economic carboxylic acid precursors. The CAR enzyme class as a whole, however, is still poorly understood. To date, relatively few CAR sequences have been reported, especially from fungal sources. Here, we sought to increase the diversity of the CAR enzyme class. Six new CAR sequences from the white-rot fungus Pycnoporus cinnabarinus were identified from genome-wide mining. Genome and gene clustering analysis suggests that these Pc CAR enzymes play different natural roles in Basidiomycete systems, compared to their type II Ascomycete counterparts. The cDNA sequences of all six Pccar genes were deduced and analysis of their corresponding amino acid sequence showed that they encode for proteins of similar properties that possess a conserved modular functional tri-domain arrangement. Phylogenetic analyses showed that all Pc CAR enzymes cluster together with the other type IV CARs. One candidate, Pc CAR4, was cloned and over-expressed recombinantly in Escherichia coli. Subsequent biotransformation-based screening with a panel of structurally-diverse carboxylic acid substrates suggest that Pc CAR4 possessed a more pronounced substrate specificity compared to previously reported CARs, preferring to reduce sterically-rigid carboxylic acids such as benzoic acid. These findings thus present a new functionally-distinct member of the CAR enzyme class. [ABSTRACT FROM AUTHOR]

• Identification and expression of novel carboxylate reductase (CAR) from the thermophilic fungus Thermothelomyces thermophila. • Characterization of the second subtype III CAR focusing on stability attributes. • Thermothelomyces thermophila CAR is slightly more thermotolerant than Neurospora crassa CAR and has a different pH optimum. • Successful in vitro application of the Tt CAR reducing vanillic acid to vanillin with cell-free regeneration of ATP and NADPH. A novel type III fungal CAR was identified from the organism Thermothelomyces thermophila. High expression levels were observed in E. coli using the pETDuet-1 plasmid system in combination with an autoinduction protocol. A broad substrate scope ranging from aromatic to aliphatic carboxylic acids was tested and Tt CAR showed activity for all substrates. High specific activities for aromatic substrates and short chain aliphatic substrates were observed, comparable to those of Nc CAR, the first type III fungal CAR. Tt CAR's pH and temperature optima were at 6.5 and 30 °C, respectively. Up to 20% (v/v) cosolvents did not show a decrease in specific activity of Tt CAR using (E)-cinnamic acid as a substrate. Its half-life at 40 °C was determined to be 8.25 h and its melting temperature (T m) was 56 °C. In vitro reactions with Tt CAR reduced 95.2% of 10 mM vanillic acid, which correlated to a titer of 1.4 g L−1 of vanillin. The space time yield of 0.029 g L-1 h-1 indicates that further improvements would be necessary for an industrially relevant application. This would be especially important when competing against de novo synthesis of bio vanillin by microbial strains producing >30 g L-1. In de novo and in vivo biosynthesis systems, by-products are fairly common. By contrast, we were pleased to observe less than 0.7% of vanillyl alcohol formed, making the cell-free acid reduction in the envisaged sequential two-step bioconversion from eugenol to vanillin very attractive. [ABSTRACT FROM AUTHOR]

Abstract: Isolated from pig liver as a crude, inhomogeneous enzyme fraction, pig liver esterase (PLE) was found to metabolize a wide range of substrates; often in a highly stereoselective manner. This crude esterase preparation, however, contains several iso-enzymes at proportions varying from batch to batch. Racemic methyl-(4E)-5-chloro-2-isopropyl-4-pentenoate is cleaved enantioselectively by crude PLE, but not by recombinantly expressed γ-isoform of PLE. Concluding that another PLE iso-enzyme must carry the relevant activity, we cloned and sequenced cDNAs of several PLE isoforms and functionally expressed them in Pichia pastoris. One novel isoform termed alternative pig liver esterase (APLE) was found to hydrolyze methyl-(2R,4E)-5-chloro-2-isopropyl-4-pentenoate in a highly stereoselective manner (E >200). When heterologously expressed and directed for secretion in P. pastoris, APLE was found to be localized in the periplasm. The presence or absence of a putative C-terminal ER retention signal did neither influence functional expression nor cellular localization. The recombinant enzyme, purified by ion exchange chromatography, had a specific activity of 36U (mg protein)−1 towards racemic methyl-(4E)-5-chloro-2-isopropyl-4-pentenoate. [Copyright &y& Elsevier]