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A new and efficient sulfide monooxygenase-producing strain, ECU0066, was isolated and identified as a Rhodococcus sp. that could transform phenylmethyl sulfide (PMS) to (S)-sulfoxide with 99% enantiomeric excess via two steps of enantioselective oxidations. Its enzyme activity could be effectively
induced by adding PMS or phenylmethyl sulfoxide (PM...
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To expand the available set of Baeyer–Villiger monooxygenases (BVMOs), we have created expression constructs for producing 22 Type I BVMOs that are present in the genome of Rhodococcus jostii RHA1. Each BVMO has been probed with a large panel of potential substrates. Except for testing their substrate acceptance, also the enantioselectivity of some...
Styrene monooxygenases (SMOs) are flavoenzymes catalyzing the epoxidation of styrene into styrene oxide. SMOs are composed of a monooxygenase (StyA) and a reductase (StyB). The latter delivers reduced FAD to StyA on the expense of NADH. We identified Rhodococcus opacus 1CP as the first microorganism to possess three different StyA isoforms occurrin...
Enantiopure sulfoxides can be prepared via the asymmetric oxidation of sulfides using sulfide monooxygenases. The n-octane-water biphasic system was chosen for the bio-oxidation of a water-insoluble phenyl methyl sulfide (PMS) by Rhodococcus sp. CCZU10-1. In this n-octane-water system, the optimum reaction conditions were obtained. (S)-phenyl methy...
Citations
... [38] In 2009, Li et al. isolated Rhodococcus sp. ECU0066, which converted the thioanisole as a sulfide model substrate to the S-isomer of phenyl methyl sulfoxide at a yield of 44.2 % and an enantiomeric excess of 99 % [39] via its whole cell. The whole cells of Rhodococcus sp. ...
... ECU0066 also exhibited good activities toward other substituted model sulfides, producing the S-enantiomer of the corresponding sulfoxides (Scheme F), whereas they exclusively produced the R-enantiomer in the case of the methoxysubstituted model. [39] The reaction conditions were then optimized in subsequent research to improve the product yield and convert higher concentrations of the substrate. [40] Among the tested organic-aqueous biphasic systems, the isooctaneaqueous system was found to be the most effective when employed with resting cells of Rhodococcus sp. ...
Actinobacteria are one of the most intriguing bacterial phyla in terms of chemical diversity and bioactivities of their reported biomolecules and natural products, including various types of chiral molecules. Actinobacterial genera such as Detzia, Mycobacterium, and Streptomyces are among the microbial sources targeted for selective reactions such as asymmetric biocatalysis catalyzed by whole cells or enzymes induced in their cell niche. Remarkably, stereoselective reactions catalyzed by actinobacterial whole cells or their enzymes include stereoselective oxidation, stereoselective reduction, kinetic resolution, asymmetric hydrolysis, and selective transamination, among others. Species of actinobacteria function with high chemo‐, regio‐, and enantio‐selectivity under benign conditions, which could help current industrial processing. Numerous selective enzymes were either isolated from actinobacteria or expressed from actinobacteria in other microbes and hence exploited in the production of pure organic compounds difficult to obtain chemically. In addition, different species of actinobacteria, especially Streptomyces species, function as natural producers of chiral molecules of therapeutic importance. Herein, we discuss some of the most outstanding contributions of actinobacteria to asymmetric biocatalysis, which are important in the organic and/or pharmaceutical industries. In addition, we highlight the role of actinobacteria as microbial cell factories for chiral natural products with insights into their various biological potentialities.
... Chiral sulfoxides may also be formed by whole cell bio-oxidation of prochiral sulfides, which has many benefits, such as cheap costs and no requirement of expensive cofactor regeneration. In recent years, whole cell bio-oxidation of prochiral sulfides in single water phase system has attracted a lot of attention [21][22][23][24][25][26][27][28][29][30][31]. As an excellent alternative to reactions catalyzed using pure enzymes, the use of whole cells including bacteria, fungi, and yeast in the asymmetric oxidation of sulfides can be cheap and simple to perform because of avoiding the use of expensive cofactors [32]. ...
(S)-Omeprazole is a very effective anti-ulcer medicine that is difficult to be prepared using whole cells at elevated substrate concentrations. In the chloroform–water biphasic system, resting cells of the mutant QZ-3 of Rhodococcus rhodochrous (R. rhodochrous) ATCC 4276 were used to catalyze the bio-oxidation of omeprazole sulfide for preparation of (S)-omeprazole. Using response surface methodology (RSM), the reaction was optimized to work at a substrate concentration of 180 mM and a cell concentration of 100 g/L. The optimal yield of (S)-omeprazole obtained was 92.9% with enantiomeric excess (ee) (> 99%), and no sulfone by-product was detected under the optimal working conditions; reaction temperature 37 °C, pH 7.3 and reaction time, 43 h. A quadratic polynomial model was established, which predicts the experimental data with very high accuracy (R2 = 0.9990). The chloroform–water biphasic system may contribute to the significant improvement in substrate tolerance because almost all substrates are partitioned in the organic phase (water solubility of omeprazole sulfide is only about 0.5 mg/mL), resulting in little damage and inhibition to cells by substrates. The mutant QZ-3 of R. rhodochrous ATCC 4276 exhibited high enantioselectivity, activity and substrate and product tolerance. The aerated flask provides enough oxygen for a high concentration of cells. Accordingly, bio-oxidation is thus more promising for efficient preparation of chiral sulfoxides.
... In contrast, biocatalyst-mediated exquisite asymmetric sulfoxidation using molecular oxygen as the oxidant under environment-friendly conditions, typically with high chemo-, regio-, and/or enantioselectivity, is emerging as an attractive green alternative to conventional chemical synthesis methods (Wenning et al. 2016;He et al. 2013;Li et al. 2009;Nikodinovic-Runic et al. 2013;Zhang et al. 2020). Unfortunately, only a very few native biocatalysts can be used to oxidize prazole-family sulfides because of their low activity. ...
... The sulfoxidation reactions of prazolefamily sulfides were analyzed by chiral HPLC as mentioned before. The product analysis for aromatic sulfides oxidation was detected as described by Li et al. (2009), and the oxygenation products of aliphatic ketones were identified by GC-MS as described by Zhang et al. (2018). ...
Biocatalytic synthesis of pharmaco-chiral sulfoxides has gained interest in recent years for its environmental friendliness. However, only a few natural biocatalysts can be used for the efficient synthesis of pharmaco-sulfoxides, including (R)-lansoprazole, a chiral proton pump inhibitor used to treat gastrointestinal diseases. In this study, the sequence of BoBVMO (Baeyer-Villiger monooxygenase from Bradyrhizobium oligotrophicum) was used as a probe to identify BVMOs via genomic mining for the highly efficient synthesis of (R)-lansoprazole and other pharmaco-sulfoxides. After virtual sequence filtering, target gene cloning, heterologous expression, and activity screening for lansoprazole sulfide (LPS) monooxygenation, seven new BVMOs were identified among more than 10,000 homologous BVMOs. According to the conserved sequence and phylogenetic tree analysis, these discovered enzymes belong to the family of type I BVMOs and the ethionamide monooxygenase subtype. Among them, CbBVMO, Baeyer-Villiger monooxygenase from Cupriavidus basilensis, showed the highest efficiency and excellent enantioselectivity for converting LPS into (R)-lansoprazole. Moreover, CbBVMO showed a wide substrate spectrum toward other bulky prazole-family sulfides. The results indicate that CbBVMO is a potential enzyme for extending the application of BVMOs in pharmaceutical industry.
Key points
• CbBVMO is the most efficient biocatalyst for (R)-lansoprazole biosynthesis.
• CbBVMO catalyzes the conversion of various bulky prazole sulfides.
• CbBVMO is a promising enzyme for the biosynthesis of pharmaco-sulfoxides.
... Rhodococci are also able to degrade various organic compounds, including ones highly toxic and recalcitrant; moreover, they are equipped with enzymes that catalyze biologically relevant reactions such as the biodesulfurization of fossil fuels [21], the degradation of polychlorinated biphenyls (PCBs) [22], and the use of a large variety of other organic compounds as sources of energy [19]. They are also able to degrade halogenated hydrocarbons with long or short chains [23] and to metabolize numerous halogenated differently substituted aromatic and heteroaromatic compounds [24], bioactive steroids [25], and acrylamide [26]. ...
Cortisone is a steroid widely used as an anti-inflammatory drug able to suppress the immune system, thus reducing inflammation and attendant pain and swelling at the site of an injury. Due to its numerous side effects, especially in prolonged and high-dose therapies, the development of the pharmaceutical industry is currently aimed at finding new compounds with similar activities but with minor or no side effects. Biotransformations are an important methodology towards more sustainable industrial processes, according to the principles of “green chemistry”. In this work, the biotransformation of cortisone with Rhodococcus rhodnii DSM 43960 to give two new steroids, i.e., 1,9β,17,21-tetrahydoxy-4-methyl-19-nor-9β-pregna-1,3,5(10)-trien-11,20-dione and 1,9β,17,20β,21-pentahydoxy-4-methyl-19-nor-9β-pregna-1,3,5(10)-trien-11-one, is reported. These new steroids have been fully characterized.
... To date, most characterized CYP116Bs have been isolated from bacteria of the genus Rhodococcus, including CYP116B2, which is one of the first class VII P450 discovered [15], the self-sufficient CYP116B3 [16], and the so-called P450 SMO [17]. This genus also harbors P450s of the subfamily CYP116A, which includes non-self-sufficient members [18]. ...
Members of class VII cytochromes P450 are catalytically self-sufficient enzymes containing a phthalate dioxygenase reductase-like domain fused to the P450 catalytic domain. Among these, CYP116B46 is the first enzyme for which the 3D structure of the whole polypeptide chain has been solved, shedding light on the interaction between its domains, which is crucial for catalysis. Most of these enzymes have been isolated from extremophiles or detoxifying bacteria that can carry out regio- and enantioselective oxidation of compounds of biotechnological interest. Protein engineering has generated mutants that can perform challenging organic reactions such as the anti-Markovnikov alkene oxidation. This potential, combined with the detailed 3D structure, forms the basis for further directed evolution studies aimed at widening their biotechnological exploitation.
... strain ECU066 primarily producing (S)-sulfoxides at rather low rates (< 1 U g cdw À 1 ). [18] For Rhodococcus sp. CCZU10-1, a high (S)selectivity for the sulfoxidation of sulfides 1-4 (e.e. > 99.9 %) was reported, but specific activities remained low (< 1 U g cdw À 1 , increased up to 6.4 U g cdw À 1 upon application of n-octane as second liquid organic phase). ...
... [36] Moreover, no overoxidation to sulfones was observed, which poses a common drawback among asymmetric sulfoxidations employing chemical [2] or biological catalysts such as Baeyer-Villiger monooxygenases, [13,37] P450 monooxygenases, peroxygenases, and peroxidases. [12,18,38,39] The specific sulfoxidation catalysts described so far mainly give access to (R)enantiomers at lower rates emphasizing the attractiveness of the whole-cell biocatalyst reported here. [40] Interestingly, the BVMO designated 4-hydroxyacetophenone monooxygenase (HAPMO) has been reported to produce highly pure (S)-and (R)enantiomers in dependence of the nature of the sulfide supplied as substrate. ...
Chiral sulfoxides have gained attention as synthons and precursors for API synthesis. Flavoproteins such as Baeyer‐Villiger or styrene monooxygenases mainly provide access to (R)‐sulfoxides and often suffer from low selectivity, activity, and/or limited substrate scope. The flavoprotein monooxygenase AbIMO from Acinetobacter baylyi ADP1 initiates indole degradation. Here, AbIMO was expressed recombinantly in E. coli and characterized for its sulfoxidation activity and substrate spectrum. Next to indole and styrene, AbIMO was found to accept numerous alkyl aryl sulfides as substrates, transforming them to (S)‐sulfoxides with high enantioselectivity (95 % to >99 % for most sulfides). The formulation as a whole‐cell biocatalyst allowed specific production rates of up to 370 U gcdw⁻¹ – the highest specific oxygenase activity achieved in whole cells so far – and the preparative synthesis of enantiopure (S)‐aryl alkyl sulfoxides. With its extraordinarily high specific activity, high specificity, ease of handling, and high stability (catalyst is stable for >16 days at 4 °C), the designed whole‐cell biocatalyst adds enormous value to the portfolio of chemical and biological catalysts for asymmetric sulfoxide synthesis.
... Phenyl methyl sulfide (PMS) was oxidized into (S)-phenyl methyl sulfoxide (PMSO) by whole-cells of R. sp. ECU0066 [234,235] and R. sp. CCZU10-1 [233]. ...
The application of purified enzymes as well as whole-cell biocatalysts in synthetic organic chemistry is becoming more and more popular, and both academia and industry are keen on finding and developing novel enzymes capable of performing otherwise impossible or challenging reactions. The diverse genus Rhodococcus offers a multitude of promising enzymes, which therefore makes it one of the key bacterial hosts in many areas of research. This review focused on the broad utilization potential of the genus Rhodococcus in organic chemistry, thereby particularly highlighting the specific enzyme classes exploited and the reactions they catalyze. Additionally, close attention was paid to the substrate scope that each enzyme class covers. Overall, a comprehensive overview of the applicability of the genus Rhodococcus is provided, which puts this versatile microorganism in the spotlight of further research.
... A recent study proposes that the transformation of IBP and other drugs is probably the outcome of the enzymatic activities of the bacterial strain. For dealkylation, hydroxylation and oxidation processes, the monooxygenase enzymes produced by bacteria are accountable for transformation of drugs (Li et al., 2009). Hydrolysis and decarboxylation occurs due to hydrolase and decarboxylase enzymes (Shimazu et al., 2001;Young et al., 2010). ...
Pharmaceutical effluents released from industries are accountable to deteriorate the aquatic and soil environment through indirect toxic effects. Microbes are adequately been used to biodegrade pharmaceutical industry wastewater and present study was envisaged to determine biodegradation of pharmaceutical effluent by Micrococcus yunnanensis. The strain showed 42.82% COD (Chemical oxygen demand) reduction before optimization. After applying Taguchi's L8 array as an optimization technique, the biodegradation rate was enhanced by 82.95% at optimum conditions (dextrose- 0.15%, peptone 0.1%, inoculum size 4% (wv-1), rpm 200, pH 8 at 25 °C) within 6 h. The confirmation of pharmaceuticals degradation was done by 1H NMR (Nuclear magnetic resonance) studies followed by elucidation of transformation pathways of probable drugs in the effluent through Q-Tof-MS (Quadrupole Time of Flight- Mass Spectrometry). The cytotoxicity evaluation of treated and untreated wastewater was analyzed on Human Embryonic Kidney (HEK 293) cells using Alamar Blue assay, which showed significant variance.
... Xu and coworkers [55] reported the catalytic performance of a newly isolated bacterium, Rhodococcus sp. strain ECU0066, in the asymmetric oxidation of aryl sulfides 92 to the corresponding sulfoxides (93). ...
Currently, the power and usefulness of biocatalysis in organic synthesis is undeniable, mainly due to the very high enantiomeric excess reached using enzymes, in an attempt to emulate natural processes. However, the use of isolated enzymes has some significant drawbacks, the most important of which is cost. The use of whole cells has emerged as a useful strategy with several advantages over isolated enzymes; for this reason, modern research in this field is increasing, and various reports have been published recently. This review surveys the most recent developments in the enantioselective reduction of carbon-carbon double bonds and prochiral ketones and the oxidation of prochiral sulfides using whole cells as biocatalytic systems.
... Biocatalytic procedures present some advantages, such as the use of mild and environmentally friendly reaction conditions and oxidants [7,8]. Several types of oxidative enzymes can be used for the synthesis of chiral sulfoxides, which include peroxidases [9][10][11], dioxygenases [12,13] and different types of monooxygenases [14,15]. BVMOs represent a class of flavin-containing monooxygenases that catalyze the Baeyer-Villiger oxidation of aldehydes and ketones, but they are also able to perform epoxidations and the oxygenation of sulfides and other heteroatom-containing compounds, often with high chemo-, regio-and/or enantioselectivity [16][17][18][19]. ...
... As previously reported, PockeMO showed a moderate tolerance to organic cosolvents in the Baeyer-Villiger oxidation of ketones. In addition, certain BVMOs have shown interesting properties in the biocatalyzed sulfoxidation of prochiral sulfides in presence of organic cosolvents [14]. For these reasons, we decided to test the effect of organic cosolvents with different properties, using 2a as model sulfide substrate, a substrate which was oxidized with good activity and selectivity to (R)-2b in 50 mM Tris/HCl, pH 8.0. ...
A recently discovered, moderately thermostable Baeyer-Villiger monooxygenase, polycyclic ketone monooxygenase (PockeMO), from Thermothelomyces thermophila has been employed as a biocatalyst in a set of asymmetric sulfoxidations. The enzyme was able to catalyze the oxidation of various alkyl aryl sulfides with good selectivities and moderate to high activities. The biocatalytic performance was able to be further increased by optimizing some reaction parameters, such as the addition of 10% v v−1 of water miscible solvents or toluene, or by performing the conversion at a relatively high temperature (45 °C). PockeMO was found to display an optimum activity at sulfide concentrations of 50 mM, while it can also function at 200 mM. Taken together, the data show that PockeMO can be used as robust biocatalyst for the synthesis of optically active sulfoxides.
