Aqueous-organic Two-phase System Research Articles

Bioproduction of 4-vinylphenol from corn cob alkaline hydrolyzate in two-phase extractive fermentation using free or immobilized recombinant E. coli expressing pad gene

In situ extractive fermentation was used to produce 4-vinyl derivatives from hydroxycinnamic acids extracted from corn cobs by recombinant Escherichia coli cells expressing Lactobacillus plantarum phenolic acid descarboxylase (PAD) gene. This microorganism mainly produced 4-vinylphenol (4VP) from p-coumaric acid (p-CA). In the first study , we observed that the concentrations of 4VP are higher than 1g/L which had a negative impact on decarboxylation of p-CA to 4VP by recombinant E. coli cells. Because of this, and in order to improve the downstream process, a two-phase aqueous-organic solvent system was developed. The results of the extractive fermentation indicated that it was possible to use hydrolyzates as aqueous phase to bioproduce 4VP, and recover simultaneously the product in the organic phase containing hexane. The detoxification of pre-treated corn cob alkaline hydrolyzate improved 4VP production up to 1003.5mg/L after 24h fermentation (QP=41.813mg/Lh). Additionally, preliminary experiments using cells immobilized in calcium alginate showed to be a good system for the biotransform of p-CA to 4VP in extractive fermentation, although the process hindered partially the recovery of 4VP in the organic phase.

Enhanced 3-methylcatechol production by Pseudomonas putida TODE1 in a two-phase biotransformation system.

In this study, genetically engineered Pseudomonas putida TODE1 served as a biocatalyst for the bioproduction of valuable 3-methylcatechol (3MC) from toluene in an aqueous-organic two-phase system. The two-phase system was used as an approach to increase the biocatalyst efficiency. Among the organic solvent tested, n-decanol offered several benefits including having the highest partitioning of 3MC, with a high 3MC yield and low cell toxicity. The effect of media supplementation with carbon/energy sources (glucose, glycerol, acetate and succinate), divalent metal cations (Mg(2+), Ca(2+), Mn(2+) and Fe(2+)), and short-chain alcohols (ethanol, n-propanol and n-butanol) as a cofactor regeneration system on the toluene dioxygenase (TDO) activity, cell viability, and overall 3MC yield were evaluated. Along with the two-step cell preparation protocol, supplementation of the medium with 4 mM glycerol as a carbon/energy source, and 0.4 mM Fe(2+) as a cofactor for TDO significantly enhanced the 3MC production level. When in combination with the use of n-decanol and n-butanol as the organic phase, a maximum overall 3MC concentration of 31.8 mM (166 mM in the organic phase) was obtained in a small-scale production, while it was at 160.5 mM (333.2 mM in the organic phase) in a 2-L scale. To our knowledge, this is the highest 3MC yield obtained from a TDO-based system so far.

Hydrolysis of isoflavone glycoside by immobilization of β-glucosidase on a chitosan-carbon in two-phase system

We explored a method to examine the hydrolysis of isoflavone glycoside by immobilizing β-glucosidase on chitosan-carbon beads in an aqueous–organic two-phase system. The chitosan-carbon beads were cross-linked with glutaraldehyde to immobilize β-glucosidase from Exiguobacterium sp. DAU5. The optimal pH and temperature were 9.0 and 55°C, respectively. Under the optimized conditions, crude and purified enzymes immobilized onto chitosan-carbon beads gave yields of 16.7% and 60%, respectively. The specific activities of immobilized crude and purified enzymes were 4.3U/g and 6U/g, respectively. The immobilized enzyme retained more than 80% of its maximum activity at pH 7.0–11.0, while temperature was more influential (80% activity after 40°C for 1.5h, but only 40% activity after 55°C for 0.5h, respectively. The immobilized enzyme was able to hydrolyze isoflavone glycoside in an aqueous–organic two-phase system, and the hydrolyzed products were enriched in the organic phase, making it easy to recover the products, i.e., genistein and daidein from the reaction system. These results suggest that immobilized β-glucosidase may be applicable for the industrial-scale hydrolysis of isoflavone glycoside.

Fusion of a Flavin-Based Fluorescent Protein to Hydroxynitrile Lyase from Arabidopsis thaliana Improves Enzyme Stability

Hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) was fused to different fluorescent reporter proteins. Whereas all fusion constructs retained enzymatic activity and fluorescence in vivo and in vitro, significant differences in activity and pH stability were observed. In particular, flavin-based fluorescent reporter (FbFP) fusions showed almost 2 orders of magnitude-increased half-lives in the weakly acidic pH range compared to findings for the wild-type enzyme. Analysis of the quaternary structure of the respective FbFP-AtHNL fusion proteins suggested that this increased stability is apparently caused by oligomerization mediated via the FbFP tag. Moreover, the increased stability of the fusion proteins enabled the efficient synthesis of (R)-mandelonitrile in an aqueous-organic two-phase system at a pH of <5. Remarkably, (R)-mandelonitrile synthesis is not possible using wild-type AtHNL under the same conditions due to the inherent instability of this enzyme below pH 5. The fusion strategy presented here reveals a surprising means for the stabilization of enzymes and stresses the importance of a thorough in vitro characterization of in vivo-employed fluorescent fusion proteins.

Flooding characteristics of an aqueous two-phase system in a counter-current Kühni-type column

The flooding behavior of an aqueous two-phase system was investigated using a laboratory-scale counter-current agitated Kühni-type column. Experimental flooding limits were compared to the literature which has been established for aqueous–organic two-phase systems. Although the density difference and interfacial tension in the aqueous two-phase system studied were beyond the values typically encountered in aqueous-organic systems, comparison to the existing literature allowed for a prediction of the onset of flooding. Thus existing estimation procedures of the mean Sauter droplet diameter and terminal droplet velocity can be applied to aqueous two-phase systems.

Microgel‐Stabilized Smart Emulsions for Biocatalysis

Emulsions stabilized by stimuli-responsive microgels were used to perform enzyme catalysis. Many substrates are poorly water-soluble, while enzymes naturally require aqueous environments, thus resulting in a two-phase aqueous-organic system. Smart microgels allow an enzyme-catalyzed reaction to be performed in an emulsion that can be broken under controlled conditions to separate the reaction product and to recycle the enzyme (E) and the microgel.

Continuous aqueous two-phase extraction of human antibodies using a packed column

The performance of a pilot scale packed differential contactor was evaluated for the continuous counter-current aqueous two-phase extraction (ATPE) of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cells supernatant (CS) enriched with pure protein. Preliminary studies have been firstly performed in order to select the dispersed phase (phosphate-rich or polyethylene glycol 3350Da (PEG)-rich phase) and the column packing material. The PEG-rich phase has been selected as the dispersed phase and the stainless steel as the preferred material for the column packing bed since it was not wetted preferentially by the selected dispersed phase. Hydrodynamic studies have been also performed, and the experimental results were successfully adjusted to the Richardson–Zaki and Mísek equations, typically used for the conventional organic-aqueous two-phase systems. An experimental set-up combining the packed column with a pump mixer-settler stage showed to have the best performance and to be advantageous when compared to the IgG batch extraction. An IgG recovery yield of 85% could be obtained with about 50% of total contaminants and more than 85% of contaminant proteins removal. Mass transfer studies have revealed that the mass transfer was controlled by the PEG-rich phase. A higher efficiency could be obtained when using an extra pump mixer-settler stage and higher flow rates.

Continuous steroid biotransformations in microchannel reactors

The use of microchannel reactor based technologies within the scope of bioprocesses as process intensification and production platforms is gaining momentum. Such trend can be ascribed a particular set of characteristics of microchannel reactors, namely the enhanced mass and heat transfer, combined with easier handling and smaller volumes required, as compared to traditional reactors. In the present work, a continuous production process of 4-cholesten-3-one by the enzymatic oxidation of cholesterol without the formation of any by-product was assessed. The production was carried out within Y-shaped microchannel reactors in an aqueous-organic two-phase system. Substrate was delivered from the organic phase to aqueous phase containing cholesterol oxidase and the product formed partitions back to the organic phase. The aqueous phase was then forced through a plug-flow reactor, containing immobilized catalase. This step aimed at the reduction of hydrogen peroxide formed as a by-product during cholesterol oxidation, to avoid cholesterol oxidase deactivation due to said by-product. This setup was compared with traditional reactors and modes of operation. The results showed that microchannel reactor geometry outperformed traditional stirred tank and plug-flow reactors reaching similar conversion yields at reduced residence time. Coupling the plug-flow reactor containing catalase enabled aqueous phase reuse with maintenance of 30% catalytic activity of cholesterol oxidase while eliminating hydrogen peroxide. A final production of 36 m of cholestenone was reached after 300 hours of operation.

ChemInform Abstract: Cyclodextrins and Their Applications in Aqueous‐Phase Metal‐Catalyzed Reactions

AbstractReview: 132 refs.

Transformation of Geniposide into Genipin by Immobilized β-Glucosidase in a Two-Phase Aqueous-Organic System

Genipin is the bioactive compound of geniposide and a natural cross-linking agent. In order to improve the preparation process of genipin, the hydrolysis of geniposide to genipin by immobilized β-glucosidase in an aqueous-organic two-phase system was studied. β-Glucosidase was immobilized by the crosslinking-embedding method using sodium alginate as the carrier. The optimum reaction temperature, pH value and time were 55 °C, 4.5 and 2.5 h, respectively. To reduce genipin hydrolysis and byproduct production the reaction was carried out in an aqueous-organic two-phase system comprising ethyl acetate and sodium acetate buffer. The product was analyzed by HPLC, UV, IR, and NMR. The yield of genipin was 47.81% and its purity was over 98% (HPLC).

Hydrodynamic studies of liquid–liquid slug flows in circular microchannels

The aim of the work presented was to clarify the existence of a wall film and its influence on the hydrodynamics of liquid–liquid slug flow capillary microreactor. The methodology of the laser induced fluorescence (LIF) was adopted for visualisation purposes. The measurement of the light intensity profiles revealed a fully developed wall film for a variety of aqueous–organic two-phase systems in glass and PTFE capillaries of 1 mm internal diameter. In addition an acid as a quenching agent enabled the observation of the internal circulation patterns within the liquid slugs, as the fluorescent dye was deactivated by the acid diffusing in from the dye-free phase. A well-defined internal circulation pattern was always present in the wetting phase, i.e. that forming the wall film, leading to uniform mixing in the slugs of this phase. Stagnant zones and local circulation vortices, indicated by variations in the concentrations of the quenched dye, were observed in the non-wetting dispersed phase. These more complex flow structures varied little with the slug velocity, but were strongly dependent on the physical properties of the liquid–liquid system. To predict slug shape and hydrodynamics within the liquid slugs, CFD simulations were carried out using the volume-of-fluid method (VOF) based on the incompressible Navier–Stokes equation with appropriate boundary conditions between the two phases. The slug generation process was studied in a T-junction with 1 mm internal diameter inlets. The implementation of the wetting contact angle, measured in the visualisation experiments for the various systems, led to realistic slug lengths and shapes. The velocity vector plot indicated a fully developed internal circulation pattern within the simulated slugs. Calculations for a single slug with a non-wetting condition gave rise to a wall film in the simulated system. The results obtained demonstrate the significance of the wall film in the hydrodynamics and mass transfer liquid–liquid slug flow and reveal the presence of hitherto unsuspected complex patterns in place of simple single Taylor vortex flow assumed in the past.

Noble Metal Nanoparticles Stabilized by Cyclodextrins: A Pertinent Partnership for Catalytic Applications

For economic and ecological purposes, the design of stable and reusable noble metal nanoparticles in water has become a concern over the past few years. Among the various water-soluble protective agents, cyclodextrins proved to be attractive candidates to efficiently stabilize catalytically active nanospecies in aqueous media and also to improve the mass transfer in aqueous-organic two-phase systems. Various catalytic reactions such as arenes hydrogenations, olefins reduction or cross-coupling reactions were successfully achieved with good yields and selectivities according to the relevant choice of the cyclodextrins (cavity or functionalization). Keywords: Nanoparticles, Noble metal, Cyclodextrins, Catalysis, Water

Synthesis, Characterization and Application of a Multi-Site Phase Transfer Catalyst in Radical Polymerization of n-Butyl Methacrylate—A Kinetic Study

The multi-site phase transfer catalyzed radical polymerization of n-butyl methacrylate (n-BMA) using newly synthesized and characterized 1,1,2,2-tetramethyl-1-benzyl-2-n-propylethylene-1,2-diammonium bromide chloride (TMBPEDBC) as a multi-site phase transfer catalyst was investigated in an aqueous-organic two-phase system at 60 ± 1°C under nitrogen circumstances. The kinetics and effects of various operating variables (monomer, initiator, catalyst, temperature, acid, and ionic strength) on the rate of polymerization (Rp) were examined in detail. The order with respect to monomer, initiator and multi-site phase transfer catalyst was found to be 0.50. A suitable kinetic reaction scheme has been proposed to account for experimental observations, and its significance is discussed.

ChemInform Abstract: Enzymatic Synthesis of Glycosides in Aqueous-Organic Two-Phase Systems and Supersaturated Substrate Solutions

ChemInform Abstract: Enzymatic Synthesis of Glycosides in Aqueous-Organic Two-Phase Systems and Supersaturated Substrate Solutions

Cyclodextrins and their applications in aqueous-phase metal-catalyzed reactions

Abstract During these last years, cyclodextrins have greatly contributed to the development of catalytic processes in water. These cyclic oligosaccharides composed of 6(α), 7(β) or 8(γ) glucopyranose units improve the mass transfer in aqueous-organic two-phase systems and are useful compounds to design new catalysts. Thus, chemically modified cyclodextrins can be used to: (1) significantly increase the rate and selectivity of reactions catalyzed by water-soluble organometallic complexes; (2) design new water-soluble ligands for aqueous organometallic catalysis; (3) stabilize catalytically active noble metal nanoparticles in water; (4) facilitate reactions catalyzed by supported metals or metallic powder in water.

Scaling‐up of complex whole‐cell bioconversions in conventional and non‐conventional media

The use of whole cells is becoming a more common approach in pharmaceutical and agrochemical industries in order to obtain pure compounds with fewer production steps, higher yields, and cleaner processes, as compared to those achieved with traditional strategies. Whole cells are often used as enzymes pools, in particular when multi-step reactions and/or co-factor regeneration are envisaged. Nonetheless, published information on the scale-up of such systems both in aqueous and in two-phase aqueous-organic systems is relatively scarce. The present work aims to evaluate suitable scale-up criteria in conventional and non-conventional medium for a whole-cell bioconversion that uses resting cells of Mycobacterium sp. NRRL B-3805 to cleave the side chain of beta-sitosterol, a poorly water-soluble substrate. The experiments were performed in 24-well microtiter plates and in 250 mL shaken flasks as orbital stirred systems, and in 300 mL stirred tanks as mechanically stirred systems. Results show that productivity yields were similar in all scales tested, when maintaining oxygen mass transfer coefficients constant in aqueous systems, or when maintaining constant volumetric power consumption in aqueous-organic two-phase systems.

Synthesis of monoacylglycerols by enzymatic methods

Monoacylglycerols are non-ionic surfactants widely used in the food industry. They are also important in cosmetic and pharmaceutical industries as drug carriers and for the consistency improvements in creams and lotions. Current process for their production is based on the glycerolysis of natural fats and oils in the presence of inorganic catalysts at temperatures higher than 220 oC. The major drawbacks of this process include high-energy consumption, low yield, and poor product quality. The use of lipases for the monoacylglycerols production offers environmental advantages and a reduction in energy consumption. Besides, the same surfactants prepared by the enzymatic synthesis may be labeled as ?natural?. Recent progress in the application of highly-stable lipases in the organic solvents offers the possibility of employing various methods to the enzyme-catalyzed synthesis of monoacylglycerols, such as selective hydrolysis of fats and oils using 1,3-regiospecific lipases, the esterification of glycerol with fatty acids and the glycerolysis of fats or oils. In this review, different reaction systems such as aqueous-organic two-phase systems, microemulsions and reverse micelles systems, anhydrous organic solvents, solvent-free systems with free or immobilized lipases, as well as the use of two-phase membrane reactor systems are presented. We discuss some of the key factors, such as the control of water content, removing of the products from reaction system, and the effects of solvent on the lipase activity and selectivity, that must be addressed in order to obtain an efficient reaction system with high yields of monoacylglycerols. Engineering of the enzymatic monoacylglycerols synthesis processes requires also optimization of other factors as: molar ratio of substrates, temperature, type of lipase immobilization and supports (if any), reactor design and operating regime.

Chemically Modified Cyclodextrins: An Attractive Class of Supramolecular Hosts for the Development of Aqueous Biphasic Catalytic Processes

During these last years, cyclodextrins have contributed greatly to the development of catalytic processes in water. These cyclic oligosaccharides composed of 6(α), 7(β) or 8(γ) glucopyranose units improve the mass transfer in aqueous-organic two-phase systems and are useful compounds to design new catalysts. Thus, chemically modified cyclodextrins can be used to: (i) increase significantly the rate and selectivity of reactions catalyzed by water-soluble organometallic complexes, (ii) design new water-soluble ligands for aqueous organometallic catalysis, (iii) stabilize catalytically active noble metal nanoparticles in water and (iv) favour the dispersion and activation of the palladium on charcoal in water.

P450BM-3-catalyzed whole-cell biotransformation of α-pinene with recombinant Escherichia coli in an aqueous–organic two-phase system

A recombinant Escherichia coli BL21 (DE3) strain overexpressing a variant of P450(BM-3) (V26T/R47F/A74G/F87V/L188K; abbreviated: BL21 (P450(BM-3) QM)) oxyfunctionalizes the bicyclic monoterpene alpha-pinene to alpha-pinene oxide, verbenol, and myrtenol. To address the low water solubility and the toxicity of terpenoids, an aqueous-organic two-phase bioprocess was developed. Diisononyl phthalate was selected as a biocompatible organic carrier solvent capable of masking the toxic effects mediated by alpha-pinene and of efficiently extracting the products enabling scale-up to the bioreactor. With an aqueous to organic phase ratio of 3:2 and 30% (v/v) of alpha-pinene in the organic phase, a biocatalytic product formation period of more than 4 h was achieved. A comparison of the biotransformation performance of BL21 (P450(BM-3) QM) and a strain with an additional heterologous NADPH regeneration system comprising glucose facilitator and dehydrogenase, but only expressing half the amount of P450(BM-3) QM, shows comparable product concentrations of 1,020 +/- 144 and 800 +/- 61 mg l (Aq) (-1) , respectively. The total product yields Y (P/P450) (micromol micromol (P450) (-1) ) were 80% higher when the strain with the cofactor regeneration system was used. A total product concentration of over 1 g l (Aq) (-1) , corresponding to the highest value reported for microbial alpha-pinene oxyfunctionalization so far, marks a promising step forward toward a future application of recombinant microorganisms for the selective oxidation of terpenoids to value-added products.

Application of organic mono-phase and organic–aqueous two-liquid-phase systems in microalgal conversion of androst-4-en-3,17-dione by Nostoc muscorum

Organic mono-phase and organic–aqueous two-phase systems were applied for 17-carbonyl reduction of androst-4-en-3,17-dione to testosterone by whole cells of the microalga Nostoc muscorum (Nostocaceae). To investigate the correlation between solvent hydrophobicity and biotransformation yield in mono- and biphasic systems, a range of 16 organic solvents with log Poctanol values (logarithm of the solvent partition coefficient in the n-octanol/water system) between − 1.1 and 8.8 were examined. Organic solvents with log Poctanol values greater than 7, such as hexadecane and tetradecane, provided the best biocompatibility with the bioconversion by algal cells. The data also indicated that the highest yields were obtained using organic–aqueous (1:1, v/v) biphasic systems. The optimum volumetric phase ratio, reaction temperature and substrate concentration were 1:1, 30°C and 0.5 mg mL−1, respectively. Under the mentioned conditions a fourfold increase in biotransformation yield (from 7.8±2.3 to 33.4±1.8%) was observed.