Aqueous-organic Two-phase System Research Articles

Styrene Oxide Isomerase Catalyzed Meinwald Rearrangement Reaction: Discovery and Application in Single-Step and One-Pot Cascade Reactions

Styrene oxide isomerase (SOI) is a unique enzyme that catalyzes the Meinwald rearrangement of aryl epoxides into carbonyl compounds. In this review, we first summarize the scientific literature on the discovery and characterization of SOI’s catalytic properties such as activity, substrate scope, and enantioselectivity. We then discuss its potential use for biocatalytic synthesis of aldehydes via single-step reactions using cell-free extracts and whole cells. Emerging technologies, such as the use of a stability tag, could enhance SOI biotransformation to reach product concentrations as high as 3.37 M. SOI has also been utilized in the engineering of biocatalytic cascades to produce valuable compounds such as chiral acids, alcohols, and amines from styrene. The best systems developed to date could reach product concentrations up to 100 mM. Additionally, recent efforts are focused on the metabolic engineering of the “styrene-derived” pathway involving SOI for microbial production of the aroma compound 2-phenylethanol (2-PE) from renewable sources such as l-phenylalanine, glucose, or glycerol, achieving concentrations up to 85 mM. The application of two-phase aqueous–organic systems in practical biotransformation reactions involving SOI is also notable. Finally, some perspectives and recommendations for future work are given to set the stage for development of SOI research toward applications in sustainable industrial biocatalysis.

Chiral Yolk-Shell MOF as an Efficient Nanoreactor for Asymmetric Catalysis in Organic-Aqueous Two-Phase System.

It remains a great challenge to introduce large and efficient homogeneous asymmetric catalysts into MOFs and other microporous materials as well as retain their degrees of freedom. Herein, a new heterogeneous strategy of homogeneous chiral catalysts is proposed, that is, to construct a yolk-shell MOFs-confined, large-size, and highly efficient homogeneous chiral catalyst, which can be used as a nanoreactor for asymmetric catalytic reactions.

ω-Transaminase-Mediated Asymmetric Synthesis of (S)-1-(4-Trifluoromethylphenyl)Ethylamine

The pivotal role played by ω-transaminases (ω-TAs) in the synthesis of chiral amines used as building blocks for drugs and pharmaceuticals is widely recognized. However, chiral bulky amines are challenging to produce. Herein, a ω-TA (TR8) from a marine bacterium was used to synthesize a fluorine chiral amine from a bulky ketone. An analysis of the reaction conditions for process development showed that isopropylamine concentrations above 75 mM had an inhibitory effect on the enzyme. Five different organic solvents were investigated as co-solvents for the ketone (the amine acceptor), among which 25–30% (v/v) dimethyl sulfoxide (DMSO) produced the highest enzyme activity. The reaction reached equilibrium after 18 h at 30% of conversion. An in situ product removal (ISPR) approach using an aqueous organic two-phase system was tested to mitigate product inhibition. However, the enzyme activity initially decreased because the ketone substrate preferentially partitioned into the organic phase, n-hexadecane. Consequently, DMSO was added to the system to increase substrate mass transfer without affecting the ability of the organic phase to prevent inhibition of the enzyme activity by the product. Thus, the enzyme reaction was maintained, and the product amount was increased for a 62 h reaction time. The investigated ω-TA can be used in the bioconversion of bulky ketones to chiral amines for future bioprocess applications.

First Steps to Rationalize Host-Guest Interaction between α-, β-, and γ-Cyclodextrin and Divalent First-Row Transition and Post-transition Metals (Subgroups VIIB, VIIIB, and IIB).

Cyclodextrins (CDs) are cyclic oligosaccharides mainly composed of six, seven, and eight glucose units, so-called α-, β-, and γ-CDs, respectively. They own a very particular molecular structure exhibiting hydrophilic features thanks to primary and secondary rims and delimiting a hydrophobic internal cavity. The latter can encapsulate organic compounds, but the former can form supramolecular complexes by hydrogen-bonding or electrostatic interactions. CDs have been used in catalytic processes to increase mass transfer in aqueous-organic two-phase systems or to prepare catalysts. In the last case, interaction between CDs and metal salts was considered to be a key point in obtaining highly active catalysts. Up to now, no work was reported on the investigation of factors affecting the binding of metal to CD. In the study herein, we present the favorable combination of electrospray ionization coupled to mass spectrometry [ESI-MS(/MS)] and density functional theory molecular modeling [B3LYP/Def2-SV(P)] to delineate some determinants governing the coordination of first-row divalent transition metals (Mn2+, Co2+, Ni2+, Cu2+, and Fe2+) and one post-transition metal (Zn2+) with α-, β-, and γ-CDs. A large set of features concerning the metal itself (ionic radius, electron configuration, and spin state) as well as the complexes formed (the most stable conformer, relative abundance in MS, CE50 value in MS/MS, binding energy, effective coordination number, average bond lengths, binding site localization, bond dissociation energies, and natural bond orbital distribution) were screened. Taking into account all of these properties, various selectivity rankings have been delineated, portraying differential association/dissociation behaviors. Nonetheless, unique 3D topologies for each CD-metal complex were emphasized. The combination of these approaches brings a stone for building a compendium of molecular features to serve as a suitable descriptor or predictor for a better first round rationalization of catalytic activities.

Cascade Biocatalysis for Regio- and Stereoselective Aminohydroxylation of Styrenyl Olefins to Enantiopure Arylglycinols

Chiral β-amino alcohols are privileged scaffolds frequently found in pharmaceutically active molecules and natural products. Aminohydroxylation of olefins is one of the most powerful strategies to access chiral vicinal amino alcohols. However, the direct regio- and stereoselective aminohydroxylation of olefins to unprotected enantioenriched β-amino alcohols remains a long-standing challenge. Herein, we report that a novel one-pot four-enzyme [styrene monooxygenase (SMO)/epoxide hydrolase (EH)/alcohol dehydrogenase (ADH)/ω-transaminase (TA)] biocatalytic cascade efficiently catalyzes the direct transformation of readily available styrenyl olefins into unprotected 2-amino-2-phenyl ethanols in good yields and excellent enantioselectivity. In vitro cascade biocatalysis aminohydroxylation of styrenyl olefins was first investigated by the combined four enzymes (SMO/EH/ADH/TA) with a trace amount of NADH (0.02 mM) and pyridoxal-5′-phosphate (0.1 mM), affording both enantiomers of β-amino alcohols 5a–j in 13.9–98.7% conversions and 86–99% ee. Whole-cell-based cascade biocatalysis was achieved by using the constructed recombinant Escherichia coli pairwise combinations and single tailor-made whole-cell biocatalyst without an additional NADH cofactor; (R)- and (S)-β-amino alcohols 5a–j could be obtained in 14.6–99.7% conversions and 86–99% ee. Moreover, the preparative experiments of this new cascade biocatalysis were demonstrated by the single tailor-made whole-cell biocatalyst [E. coli (CGS-DEM) and E. coli (CGS-DEB)] with the substrates 1a–b and 1h in an aqueous-organic two-phase system, affording chiral β-amino alcohols [(R)- or (S)-5a–b, h] in good yields (50.9–64.3%) and excellent ee (>99%).

Extraction of Lanthanide Chlorides in Aqueous-Organic Two-Phase Systems with Salts of Tertiary and Secondary Amines

Extraction of Lanthanide Chlorides in Aqueous-Organic Two-Phase Systems with Salts of Tertiary and Secondary Amines

Rational design of halohydrin dehalogenase for efficient chiral epichlorohydrin production with high activity and enantioselectivity in aqueous-organic two-phase system

Halohydrin dehalogenases (HHDHs) represent as important biocatalysts for the production of valuable chiral epoxides and β-substituted alcohols. The HHDH from A. radiobacter (HheC) showed great potential for biosynthesis of (S)-epichlorohydrin ((S)-ECH). However, two bottlenecks including self-degradation and enzyme-mediated racemization of product in aqueous solution restricted its wide-spread applications. To solve the problems, the biocatalytic process was performed in aqueous-organic two-phase system and structure-based engineered of HheC was performed. A newly found zigzag tunnel connecting the active site region and outside space, as well as a depressed cave structure in formation of an extra three-pot herringbone channel were demonstrated to play a pivotal role affecting the activity and enantioselectivity of the enzyme. By mutation of the involved amino acid residues, two variants E255 T and E255 G showed significantly improved activity, with specific activity increased from 59.9 U/mg to 101.6 U/mg and 77.5 U/mg, respectively, while one variant Y93 L exhibited high enantioselectivity, with ee value of (S)-ECH maintained at >99 %. In addition, the synergic variant Y93 L/E255 T had both improved catalytic efficiency and enantioselectivity. The results not only facilitate the applicability of HheC for efficient production of (S)-ECH, but also give new insights into the structural-functional relationship of the enzyme.

Interphase Distribution of Lanthanide Nitrates in Aqueous Organic Two-Phase Systems with Amine and Organic Acid Salts

The extraction of lanthanide nitrates from 0.5 M NaNO3 solutions in 1 : 1 : 1 ternary aqueous organic systems using mixtures of trioctylammonium dialkyl phosphate and dicyclohexylammonium caprylate as extractants was studied. The extractability of lanthanides was shown to increase in the series La < Pr < Nd, Sm, Eu < Gd < Tb < Dy < Ho < Er < Tm, in which the atomic number of the metal increases. The selectivity of extraction in the ternary system was observed during the extraction of heavy lanthanides.

Understanding the role of the hydrogen bond donor of the deep eutectic solvents in the formation of the aqueous biphasic systems

Deep eutectic solvents (DES) have been proposed as phase-forming compounds of aqueous biphasic systems (ABS). However, due to DES nature and the high water content of the ABS, their nature and behavior remains controversial. To foster the understanding of DES-based ABS, the present work uses the relative hydrophilicity of the hydrogen bond donor (HBD) as a descriptor to clarify its role on ABS formation and phase properties. Dipotassium phosphate (K2HPO4)-based ABS phase diagrams composed of cholinium chloride ([N111(2OH)]Cl), as hydrogen bond acceptor (HBA), and several HBD (alcohols and sugars) were compared in molality units and the saturation solubility point for each binodal curve determined. The results here reported establish the HBD role as function of its relative hydrophilicity: very hydrophilic HBD act only as an adjuvant in the formation of ABS; HBD of intermediate hydrophilicity influence the ABS formation; while the most hydrophobic HBD tend to form organic-aqueous two-phase systems, where the HBA acts as an adjuvant to the system.

Synthesis of Furanic Polyamides and Composite Coatings from Plant Biomass

We report, for the first time, the synthesis of polyamide composite coating based on renewable plant biomass sources. 2,5-furandicarboxylic acid (FDCA) was prepared by catalytic oxidation of 5-hydroxymethylfurfural (HMF) obtained by plant biomass conversion. FDCA was used in the synthesis of aliphatic and aromatic furanic polyamides. Two approaches to furanic PAs synthesis have been investigated: (i) synthesis of hexamethylenediamine furanoate salt and its subsequent polycondensation; (ii) synthesis of FDCA dichloride and its subsequent polycondensation in a two-phase aqueous-organic system. The effect of the nature of organic solvent (tetraclormetan, dichloromethane, N-methyl-2-pyrrolidone) and the nature of diamine (hexamethylenediamine and paraphenylenediamine) on the yield and molecular weight of furanic polyamide was studied. The synthesized aliphatic polyamide was used for enamel fabrication. Colloidal graphite and activated carbon obtained from the waste of biomass conversion into HMF were used as fillers. The furanic PA composite coatings on steel provide the lower coefficient of friction and lower wear compared with that of commercial polyamide (PA6) coatings and can be considered as novel promising anti-friction coating materials.

Isoprene Synthesis Using MIL-101(Cr) Encapsulated Silicotungstic Acid Catalyst

A single-stage synthesis of isoprene from methyl tert-butyl ether (MTBE) and formalin in an organic-aqueous two-phase system was studied by using solid acid catalysts, i.e., USY zeolite, silicotungstic acid (STA) 25 wt% encapsulated in MIL-101(Cr) (STA25@MIL-101), and STA 25 wt% encapsulated in SBA-15 (STA25@SBA-15). From preliminary experiments, the catalytic activity decreased in the order: STA25@MIL-101 > SBA25@SBA-15 > USY zeolite. This suggested that isoprene formation was favored with high surface area and high acid strength of catalyst. Then, the porous hybrid material of a MIL-101 metal organic framework and STA was studied in more detail. MIL-101 was not efficient for isoprene synthesis at mild reaction condition. On increasing STA loading, which was well correlated with the Brӧnsted acid property, the catalyst activity increased in the order: MIL-101 < STA30@MIL-101 < STA60@MIL-101. The high acidity catalyst gave high isoprene yield at optimum low temperature and low side reaction products. For the STA30@MIL-101 and STA60@MIL-101 catalysts, the isoprene yield could be sustained at 18.5% (0.4% SD) and 30.0% (1.5% SD), respectively over three recycling runs. It is apparent that no STA leaching from the low STA loading catalyst occurred.

Reactive Extraction of Zn and Cu with Bis(2-Ethylhexyl)Phosphate: Optimization using Taguchi Orthogonal Design

A comprehensive study on the reactive extraction of Zn and Cu has been established in an aqueous-organic two-phase system using bis(2-ethylhexyl)phosphate (HDEHP) to separate both the metals with the single extractant. Taguchi’s experimental design with orthogonal array L25 has been employed to evaluate the effect of most influencing parameters such as aqueous phase pH, carrier concentration, and contact time; experiments have been conducted accordingly. The optimum conditions have been obtained as following: aqueous phase pH 3.5 and 5.5 for Zn and Cu, respectively; carrier concentration, 15%; equilibrium time, 30 min. Under these optimum conditions, a quantitative extraction of Zn (91.8%) and Cu (90%) has been observed. Extraction equilibrium constant Keq has been estimated as 3 × 10−3 (mol/L)1/2 and 2.78 × 10−3 (mol/L)−1/2 for Zn and Cu, respectively. Herein, a complementary kinetic study has been also performed and the extraction process has been found to conform to the chemical reaction controlled mechanism.

Effects of limonene, n-decane and n-decanol on growth and membrane fatty acid composition of the microalga Botryococcus braunii

Botryococcus braunii is a promising microalga for the production of biofuels and other chemicals because of its high content of internal lipids and external hydrocarbons. However, due to the very thick cell wall of B. braunii, traditional chemical/physical downstream processing very often is not as effective as expected and requires high amounts of energy. In this cases, the application of two-phase aqueous-organic solvent systems could be an alternative to cultivate microalgae allowing for a simultaneous extraction of the valuable compounds without significant negative effects on cell growth. Two-phase systems have been applied before, however, there are no studies so far on the mechanisms used by microalgae to survive in contact with solvents present as a second-phase. In this study, the effects of the solvents limonene, n-decane and n-decanol on growth of the microalga B. braunii as well as the adaptive cell response in terms of their phospholipid fatty acid contents were analized. A concentration-dependent negative effect of all three solvents on cell growth was observed. Effects were accompanied by changes of the membrane fatty acid composition of the alga as manifested by a decrease of the unsaturation . In addition, an association was found between the solvent hydrophobicity (given as log octanol–water partition coefficient (text {P}_{O-W}) values) and their toxic effects, whereby n-decanol and n-decane emerged as the most and least toxic solvent respectively. Among the tested solvents, the latter promises to be the most suitable for a two-phase extraction system.

Phase Transfer of the Organic Substrate in the Epoxidation Reaction of Allyl Chloride in Two-Phase Aqueous–Organic Systems

The mechanism of synergism for mixtures of phase-transfer carriers QХ with tertiary amines and pyridine in the epoxidation reaction of allyl chloride has been established. It has been shown that tertiary amines (triethylamine, tributylamine, N,N-dimethylaniline, and N-methyldiethanolamine) and pyridine oxidizable in situ to N-oxides promote the transfer of the organic substrate (allyl chloride) to the interface (PB). It is assumed that the synergism of mixtures of phase-transfer carriers QХ with a tertiary amine oxide or pyridine oxide can also be due to the formation of a mixture of two catalysts, Q3[PW4O24] and WO(O2)2L (where L = a tertiary amine oxide or pyridine oxide). The WO(O2)2L complex will provide the stage of reoxidation of the complex Q3[PW4O24 −х] in the organic phase and, hence, the possibility for the development of the process of epoxidation not only at the interface but also in the bulk of the organic phase. The maximum coefficient of synergistic effect (ks = 2) is observed for a mixture of cetylpyridinium bromide (90 mol %) and pyridine N-oxide (10 mol %).

Liquid-phase synthesis of isoprene from MTBE and formalin using cesium salts of silicotungstic acid

A single-stage synthesis of isoprene from methyl tertiary-butyl ether (MTBE) and formalin in an organic-aqueous two-phase system were studied by using Cs-exchanged silicotungstic acid catalysts (CsXH4-XSiW12O40, abbreviated as CsX-STA). The CsX-STA catalysts (x=2.0, 2.5, 3.0, and 3.5) were prepared using Cs2CO3 as Cs source and water as solvent. The catalysts were characterized by using XRF, FTIR, FT-Raman, XRD, N2 sorption, light scattering, TGA, and potentiometric titration techniques to relate catalytic activities to catalyst properties. The characterization results showed that the Keggin structure and cubic unit cell of all Cs-STA catalyst samples were well preserved. The increase in acid strength and total acid sites with decreasing Cs content (x) resulted in increasing formaldehyde conversion and isoprene selectivity. However, at low Cs content (x≤3.0), the high contribution of homogeneous catalysis also played a role in the catalytic activities. The highest activity (isoprene formation normalized with total acid sites) of Cs2.5-STA catalyst was found to be attributed to its high surface area. For the organic solvent effect, the Cs2.5-STA catalyst activity increased with the increasing solvent polarity in the order: n-hexane < cyclohexane < toluene.

Multi-site phase transfer catalyzed radical polymerization of methyl methacrylate in mixed aqueous\u2013organic medium: a kinetic study

This work establishes the kinetics of radical polymerization of methyl methacrylate in an aqueous–organic two-phase system using 1,4-bis (triethylmethylammonium) benzene dichloride (TEMABDC) as multi-site phase transfer catalyst and potassium peroxydisulphate (K2S2O8) as water-soluble initiator at 60 ± 1 °C under nitrogen atmosphere. The role of concentrations of monomer, initiator, catalyst, acid and ionic strength, temperature and volume fraction of aqueous phase on the rate of polymerization (Rp) was investigated. The rate of polymerization (Rp); Rp α [MMA]0.64, [TEMABDC]1.24 and [K2S2O8]1.50. The rate of polymerization increases with an increase in the concentration of monomer, initiator, catalyst and temperature. A generalized reaction model was developed to explain the phase transfer catalyzed polymerization reaction. Based on the kinetic results, radical mechanism has been derived. The activation energy and other thermodynamic parameters were calculated. The FT-IR spectroscopy validates a band of 1732 cm−1 of ester group of the obtained polymer. The viscosity average molecular weight of the PMMA was found 1.6955 × 104 g/mol.

Removal of the product from the culture medium strongly enhances free fatty acid production by genetically engineered Synechococcus elongatus

BackgroundCyanobacterial mutants engineered for production of free fatty acids (FFAs) secrete the products to the medium and hence are thought to be useful for biofuel production. The dAS1T mutant constructed from Synechococcus elongatus PCC 7942 has indeed a large capacity of FFA production, which is comparable to that of triacylglycerol production in green algae, but the yield of secreted FFAs is low because the cells accumulate most of the FFAs intracellularly and eventually die of their toxicity. To increase the FFA productivity, enhancement of FFA secretion is required.ResultsGrowth of dAS1T cells but not WT cells was inhibited in a liquid medium supplemented with 0.13 g L−1 of palmitic acid. This suggested that when FFA accumulates in the medium, it would inhibit the release of FFA from the cell, leading to FFA accumulation in the cell to a toxic level. To remove FFAs from the medium during cultivation, an aqueous-organic two-phase culture system was developed. When the dAS1T culture was overlaid with isopropyl myristate (IM), the final cell density, cellular chlorophyll content, and the photosynthetic yield of PSII were greatly improved. The total amount of extracellular FFA was more than three times larger than that in the control culture grown without IM, with most of the secreted FFAs being recovered in the IM layer. The cellular FFA content was decreased by more than 85% by the presence of the IM layer. Thus, the two-phase culture system effectively facilitated FFA secretion out of the cell. An average FFA excretion rate of 1.5 mg L−1 h−1 was attained in the 432 h of cultivation, with a total amount of excreted FFA being 0.64 g L−1 of culture. These figures were more than three times higher than those reported previously for the cyanobacteria-based FFA production systems.ConclusionsRemoval of FFA from the culture medium is important for improving the productivity of the FFA production system using cyanobacteria. Further increase in productivity would require an increase in both the rates of FFA production in the cell and active FFA export across the plasma membrane.

Engineering Escherichia coli for high-yield geraniol production with biotransformation of geranyl acetate to geraniol under fed-batch culture.

BackgroundGeraniol is an acyclic monoterpene alcohol, which exhibits good prospect as a gasoline alternative. Geraniol is naturally encountered in plants at low concentrations and an attractive target for microbial engineering. Geraniol has been heterologously produced in Escherichia coli, but the low titer hinders its industrial applications. Moreover, bioconversion of geraniol by E. coli remains largely unknown.ResultsRecombinant overexpression of Ocimum basilicum geraniol synthase, Abies grandis geranyl diphosphate synthase, and a heterotic mevalonate pathway in E. coli BL21 (DE3) enabled the production of up to 68.6 ± 3 mg/L geraniol in shake flasks. Initial fed-batch fermentation only increased geraniol production to 78.8 mg/L. To further improve the production yield, the fermentation conditions were optimized. Firstly, 81.4 % of volatile geraniol was lost during the first 5 h of fermentation in a solvent-free system. Hence, isopropyl myristate was added to the culture medium to form an aqueous-organic two-phase culture system, which effectively prevented volatilization of geraniol. Secondly, most of geraniol was eventually biotransformed into geranyl acetate by E. coli, thus decreasing geraniol production. For the first time, we revealed the role of acetylesterase (Aes, EC 3.1.1.6) from E. coli in hydrolyzing geranyl acetate to geraniol, and production of geraniol was successfully increased to 2.0 g/L under controlled fermentation conditions.ConclusionsAn efficient geraniol production platform was established by overexpressing several key pathway proteins in engineered E. coli strain combined with a controlled fermentation system. About 2.0 g/L geraniol was obtained using our controllable aqueous-organic two-phase fermentation system, which is the highest yield to date. In addition, the interconversion between geraniol and geranyl acetate by E. coli was first elucidated. This study provided a new and promising strategy for geraniol biosynthesis, which laid a basis for large-scale industrial application.

Comparative study on hydrolysis of oils by lipase immobilized biocatalytic PS membranes using biphasic enzyme membrane reactor

In the present study the hydrolysis of olive, palm and castor oil had been carried out to yield valuable free fatty acids by using immobilized Candida rugosa lipase. This study was designed to achieve high yield of lipase immobilization by investigating different factors (applied pressure, time, and lipase concentration) for ultrafiltration process of immobilization. A biphasic enzyme membrane bioreactor was fabricated having aqueous-organic two-phase system with lipase immobilized polysulfone. Effect of operating variables on the performance of this biphasic EMR was investigated with the hydrolysis of olive oil, palm oil and castor oil.It was found that the optimal conditions for highest degree of hydrolysis (%) were temperature 37°C, pH 8.0, at concentration of 0.05M in iso-octane and initial reaction time was 24h. In optimal conditions, degree of hydrolysis for olive oil, palm oil and castor oil was 38.5%, 35.4% and 21.2% respectively. Reusability feature was also studied and only ∼20% decrease in activity was observed after five cycles.

Enzymatic hydrolysis in an aqueous organic two-phase system using centrifugal partition chromatography

Multi-phase reaction systems, mostly aqueous organic systems, are used in enzyme catalysis to convert hydrophobic substrates which are almost insoluble in aqueous media. In this study, a Centrifugal Partition Chromatograph is used as a compact device for enzymatic multi-phase reaction that combines efficient substrate supply to the aqueous phase and separation of both phases in one apparatus. A process design procedure to systematically select the aqueous and organic phase to achieve stable and efficient reaction rates and operation conditions in Centrifugal Partition Chromatography for efficient mixing and separation of the phases is presented. The procedure is applied to the hydrolysis of 4-nitrophenyl palmitate with a lipase derived from Candida rugosa. It was found that the hydrolysis rate of 4-nitrophenyl palmitate was two times higher in Centrifugal Partition Chromatography than in comparable stirred tank reactor experiments.