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Optically pure sulfoxides are valuable organosulfur compounds extensively employed in medicinal and organic synthesis. In this study, we present a biocatalytic oxidation‐reduction cascade system designed for the preparation of enantiopure sulfoxides. The system involves the cooperation of a low‐enantioselective chimeric oxidase SMO (styrene monooxygenase) with a high‐enantioselective reductase MsrA (methionine sulfoxide reductase A), facilitating "non‐selective oxidation and selective reduction" cycles for prochiral sulfide oxidation. The regeneration of requisite cofactors for MsrA and SMO was achieved via a cascade catalysis process involving three auxiliary enzymes, sustained by cost‐effective D‐glucose. Under the optimal reaction conditions, a series of heteroaryl alkyl, aryl alkyl and dialkyl sulfoxides in R configuration were synthesized through this "one‐pot, one step" cascade reaction. The obtained compounds exhibited high yields of >90 % and demonstrated enantiomeric excess (ee) values exceeding 90 %. This study represents an unconventional and efficient biocatalytic way in utilizing the low‐enantioselective oxidase for the synthesis of enantiopure sulfoxides. [ABSTRACT FROM AUTHOR]

Optically pure sulfoxides are noteworthy compounds applied in a wide range of industrial fields; however, the biocatalytic deracemization of racemic sulfoxides is challenging. Herein, a high‐enantioselective methionine sulfoxide reductase A (MsrA) was combined with a low‐enantioselective styrene monooxygenase (SMO) for the cyclic deracemization of sulfoxides. Enantiopure sulfoxides were obtained in >90 % yield and with >90 % enantiomeric excess (ee) through dynamic "selective reduction and non‐selective oxidation" cycles. The cofactors of MsrA and SMO were subsequently regenerated by the cascade catalysis of three auxiliary enzymes through the consumption of low‐cost D‐glucose. Moreover, this "one‐pot, one‐step" cyclic deracemization strategy exhibited a wide substrate scope toward various aromatic, heteroaromatic, alkyl and thio‐alkyl sulfoxides. This system proposed an efficient strategy for the green synthesis of chiral sulfoxide. [ABSTRACT FROM AUTHOR]

Enantiopure organic compounds are of major importance for the chemical and pharmaceutical industry. Flavin‐dependent group E monooxygenases, composed of monooxygenase and reductase, are known to perform epoxidation of substituted alkenes as well as sulfoxidation in a regio‐ and enantioselective fashion. Group E is divided into styrene monooxygenases (SMO) and indole monooxygenases (IMO). Hitherto mainly SMOs have been characterized. In this study, we assayed 31 monooxygenases from both types, while 23 of which showed activity. They almost exclusively produced (S)‐styrene oxide at high enantiomeric excess with maximum activities of 0.73 μmol min−1 mg−1 (kcat=0.54 s−1). In case of sulfoxidation, we found that the enantioselectivity is contrary between both types. IMOs preferably produce the (S)‐enantiomer while SMOs have a tendency to produce the (R)‐enantiomer. Sequence analysis and molecular docking of substrates allowed identifying fingerprint motives: SMO N46‐V48‐H50‐Y73‐H76‐S96 and IMO S46‐Q48‐M50‐V/I73‐I76‐A96. These form an essential part of the active site while the loop (AS44‐51) interacts with the co‐substrate and other amino acids direct the substrate. The motives clearly distinguish group E monooxygenases and define the enantioselectivity and thus direct biotechnological applications. Two‐hour biotransformations with several sulfides in conjunction with upscale experiments (10 and 100 mg scale) resulted in the identification of promising candidates for the realization of biocatalytic processes. [ABSTRACT FROM AUTHOR]

Since ancient times, people have been attracted by dyes and they were a symbol of power. Some of the oldest dyes are indigo and its derivative Tyrian purple, which were extracted from plants and snails, respectively. These 'indigoid dyes' were and still are used for coloration of textiles and as a food additive. Traditional Chinese medicine also knows indigoid dyes as pharmacologically active compounds and several studies support their effects. Further, they are interesting for future technologies like organic electronics. In these cases, especially the indigo derivatives are of interest but unfortunately hardly accessible by chemical synthesis. In recent decades, more and more enzymes have been discovered that are able to produce these indigoid dyes and therefore have gained attention from the scientific community. In this study, group E monooxygenases (styrene monooxygenase and indole monooxygenase) were used for the selective oxygenation of indole (derivatives). It was possible for the first time to show that the product of the enzymatic reaction is an epoxide. Further, we synthesized and extracted indigoid dyes and could show that there is only minor by-product formation (e.g. indirubin or isoindigo). Thus, group E monooxygenase can be an alternative biocatalyst for the biosynthesis of indigoid dyes. [ABSTRACT FROM AUTHOR]

Two styrene monooxygenase types, StyA/StyB and StyA1/StyA2B, have been described each consisting of an epoxidase and a reductase. A gene fusion which led to the chimeric reductase StyA2B and the occurrence in different phyla are major differences. Identification of SMOA/SMOB-ADP1 of Acinetobacter baylyi ADP1 may enlighten the gene fusion event since phylogenetic analysis indicated both proteins to be more related to StyA2B than to StyA/StyB. SMOB-ADP1 is classified like StyB and StyA2B as HpaC-like reductase. Substrate affinity and turnover number of the homo-dimer SMOB-ADP1 were determined for NADH (24 µM, 64 s) and FAD (4.4 µM, 56 s). SMOB-ADP1 catalysis follows a random sequential mechanism, and FAD fluorescence is quenched upon binding to SMOB-ADP1 ( K = 1.8 µM), which clearly distinguishes that reductase from StyB of Pseudomonas. In summary, this study confirmes made assumptions and provides phylogenetic and biochemical data for the differentiation of styrene monooxygenase-related flavin reductases. [ABSTRACT FROM AUTHOR]

We describe the enantioselective epoxidation of straight-chain aliphatic alkenes using a biocatalytic system containing styrene monooxygenase from Rhodococcus sp. ST-10 and alcohol dehydrogenase from Leifsonia sp. S749. The biocatalyzed enantiomeric epoxidation of 1-hexene to ( S)-1,2-epoxyhexane (>44.6 mM) using 2-propanol as the hydrogen donor was achieved under optimized conditions. The biocatalyst had broad substrate specificity for various aliphatic alkenes, including terminal, internal, unfunctionalized, and di- and tri-substituted alkenes. Here, we demonstrate that this biocatalytic system is suitable for the efficient production of enantioenriched ( S)-epoxyalkanes. [ABSTRACT FROM AUTHOR]

Nine different sulfur-containing compounds were biotransformed to the corresponding sulfoxides by Escherichia coli Bl21(DE3) cells expressing styrene monooxygenase (SMO) from Pseudomonas putida CA-3. Thioanisole was consumed at 83.3 μmoles min g cell dry weight resulting mainly in the formation of R-thioanisole sulfoxide with an enantiomeric excess (ee) value of 45 %. The rate of 2-methyl-, 2-chloro- and 2-bromo-thioanisole consumption was 2-fold lower than that of thioanisole. Surprisingly, the 2-methylthioanisole sulfoxide product had the opposite ( S) configuration to that of the other 2-substituted thioanisole derivatives and had a higher ee value (84 %). The rate of oxidation of 4-substituted thioanisoles was higher than the corresponding 2-substituted substrates but the ee values of the products were consistently lower (10-23 %). The rate of benzo[ b]thiophene and 2-methylbenzo[ b]thiophene sulfoxidation was approximately 10-fold lower than that of thioanisole. The ee value of the benzo[ b]thiophene sulfoxide could not be determined as the product racemized rapidly. E. coli cells expressing an engineered SMO (SMOeng R3-11) oxidised 2-substituted thioanisoles between 1.8- and 2.8-fold faster compared to cells expressing the wild-type enzyme. SMOeng R3-11 oxidised benzo[ b]thiophene and 2-methylbenzo[ b]thiophene 10.1 and 5.6 times faster that the wild-type enzyme. The stereospecificity of the reaction catalysed by SMOeng was unchanged from that of the wild type. Using the X-ray crystal structure of the P. putida S12 SMO, it was evident that the entrance of substrates into the SMO active site is limited by the binding pocket bottleneck formed by the side chains of Val-211 and Asn-46 carboxyamide group. [ABSTRACT FROM AUTHOR]

Abstract: Styrene monooxygenase (SMO) catalyzes the first step of styrene degradation, and also serves as an important enzyme for the synthesis of enantiopure epoxides. To enhance its activity, molecular docking of styrene was performed based on the X-ray crystal structure of the oxygenase subunit of SMO to identify three amino acid residues (Tyr73, His76 and Ser96) being adjacent to the phenyl ring of styrene. Variants at those positions were constructed and their enzymatic activities were analyzed. Three mutants (Y73V, Y73F, and S96A) were found to exhibit higher enzymatic activities than the wild-type in the epoxidation of styrene, while retaining excellent stereoselectivity. The specific epoxidation activity of the most active mutant S96A toward styrene and trans-β-methyl styrene were 2.6 and 2.3-fold of the wild-type, respectively. In addition, the Y73V mutant showed an unexpected reversal of enantiomeric preference toward 1-phenylcyclohexene. [Copyright &y& Elsevier]

Abstract: Styrene metabolism genes were isolated from styrene-assimilating bacteria Rhodococcus sp. ST-5 and ST-10. Strain ST-5 had a gene cluster containing four open reading frames which encoded styrene degradation enzymes. The genes showed high similarity to styABCD of Pseudomonas sp. Y2. On the other hand, strain ST-10 had only two genes which encoded styrene monooxygenase and flavin oxidoreductase (styAB). Escherichia coli transformants possessing the sty genes of strains ST-5 and ST-10 produced (S)-styrene oxide from styrene, indicating that these genes function as styrene degradation enzymes. Metabolite analysis by resting-cell reaction with gas chromatography–mass spectrometry revealed that strain ST-5 converts styrene to phenylacetaldehyde via styrene oxide by styrene oxide isomerase (styC) reaction. On the other hand, strain ST-10 lacked this enzyme, and thus accumulated styrene oxide as an intermediate. HPLC analysis showed that styrene oxide was spontaneously isomerized to phenylacetaldehyde by chemical reaction. The produced phenylacetaldehyde was converted to phenylacetic acid (PAA) in strain ST-10 as well as in strain ST-5. Furthermore, phenylacetic acid was converted to phenylacetyl-CoA by the catalysis of phenylacetate-CoA ligase in strains ST-5 and ST-10. This study proposes possible styrene metabolism pathways in Rhodococcus sp. strains ST-5 and ST-10. [Copyright &y& Elsevier]

A straightforward one-pot procedure combining enrichment and immobilization of recombinantely expressed FADH2 dependent styrene monooxygenase (StyA) directly from Escherichia coli cell extracts was investigated. Sepabeads EC-EA and EC-Q1A anion-exchange carriers were employed to non-covalently adsorb StyA from the cell extracts depending on basic parameters such as varying initial protein concentrations and pH. The protein fraction of the cell extract contained around 25% StyA. At low initial protein concentrations (2.5 mg mL-1) and pH 6, the enzyme could be enriched up to 52.4% on Sepabeads EC-EA and up to 46.0% on Sepabeads EC-Q1A, accounting for an almost complete StyA adsorption from the cell extracts. Higher initial protein concentrations were necessary to exploit the high loading capacity of the beads. At 20 mg mL-1, up to 37.6% of the theoretical bead loading capacity could be utilized for StyA binding using Sepabeads EC-EA, and 34.0% using Sepabeads EC-Q1A. For both carriers, protein leakage under reaction conditions could be reduced to less than 2%. During assays, the FADH2 cofactor necessary for StyA activity was supplied by the NADH-FAD reductase component styrene monooxygenase B (StyB). StyA immobilized on Sepabeads EC-Q1A displayed twice as high styrene epoxidation rates (0.2 U mgStyA -1) as compared to Sepabeads EC-EA. This activity could be increased to 0.7 U mgStyA -1 by co-immobilizing StyB on Sepabeads EC-Q1A, which corresponds to 33% of the soluble StyA activity. [ABSTRACT FROM AUTHOR]

Styrene monooxygenase catalyzes the enantioselective epoxidation of styrene but displays significantly decreased activity toward styrene derivatives with an α- or β-substituent. Based on the X-ray crystal structure of the oxygenase subunit of styrene monooxygenase, molecular docking of α-ethylstyrene was performed to identify adjacent residues. Four amino acid substitutions (R43A, L44A, L45A, and N46A) were introduced into the enzyme by site-directed mutagenesis. All four mutations led to a change of substrate preference. The mutant L45A, in particular, exhibited an altered substrate preference toward the bulkier substrate α-ethylstyrene. [ABSTRACT FROM AUTHOR]

Abstract: (S)-styrene oxide, a useful chiral intermediate, has been synthesized using an electroenzymatic method with direct electrochemical FADH2 regeneration. Low electroenzymatic efficiency arising from the fast FADH2 reoxidation could be overcome by employing a zinc oxide/carbon black composite electrode. The attractive interaction between zinc oxide and styrene monooxygenase kept the local enzyme concentration high near the electrode surface, thereby increasing the accessibility of FADH2 from the electrode surface to the enzyme. By adjusting the reaction conditions such as oxygen solubility, a high electroenzymatic efficiency of 65% was obtained. As a result, the reaction rate was increased while the amount of the side-products from the cofactor reoxidation process was decreased. The metal oxide/carbon black composite electrode can be efficiently used for electroenzymatic syntheses using diffusible-flavin dependent monooxygenases. [ABSTRACT FROM AUTHOR]

Abstract: A novel styrene monooxygenase (SMO) was isolated from Pseudomonas sp. LQ26, a styrene degrader from activated sludge. Sequence alignment demonstrated that it was the most distant member of all SMOs originating from the genus of Pseudomonas. The substrate spectrum of this enzyme extended beyond typical SMO substrates to 1-allylbenzene analogues, previously reported as non-substrates for the SMO from Pseudomonas fluorescens ST. The results demonstrate for the first time the asymmetric epoxidation of both conjugated and unconjugated alkenes catalyzed by SMO and suggest that a much broader substrate spectrum is expected for SMOs. [ABSTRACT FROM AUTHOR]

Abstract: A highly active recombinant whole-cell biocatalyst, Escherichia coli pETAB2/pG-KJE1, was developed for the efficient production of (S)-styrene oxide from styrene. The recombinant E. coli overexpressed styAB the genes of styrene monooxygenase of Pseudomonas putida SN1 and coexpressed the genes encoding chaperones (i.e., GroEL–GroES and DnaK–DnaJ–GrpE). The styrene monooxygenases were produced to ca. 40% of the total soluble proteins, enabling the whole-cell activity of the recombinant of 180U/gCDW. The high StyAB activity in turn appeared to direct cofactors and molecular oxygen to styrene epoxidation. The product yield on energy source (i.e., glucose) reached ca. 40%. In addition, biotransformation in an organic/aqueous two-liquid phase system allowed the product to accumulate to 400mM in the organic phase within 6h, resulting in an average specific and volumetric productivity of 6.4mmol/gdrycells/h (106U/gdrycells) and 67mM/h (1110U/Laq), respectively, under mild reaction conditions. These results indicated that the high productivity and the high product yield on energy source were driven by the high enzyme activity. Therefore, it was concluded that oxygenase activity of whole-cell biocatalysts is one of the critical factors to determine their catalytic performance. [Copyright &y& Elsevier]

Styrene and indole monooxygenases (SMO and IMO) are two-component flavoprotein monooxygenases composed of a nicotinamide adenine dinucleotide (NADH)-dependent flavin adenine dinucleotide (FAD)-reductase (StyB or IndB) and a monooxygenase (StyA or IndA). The latter uses reduced FAD to activate oxygen and to oxygenate the substrate while releasing water. We circumvented the need for the reductase by direct FAD reduction in solution using the NAD(P)H-mimic 1-benzyl-1,4-dihydronicotinamide (BNAH) to fuel monooxygenases without NADH requirement. Herein, we report on the hitherto unknown solvent tolerance for the indole monooxygenase from Gemmobacter nectariphilus DSM15620 (GnIndA) and the styrene monooxygenase from Gordonia rubripertincta CWB2 (GrStyA). These enzymes were shown to convert bulky and rather hydrophobic styrene derivatives in the presence of organic cosolvents. Subsequently, BNAH-driven biotransformation was furthermore optimized with regard to the applied cosolvent and its concentration as well as FAD and BNAH concentration. We herein demonstrate that GnIndA and GrStyA enable selective epoxidations of allylic double bonds (up to 217 mU mg−1) in the presence of organic solvents such as tetrahydrofuran, acetonitrile, or several alcohols. Notably, GnIndA was found to resist methanol concentrations up to 25 vol.%. Furthermore, a diverse substrate preference was determined for both enzymes, making their distinct use very interesting. In general, our results seem representative for many IMOs as was corroborated by in silico mutagenetic studies. [ABSTRACT FROM AUTHOR]

Background: Styrene monooxygenase (SMO) catalyzes the first step of aromatic alkene degradation yielding the corresponding epoxides. Because of its broad spectrum of substrates, the enzyme harbors a great potential for an application in medicine and chemical industries. Results: In this study, we achieved higher enzymatic activity and better stability towards styrene by enlarging the ligand entrance tunnel and improving the hydrophobicity through error-prone PCR and site-saturation mutagenesis. It was found that Asp305 (D305) hindered the entrance of the FAD cofactor according to the model analysis. Therefore, substitution with amino acids possessing shorter side chains, like glycine, opened the entrance tunnel and resulted in up to 2.7 times higher activity compared to the wild-type enzyme. The half-lives of thermal inactivation for the variant D305G at 60 °C was 28.9 h compared to only 3.2 h of the wild type SMO. Moreover, overexpression of SMO in Pseudomonas putida KT2440 with NADH regeneration was carried out in order to improve biotransformation efficiency for epoxide production. A hexadecane/buffer (v/v) biphasic system was applied in order to minimize the inactivation effect of high substrate concentrations on the SMO enzyme. Finally, SMO activities of 190 U/g CDW were measured and a total amount of 20.5 mM (S)-styrene oxide were obtained after 8 h. Conclusions: This study offers an alternative strategy for improved SMO expression and provides an efficient biocatalytic system for epoxide production via engineering the entrance tunnel of the enzyme's active site. [ABSTRACT FROM AUTHOR]