Direct imaging of micrometer-thick interfaces in salt-salt aqueous biphasic systems.

Unlike the interface between two immiscible electrolyte solutions (ITIES) formed between water and polar solvents, molecular understanding of the liquid-liquid interface formed for aqueous biphasic systems (ABSs) is relatively limited and mostly relies on surface tension measurements and thermodynamic models. Here, high-resolution Raman imaging is used to provide spatial and chemical resolution of the interface of lithium chloride - lithium bis(trifluoromethanesulfonyl)imide - water (LiCl-LiTFSI-water) and HCl-LiTFSI-water, prototypical salt-salt ABSs found in a range of electrochemical applications. The concentration profiles of both TFSI anions and water are found to be sigmoidal thus not showing any signs of a positive adsorption for both salts and solvent. More striking, however, is the length at which the concentration profiles extend, ranging from 11 to 2 µm with increasing concentrations, compared to a few nanometers for ITIES. We thus reveal that unlike ITIES, salt-salt ABSs do not have a molecularly sharp interface but rather form an interphase with a gradual change of environment from one phase to the other. This knowledge represents a major stepping-stone in the understanding of aqueous interfaces, key for mastering ion or electron transfer dynamics in a wide range of biological and technological settings including novel battery technologies such as membraneless redox flow and dual-ion batteries.

Improved Electrochemical Peptide Synthesis Enabled by Electron-Rich Triaryl Phosphines.

While remarkable progress has been made in the development of peptide medicines, many problems related to peptide synthesis remain unresolved. Previously, we reported electrochemical peptide synthesis using a phosphine as a potentially recyclable coupling reagent. However, there was room for improvement from the point of view of reaction efficiency, especially in the carboxylic acid activation step and the peptide bond formation step. To overcome these challenges, we searched for the optimal phosphine. Among phosphines with various electronic properties, we found that electron-rich triaryl phosphines improved the reaction efficiency. Consequently, we successfully performed electrochemical peptide synthesis on sterically hindered and valuable amino acids. We also synthesized oligopeptides that were challenging with our previous method. Finally, we examined the effect of substituents on the phosphine cations, and gained some insights into reactivity, which will aid researchers designing reactions involving phosphine cations.

Enhancing the biodesulphurization capacity of Rhodococcus sp. FUM94 in a biphasic system through optimization of operational factors.

The biodesulfurization activity of bacteria through the 4S pathway in aqueous-oil emulsions is affected by various operational factors. These factors also demonstrate interacting effects that influence the potential for field applications of biodesulfurization technology and can solely be deciphered through multi-variable experiments. In this study, the effects of the influential factors and their interactions on the desulfurizing activity of a newly identified desulfurizing bacterium, Rhodococcus sp, FUM94 were quantitatively investigated. The capacity improvement achieved through optimized values obtained in this study is significant due to its simple implementation to large scale processes. This is the most simple and the most cost-effective way to scale-up a biodesulfurization process.Using response surface methodology (RSM). Optimum values of the factors were identified with the objective of maximizing biodesulfurization activity. Results revealed that the desulfurization activity of the biocatalyst increased from 0.323 ± 0.072 to 46.57 ± 4.556 mmol 2-Hydroxybiphenyl (kg dry cell weight)-1 h-1 at the optimized conditions of 6 h reaction time, 2 g.L-1 biocatalyst concentration, 0.54 mM (100 ppm) dibenzothiophene (DBT) concentration (sulfur source), and 25% oil phase fraction. Desirability analysis proved that the selected conditions are the most desirable combination of factors (desirability value = 0.896) to achieve the highest biodesulfurization activity of the biocatalyst. A comparison between the biodesulfurization capacity achieved in this study and the capacities reported in similar studies published in the past two decades revealed that biodesulfurization under optimized operational conditions outperforms previously proposed techniques.

Extraction and purification of Aspergillus tamarii ß-fructofuranosidase with transfructosylating activity using aqueous biphasic systems (PEG/phosphate) and magnetic field.

ß-fructofuranosidases (FFases) are enzymes involved in sucrose hydrolysis and fructo-oligosaccharides' production which are of great interest for the food industry. FFase from Aspergillus tamarii URM4634 was extracted using PEG/Phosphate Aqueous Biphasic Systems (ABS), and the impact of magnetic field on the extraction behavior was evaluated. A 24-full experimental design was employed to study the influence of molar mass of PEG, concentrations of PEG and phosphate and pH on the selected response variables, i.e., partition coefficient (K), purification factor (PF), activity yield (Y) and selectivity (S). The influence of magnetic field during partition and NaCl concentration on the same responses was also studied. The best results of FFase extraction without magnetic field (K = 0.50, PF = 4.05, Y = 72.66% and S = 0.06) were observed at pH 8.0 using 12.5% (w/w) PEG 400 and 25% (w/w) NaH2PO4/K2HPO4. Application of the magnetic field allowed improving the performance, with the best results being obtained at the longest distance between magnets (lowest magnetic field) and absence of NaCl (K = 0.93, PF = 4.22, Y = 83.79% and S = 0.09). The outcomes obtained demonstrate that ABS combination with low intensity magnetic field can be used as an efficient FFase pre-purification method.

Evaluation of Acidic Ionic Liquids as Catalysts for Furfural Production from Eucalyptus nitens Wood.

Eucalyptus nitens wood samples were subjected to hydrothermal processing to obtain soluble saccharides from the hemicellulosic fraction. The hemicellulose-derived saccharides were employed as substrates for furfural production in biphasic media made up of water, methyl isobutyl ketone, and one acidic ionic liquid (1-butyl-3-methylimidazolium hydrogen sulfate or 1-(3-sulfopropyl)-3-methylimidazolium hydrogen sulfate). The reactions were carried out in a microwave-heated reactor to assess the effects of the most influential variables. Under selected operational conditions, the molar conversions of the precursors into furfural were within the range of 77-86%. The catalysts conserved their activity after reutilization in five consecutive reaction cycles.

Experimental Examination of Solubility and Lipophilicity as Pharmaceutically Relevant Points of Novel Bioactive Hybrid Compounds.

The important physicochemical properties of three novel bioactive hybrid compounds with different groups (-CH3, -F and -Cl) were studied, including kinetic and thermodynamic solubility in pharmaceutically relevant solvents (buffer solutions and 1-octanol) as well as partition coefficient in system 1-octanol/buffer pH 7.4. The aqueous solubility of these chemicals is poor and ranged from 0.67 × 10-4 to 1.98 × 10-3 mol·L-1. The compounds studied are more soluble in the buffer pH 2.0, simulating the gastrointestinal tract environment (by an order of magnitude) than in the buffer pH 7.4 modelling plasma of blood. The solubility in 1-octanol is significantly higher; that is because of the specific interactions of the compounds with the solvent. The prediction solubility behaviour of the hybrid compounds using Hansen's three-parameter approach showed acceptable results. The experimental solubility of potential drugs was successfully correlated by means of two commonly known equations: modified Apelblat and van't Hoff. The temperature dependencies of partition coefficients of new hybrids in the model system 1-octanol/buffer pH 7.4 as a surrogate lipophilicity were measured by the shake flask method. It was found that compounds demonstrated a lipophilic nature and have optimal values of partition coefficients for oral absorption. Bioactive assay manifested that prepared compounds showed antifungal activities equal to or greater than fluconazole. In addition, the thermodynamic aspects of dissolution and partition processes have been examined. Bioactive assay manifested that prepared compounds showed antifungal activities equal to or greater than the reference drug.

Enantioseparation and determination of triazole fungicides in fruit juice by aqueous biphasic system coupled with HPLC-MS/MS.

The aqueous biphasic system based on choline ionic liquids and ethylene/propylene oxide copolymer coupled with high-performance liquid chromatography-tandem mass spectrometry was developed for the simultaneous determination of the enantiomers of two fungicides: myclobutanil and tebuconazole. The influence of mass fractions of ionic liquids and the copolymer on the extraction efficiency of the target fungicides was investigated. The analytes are mainly concentrated in the bottom, copolymer-rich phase. The extraction efficiencies of the selected fungicides were significantly affected by the concentrations of the copolymer, and their extraction efficiencies decreased with the higher mass fractions of the copolymer, while the mass fraction of ionic liquids had little effect on their extraction behavior. Excellent extraction efficiency was achieved using the aqueous biphasic system based on choline L-lysinate and the copolymer. At three spiked concentrations of 0.01, 0.05, and 0.1 mg/kg, the average recoveries of the selected fungicides ranged from 80 to 89%, with the relative standard deviations in the range of 2.1-5.3%. Limits of quantitation for the enantiomers of tebuconazole and myclobutanil were 0.5 and 5.0 µg/kg, respectively. The developed system could be successfully applied to the analysis of triazole fungicides residue in real samples.

Byproduct-free geraniol glycosylation by whole-cell biotransformation with recombinant Escherichia coli.

OBJECTIVE: Geraniol, a fragrance of great importance in the consumer goods industry, can be glucosylated by the UDP-glucose-dependent glucosyltransferase VvGT14a from Vitis vinifera, yielding more stable geranyl glucoside. Escherichia coli expressing VvGT14a is a convenient whole-cell biocatalyst for this biotransformation due to its intrinsic capability for UDP-glucose regeneration. The low water solubility and high cytotoxicity of geraniol can be overcome in a biphasic system where the non-aqueous phase functions as an in situ substrate reservoir. However, the effect of different process variables on the biphasic whole-cell biotransformation is unknown. Thus, the goal of this study was to identify potential bottlenecks during biotransformation with in situ geraniol supply via isopropyl myristate as second non-aqueous phase. RESULTS: First, insufficient UDP-glucose supply could be ruled out by measurement of intracellular UDP-glucose concentrations. Instead, oxygen supply was determined as a bottleneck. Moreover, the formation of the byproduct geranyl acetate by chloramphenicol acetyltransferase (CAT) was identified as a constraint for high product yields. The use of a CAT-deficient whole-cell biocatalyst prevented the formation of geranyl acetate, and geranyl glucoside could be obtained with 100% selectivity during a biotransformation on L-scale. CONCLUSION: This study is the first to closely analyze the whole-cell biotransformation of geraniol with Escherichia coli expressing an UDP-glucose-dependent glucosyltransferase and can be used as an optimal starting point for the design of other glycosylation processes.

Phase Behavior of Aqueous Biphasic Systems with Choline Alkanoate Ionic Liquids and Phosphate Solutions: The Influence of pH.

Aqueous biphasic systems (ABS) composed of the choline alkanoate ionic liquids (ILs) choline acetate [Cho][OAc], choline propanoate [Cho][Pro], choline butyrate [Cho][But], and choline hexanoate [Cho][Hex], mixed with K3PO4 solutions at pH 7.2 and 14.5, were prepared and their phase diagrams were compared. The ability to form ABS with alkaline K3PO4 solutions decreased in the order [Cho][OAc] ≈ [Cho][Pro] > [Cho][But] > [Cho][Hex], while with neutral K3PO4 solutions, [Cho][OAc] could not form an ABS, and the other three ILs performed similarly. All of the biphasic regions of the ABS decreased with the increase in pH. 1H-NMR data indicated anion exchange between phases in ABS at neutral pH. The ABS at neutral pH were evaluated to extract the triazine herbicides simazine, cyanazine, and atrazine, and the ABS formed by [Cho][Pro] and the pH 7.2 K3PO4 solution has shown extraction recoveries higher than 90%.

A novel method for double encapsulation of C-phycocyanin using aqueous two phase systems for extension of shelf life.

Spirulina platensis is having high nutritive value due to pigments such as chlorophyll-a, phycobiliproteins (especially phycocyanins) and carotenoids. In our present work, C-phycocyanin (C-PC) was extracted from dry biomass of Spirulina platensis. C-PC being heat sensitive, reiterates the need for additional protection during drying (micro encapsulation). Accordingly, a novel method employing aqueous two phase systems (ATPSs) as carrier materials to achieve double encapsulation was studied for the first time. PEG 4000/Potassium phosphate and PEG 6000/Dextran were used at already standardized tie line length, at different volume ratios (by varying the total phase composition). ATPS at each volume ratio acted as different carrier materials offering varied degree of heat protection during double encapsulation while maltodextrin, being the conventional carrier material, was used for comparison. The best results of spray dried powders using PEG (4% w/w)/Potassium phosphate salt (18%, w/w) and PEG (6%)/Dextran (10%, w/w) phase systems as carrier materials were compared with conventional encapsulation (MDX as a carrier material) and freeze dying as control. PEG/Dextran as a carrier material with volume ratio of 0.25 resulted in the highest retention of blue colour (b*value), purity (0.43) as well as yield (YEP) of 94.99% w/w of C-PC, which could be stored for 6 months without much reduction from initial powder characteristics. From the overall results, it can be concluded that ATPS can be used as an effective carrier material for double encapsulation of biomolecules such as C-PC with additional benefit of enhancing the purity.

Recent development of unconventional aqueous biphasic system: characteristics, mechanisms and applications.

Aqueous biphasic system (ABS) is widely used in the recovery, extraction, purification and separation of proteins, enzymes, nucleic acids and antibodies. The ABS with high water content and low interfacial tension offers a biocompatible environment for the recovery of labile biomolecules. Process integration can be achieved using ABS by incorporating multiple-steps of purification, concentration and purification of biomolecules in a single-step operation which often results in high product recovery yield and purity. Conventional ABS is usually formed by aqueous solutions of two polymers or a polymer and a salt above a critical concentration. The high viscosity of polymer-based ABS causes slow phase separation and hinders the mass transfer of biomolecules, whereas polymer/salt ABS is characterized by high ionic strength resulting in the loss of bioactivity of recovered biomolecules. These limitations have encouraged the development of novel ABS which is more cost-effective for various biotechnological applications. This review discusses the characteristics and mechanisms of several types of emerging unconventional ABS using phase-forming components such as hyperbranched polymers, special salts, surfactants, magnetic fields, the addition of nanoparticles and incorporation of various solvent. Moreover, several novel applications of ABS for different separation purposes such as microfluidic-based ABS, ABS bioreactors, application of ABS as an analytical tool, and ABS micropatterning are discussed in this review. In the last section of this review, a comprehensive summary of process integration using ABS for extractive fermentations, bioconversion, crystallization and precipitation is also supplemented for the comprehensive review of various types and applications of ABS in recent years.

Cell-free protein synthesis enables one-pot cascade biotransformation in an aqueous-organic biphasic system.

Biocatalytic cascade reactions have become increasingly important and useful for chemical synthesis. However, biocatalysts are often incompatible with organic solvents, which prohibits many cascade reactions involving nonpolar substrates. In this study, we used cell-free protein synthesis (CFPS) to express enzymes in an aqueous-organic biphasic system for the construction of an artificial enzymatic pathway. CFPS-expressed enzymes without purification performed efficiently to convert styrene (below 20 mM) to (S)-1-phenyl-1,2-ethanediol (two steps in one pot) with 100% conversion. In addition, our CFPS system showed great tolerance to different organic solvents, and, importantly, the entire biocatalytic system can be consistently scaled up without a reduction of the substrate conversion rate. We, therefore, anticipate that our cell-free approach will make a possible cost-effective, high-yielding synthesis of valuable chemicals.

Efficient biosynthesis of cinnamyl alcohol by engineered Escherichia coli overexpressing carboxylic acid reductase in a biphasic system.

BACKGROUND: Cinnamyl alcohol is not only a kind of flavoring agent and fragrance, but also a versatile chemical applied in the production of various compounds. At present, the preparation of cinnamyl alcohol depends on plant extraction and chemical synthesis, which have several drawbacks, including limited scalability, productivity and environmental impact. It is therefore necessary to develop an efficient, green and sustainable biosynthesis method. RESULTS: Herein, we constructed a recombinant Escherichia coli BLCS coexpressing carboxylic acid reductase from Nocardia iowensis and phosphopantetheine transferase from Bacillus subtilis. The strain could convert cinnamic acid into cinnamyl alcohol without overexpressing alcohol dehydrogenase or aldo-keto reductase. Severe product inhibition was found to be the key limiting factor for cinnamyl alcohol biosynthesis. Thus, a biphasic system was proposed to overcome the inhibition of cinnamyl alcohol via in situ product removal. With the use of a dibutyl phthalate/water biphasic system, not only was product inhibition removed, but also the simultaneous separation and concentration of cinnamyl alcohol was achieved. Up to 17.4 mM cinnamic acid in the aqueous phase was totally reduced to cinnamyl alcohol with a yield of 88.2%, and the synthesized cinnamyl alcohol was concentrated to 37.4 mM in the organic phase. This process also demonstrated robust performance when it was integrated with the production of cinnamic acid from L-phenylalanine. CONCLUSION: We developed an efficient one-pot two-step biosynthesis system for cinnamyl alcohol production, which opens up possibilities for the practical biosynthesis of natural cinnamyl alcohol at an industrial scale.

Coupling of Bioreaction and Separation via Novel Thermosensitive Ionic Liquids Applied in the Baker's Yeast-Catalyzed Reduction of Ethyl 2-oxo-4-phenylbutyrate.

The use of baker's yeast to reduce ethyl 2-oxo-4-phenylbutyrate (EOPB) in conventional biphasic systems is hindered by low productivities due to mass transfer resistance between the biocatalyst and the substrate partitioned into two different phases. To overcome the limitation, a new reaction-separation coupling process (RSCP) was configured in this study, based on the novel thermosensitive ionic liquids (ILs) with polyoxyethylene-tail. The solubility of ILs in common solvents was investigated to configure the unique thermosensitive ionic liquids-solvent biphasic system (TIBS) in which the reduction was performed. [(CH3)2N(C2H5)(CH2CH2O)2H][PF6] (c2) in 1,2-dimethoxyethane possesses the thermosensitive function of homogeneous at lower temperatures and phase separating at higher temperatures. The phase transformation temperature (PTT) of the mixed system of c2/1,2-dimethoxyethane (v/v, 5:18) was about 33 °C. The bioreaction takes place in a "homogeneous" liquid phase at 30 °C. At the end of each reduction run, the system temperature is increased upon to the PTT, while c2 is separated from 1,2-dimethoxyethane with turning the system into two phases. The enantiomeric excesses (e.e.) of ethyl (R)-2-hydroxy-4-phenylbutyrate ((R)-EHPB) increased about 25~30% and the yield of ethyl-2-hydroxy-4-phenylbutyrate (EHPB) increased 35% in TIBS, compared with the reduction in 1,2-dimethoxyethane. It is expected that the TIBS established in this study could provide many future opportunities in the biocatalysis.

Hydrogen-Bonding Catalyzed Ring-Closing C-O/C-O Metathesis of Aliphatic Ethers over Ionic Liquid under Metal-Free Conditions.

O-heterocycles have wide applications, and their efficient and green synthesis is very interesting. Herein, we report hydrogen-bonding catalyzed ring-closing metathesis of aliphatic ethers to O-heterocycles over ionic liquid (IL) catalyst under metal- and solvent-free conditions. The IL 1-butylsulfonate-3-methylimidazolium trifluoromethanesulfonate ([SO3 H-BMIm][OTf]) is discovered to show outstanding performance, better than the reported catalysts. An interface effect plays an important role in mediating the reaction rate due to the immiscibility between the products and the IL catalyst, and the products can be spontaneously separated. NMR analysis and DFT calculation suggest that a pair of cation and anion of [SO3 H-BMIm][OTf] could form three strong H-bonds with an ether molecule, which catalyze the ether transformation via a cyclic oxonium intermediate. A series of O-heterocycles including tetrahydrofurans, tetrahydropyrans, morpholines and dioxane can be obtained from their corresponding ethers in excellent yields (e.g., >99 %). This work opens an efficient and metal-free way to produce O-heterocycles from aliphatic ethers.

Use of the aqueous biphasic system as an alternative for concentration of Ascaris lumbricoides eggs, with non-toxic separation of faecal residues and fats.

OBJECTIVES: Human enteroparasites are considered a serious public health problem in underdeveloped countries located in world regions with tropical, subtropical and equatorial climates. These parasites are commonly diagnosed by the Parasitological Examination of Faeces (PEF), performed by conventional techniques and/or commercial kits that result in tests with low-to-moderate sensitivity, due to the use of destructive chemical solvents to parasite structures, and to present excess adipose substance and digestive residues in their microscopic slides. In order to improve the efficacy of these tests/examinations, this study aimed to investigate a new alternative for the PEF, with the use of Aqueous Biphasic System (ABS). METHODS: For this, four ABSs containing poly (ethylene glycol), PEG (PEG-4000 and PEG-6000), dipotassium phosphate and sodium citrate at different concentrations in the biphasic systems were evaluated with faecal samples containing eggs of Ascaris lumbricoides. RESULTS: The ABS consisting of PEG-4000 and dipotassium phosphate, at concentrations of 55% w/w and 20% w/w, respectively, achieved 100% satisfactory results compared to the conventional TF-Test technique in terms of preservation and concentration of A. lumbricoides eggs, with adequate separation of digestive residues, without using a centrifuge or chemical solvents that may cause harm to the parasites. CONCLUSIONS: This study presents ABS as a new low-cost technical principle for the detection of parasite eggs in PEF. The new technique is simple, fast, non-toxic, not harmful to the parasite and does not require a centrifuge.

UTILISATION DU SYSTÈME BIPHASIQUE AQUEUX COMME ALTERNATIVE À LA CONCENTRATION DES ŒUFS D'ASCARIS LUMBRICOIDES, AVEC SÉPARATION NON TOXIQUE DES RÉSIDUS FÉCAUX ET DES GRAISSES: OBJECTIFS: Les entéroparasites humains sont considérés comme un sérieux problème de santé publique dans les pays sous-développés situés dans des régions du monde aux climats tropicaux, subtropicaux et équatoriaux. L'examen parasitologique des matières fécales (EPMF) permet généralement de diagnostiquer ces parasites, à l'aide de techniques classiques et/ou de kits commerciaux, conduisant à des tests de sensibilité faible à modérée, dus à l'utilisation de solvants chimiques destructeurs pour les structures du parasite et résultant en excès de substances adipeuses et de résidus digestifs dans leurs lames microscopiques. Afin d'améliorer l'efficacité de ces tests/examens, cette étude visait à étudier une nouvelle alternative à l'EPMF, avec l'utilisation du Système Biphasique Aqueux (SBA). MÉTHODES: Pour cela, quatre SBA contenant du poly (éthylène glycol), du PEG (PEG-4000 et PEG-6000), du phosphate dipotassique et du citrate de sodium à différentes concentrations ont été évalués avec des échantillons de matières fécales contenant des œufs d'Ascaris lumbricoides. RÉSULTATS: Le SBA constitué de PEG-4000 et de phosphate dipotassique, à des concentrations respectives de 55% poids/poids et 20% poids/poids, a obtenu des résultats satisfaisants à 100% par rapport à la technique conventionnelle du TF-Test, en termes de préservation et de concentration des œufs de A. lumbricoides, avec une séparation adéquate des résidus digestifs, sans utiliser de centrifugeuse ni de solvants chimiques pouvant détruire les parasites. CONCLUSIONS: Cette étude présente le SBA comme un nouveau principe technique peu coûteux pour la détection des œufs de parasites dans l'EPMF. La nouvelle technique est simple, rapide, non toxique, n'endommage pas le parasite et ne nécessite pas de centrifugeuse.

Extraction and Separation of Eight Ginsenosides from Flower Buds of Panax Ginseng Using Aqueous Ionic Liquid-Based Ultrasonic-Assisted Extraction Coupled with an Aqueous Biphasic System.

Ionic liquids (ILs) are recognized as a possible replacement of traditional organic solvents, and ILs have been widely applied to extract various compounds. The present work aims to extract ginsenosides from Panax ginseng flower buds using aqueous ionic liquid based ultrasonic assisted extraction (IL-UAE). The extraction yields of 1-alkyl-3-methylimidazolium ionic liquids with different anions and alkyl chains were evaluated. The extraction parameters of eight ginsenosides were optimized by utilizing response surface methodology (RSM). The model demonstrated that a high yield of total ginsenosides could be obtained using IL-UAE, and the optimum extraction parameters were 0.23 M [C4mim][BF4], ultrasonic time of 23 min, temperature of extraction set to 30 °C, and liquid-solid ratio of 31:1. After that, an aqueous biphasic system (ABS) was used to separate ginsenosides further. The nature and concentration of salt, as well as the value of pH in ionic liquid were evaluated, and the optimal conditions (6.0 mL IL extract, 3 g NaH2PO4, and pH 5.0) were obtained. The preconcentration factor was 2.58, and extraction efficiency reached 64.53%. The results indicate that as a simple and efficient method, an IL-UAE-ABS can be considered as a promising method for extracting and separating the natural active compounds from medicinal herbs.

Solvent-Tolerant Acyltransferase from Bacillus sp. APB-6: Purification and Characterization.

Amidase from Bacillus sp. APB-6 with very good acyltransferase activity was purified to homogeneity with a purification fold of 3.68 and 53.20% enzyme yield. The purified protein's subunit molecular mass was determined approximately 42 kDa. Hyperactivity of the enzyme was observed at pH 7.5 (150 mM, potassium-phosphate buffer) and 50 °C of incubation. An enhancement in activity up to 42% was recorded with ethylenediaminetetraacetic acid and dithiothreitol. The kinetic parameter K m values for substrates: acetamide and hydroxylamine-hydrochloride were 73.0 and 153 mM, respectively. Further, the V max for acyltransferase activity was 1667 U/mg of protein and the K i for acetamide was calculated as 37.0 mM. The enzyme showed tolerance to various organic solvents (10%, v/v) and worked well in the biphasic reaction medium. The acyltransferase activity in presence of solvents i.e. biphasic medium may prove highly favorable for the transformation of hydrophobic amides, which otherwise is not possible in simple aqueous phase.

Enhancing productivity for cascade biotransformation of styrene to (S)-vicinal diol with biphasic system in hollow fiber membrane bioreactor.

Biotransformation is a green and useful tool for sustainable and selective chemical synthesis. However, it often suffers from the toxicity and inhibition from organic substrates or products. Here, we established a hollow fiber membrane bioreactor (HFMB)-based aqueous/organic biphasic system, for the first time, to enhance the productivity of a cascade biotransformation with strong substrate toxicity and inhibition. The enantioselective trans-dihydroxylation of styrene to (S)-1-phenyl-1,2-ethanediol, catalyzed by Escherichia coli (SSP1) coexpressing styrene monooxygenase and an epoxide hydrolase, was performed in HFMB with organic solvent in the shell side and aqueous cell suspension in the lumen side. Various organic solvents were investigated, and n-hexadecane was found as the best for the HFMB-based biphasic system. Comparing to other reported biphasic systems assisted by HFMB, our system not only shield much of the substrate toxicity but also deflate the product recovery burden in downstream processing as the majority of styrene stayed in organic phase while the diol product mostly remained in the aqueous phase. The established HFMB-based biphasic system enhanced the production titer to 143 mM, being 16-fold higher than the aqueous system and 1.6-fold higher than the traditional dispersive partitioning biphase system. Furthermore, the combination of biphasic system with HFMB prevents the foaming and emulsification, thus reducing the burden in downstream purification. HFMB-based biphasic system could serve as a suitable platform for enhancing the productivity of single-step or cascade biotransformation with toxic substrates to produce useful and valuable chemicals.

Emulsion of Chloramphenicol: an Overwhelming Approach for Ocular Delivery.

BACKGROUND: Ophthalmic formulations of chloramphenicol have poor bioavailability of chloramphenicol in the ocular cavity. AIM: The present study aimed at exploring the impact of different oil mixtures in the form of emulsion on the permeability of chloramphenicol after ocular application. MATERIALS AND METHODS: Selection of oil mixture and ratio of the components was made by an equilibrium solubility method. An emulsifier was chosen according to its emulsification properties. A constrained simplex centroid design was used for the assessment of the emulsion development. Emulsions were evaluated for physicochemical properties; zone of inhibition, in-vitro diffusion and ex-vivo local accumulation of chloramphenicol. Validation of the design using check-point batch and reduced polynomial equations were also developed. Optimization of the emulsion was developed by software Design® expert 6.0.8. Assessment of the osmolarity, ocular irritation, sterility testing and isotonicity of optimized batch were also made. RESULTS: Parker Neem®, olive and peppermint oils were selected as an oil phase in the ratio 63.64:20.2:16.16. PEG-400 was selected as an emulsifier according to a pseudo-ternary phase diagram. Constrained simplex-centroid design was applied in the range of 25-39% water, 55-69% PEG-400, 5-19% optimized oil mixture, and 1% chloramphenicol. Unpaired Student's t-test showed for in-vitro and ex-vivo studies that there was a significant difference between the optimized batch of emulsion and Chloramphenicol eye caps (a commercial product) according to both were equally safe. CONCLUSION: The optimized batch of an emulsion of chloramphenicol was found to be as safe as and more effective than Chloramphenicol eye caps.