Hydrophilic Organic Solvents Research Articles

Investigation of the Composition for a Ternary Solvent System in Tube Radial Distribution Chromatography

A tube radial distribution chromatography (TRDC) system was developed based on the tube radial distribution of a ternary solvent system. The composition for the water–hydrophilic organic solvent–hydrophobic organic solvent in TRDC was investigated using phase diagrams, chromatograms, and fluorescence photographs. Mixtures of a hydrophilic organic solvent (acetonitrile, ethanol, methanol, 1-propanol, or 1,4-dioxane) and a hydrophobic solvent (ethyl acetate, hexane, 1-butanol, or chloroform) were examined as the ternary solvent system, which was the carrier solution in TRDC. Water–acetonitrile–chloroform and water–acetonitrile–ethyl acetate systems performed well in TRDC. A model analyte solution of 1-naphthol (NA) and 2,6-naphthalenedisulfonic acid was separated by TRDC using the chloroform solvent system with a range of volume ratios for the solvent components. The elution order of these two compounds could be reversed by changing the solvent system from organic solvent rich to water rich. A mixture of five analytes, NA, Eosin Y, 1-naphthalenesulfonic acid, 2,6-naphthalenedisulfonic acid, and 1,3,6-naphthalenetrisulfonic acid, was also separated using the water–acetonitrile–chloroform solvent system (32:65:3 volume ratio). These compounds eluted in the order given above.

Liquid–Liquid–Solid Triple-Phase Data for Aqueous Two-Phase Systems comprising Ethanol–1-Propanol—2-Propanol–Acetone and Salts

In this paper, the liquid–liquid–solid triple-phase data of some aqueous two-phase systems (ATPSs) containing hydrophilic organic solvents (HOS) and simple salts were explored. The systems studied comprise aqueous solutions containing ethanol, 1-propanol, 2-propanol, or acetone with (NH4)2SO4 and solutions containing 1-propanol with (NH4)2SO4, NaCl, or KCl. The Gibbs phase rule predicts that there is a linear relationship existing at such a triple-phase boundary. The linear liquid–liquid–solid boundaries were determined, and the effects of temperature, solvent, and salt on the boundary were investigated. The tie line length (TLL) of the systems distributed on the triple-phase boundary was invariant. Phase equilibrium experiments determined that the average TLL of ethanol–(NH4)2SO4 ATPSs at 298.15 K was 62.93 % with a standard deviation of 2.13 %. The linear liquid–liquid–solid triple-phase boundary was used to elucidate the two-phase region and determine the content of organic solvent or salt in an unknown sample. These results increase our understanding of HOS–salt–water aqueous two phase system (ATPS) and will be useful for those looking to develop new systems for separation science.

Screening, purification and characterization of a novel cold-active and organic solvent-tolerant lipase from Stenotrophomonas maltophilia CGMCC 4254

An extracellular organic solvent-tolerant and cold-active lipase producing bacterium was isolated from oil-contaminated soil samples, and identified taxonomically as Stenotrophomonas maltophilia. The lipase from S. maltophilia CGMCC 4254 (SML) was purified 60.5-fold to homogeneity with 38.9 U/mg specific activity. Partially purified SML displayed remarkable stability in 50% and 100% (v/v) hydrophobic organic solvents after incubation for 7 days. The enzyme also retained more than 50% of its residual activity in several pure hydrophilic organic solvents after incubation for 7 days. SML showed 57% maximum activity at 5°C, and had optimal activity at 35°C. These unique properties of SML make it promising as a biocatalyst for industrial processes.

Understanding the effect of tert-butanol on Candida antarctica lipase B using molecular dynamics simulations

The use of organic solvents as reaction media for enzymatic reactions has many advantages. Several organic solvents have been proposed as reaction media, especially for transesterifications using Candida antarctica lipase B (CalB). Among organic solvents, tert-butanol is associated with an enhanced conversion rate in bio-diesel production. Thus, it is necessary to understand the effect of tert-butanol on CalB to explain the high-catalytic efficiency compared with the reaction in other hydrophilic organic solvents. In this study, the effects of tert-butanol on the structure of CalB were investigated by MD simulations. The overall flexibility was increased in the presence of tert-butanol. The substrate entrance and the binding pocket size of CalB in tert-butanol were maintained as in TIP3P water. The distance between the catalytic residues of CalB in tert-butanol indicated a higher likelihood of forming hydrogen bonds. These structural analyses could be useful for understanding the effect of tert-butanol on lipase transesterification.

A Simple Technique of Preparing Stable CLEAs of Phenylalanine Ammonia Lyase Using Co-aggregation with Starch and Bovine Serum Albumin

Cross-linked enzyme aggregates (CLEAs) have been recently proposed as an alternative to conventional immobilization methods on solid carriers. However, the low cross-linking efficiency causes the major activity loss and instability in the conventional protocol for CLEA preparation. Herein, the effects of bovine serum albumin and starch addition on the cross-linking efficiency of CLEAs of phenylalanine ammonia lyase (PAL) from Rhodotorula glutinis were evaluated. A co-aggregation strategy was developed to improve cross-linking efficiency by adding starch and bovine serum albumin (BSA). CLEAs of PAL prepared in the presence of BSA and starch (PSB-CLEAs) retained 36 % activity, whereas CLEAs prepared without BSA and starch (PAL-CLEAs) retained only 8 % activity of the starting enzyme preparation. Compared with PAL-CLEAs, the thermal stability of PSB-CLEAs has improved considerably, maintaining 30 % residual activity after 4 h of incubation at 70 °C, whereas the PAL-CLEAs have only 13 % residual activity. PSB-CLEAs also exhibited the expected increased stability of PAL against hydrophilic organic solvents, superior operability, and higher storage stability. The proposed technique of preparing CLEAs using co-aggregation with starch and BSA would rank among the potential strategies for efficiently preparing robust and highly stable enzyme aggregates.

Recovery of lipase derived from Burkholderia cenocepacia ST8 using sustainable aqueous two-phase flotation composed of recycling hydrophilic organic solvent and inorganic salt

Recycling hydrophilic organic solvent/inorganic salt aqueous two-phase flotation (ATPF) is a novel, low cost, green and high efficient technique for recovery of biomolecules. Recycling ATPF composed of 2-propanol and potassium phosphate was developed for sustainable separation, concentration and purification of Burkholderia cenocepacia ST8 lipase from liquid fermentation broth. Thirteen parameters upon recycling hydrophilic organic solvent/inorganic salt ATPF performance were investigated. The optimum conditions for this recycling ATPF were determined to be 40mL volume of 50% (w/w) 2-propanol, 1.0L of 250g/L of potassium phosphate, pH 8.5, 100% (v/v) of crude feedstock, 30mL/min of N2 flow rate for 30min in a 8cm radius of colorimeter tube with G4 porosity (5–15μm) sintered glass disk. A purification factor of 14.4±0.04 and a lipase yield of 99.2±0.03% were achieved in this optimized ATPF. The recycling of phase-forming components employed at the end of recovery process was based on the principals of green chemistry, with high efficiency and economical viability. There was no gross variation of results during the process of scaling-up. Therefore, this novel recycling ATPF is feasible to be applied at industrial-scale.

ABC transporters and cell wall proteins involved in organic solvent tolerance in Saccharomyces cerevisiae

Pleiotropic drug resistance 1 (Pdr1p) protein is a key transcription factor in multidrug resistance. Specifically, R821S point mutation in the PDR1 gene in Saccharomyces cerevisiae diploid KK-211 strain plays an important role in tolerance to hydrophobic organic solvents, though the molecular mechanisms underlying organic solvent tolerance are unclear. In KK-211, several ABC transporters and cell wall proteins were upregulated. PDR1 R821S mutant derived from the laboratory haploid strain MT8-1 confirmatively tolerated hydrophobic organic solvents, and this study is the first to reveal its tolerance of the hydrophilic organic solvent, dimethyl sulfoxide (DMSO). To identify the genes involved in the organic solvent tolerance, we focused on the upregulated 4 ABC transporters and 6 cell wall proteins in KK-211, and demonstrated 2 ABC transporters, Pdr10p and Snq2p, and the 4 cell wall proteins, Wsc3p, Pry3p, Pir1p, and Ynl190wp were responsible for hydrophobic organic solvent (n-decane and n-undecane) tolerance. Tolerance to the hydrophilic organic solvent, DMSO, was facilitated by the overproduction of 2 ABC transporters, Snq2p and Yor1p and, 3 cell wall proteins, Wsc3p, Pry3p, and Ynl190wp. Our results suggest that overexpression of genes encoding proteins effective for tolerance to specific organic solvents would enable enhanced tolerances for practical use.

Characterization of solvent stable extracellular protease from Bacillus koreensis (BK-P21A)

A total of 18 protease producing bacterial strains were isolated from detergent effluent in South Korea using skim milk agar medium. A strain (BK-P21A) was selected and identified as Bacillus koreensis based on morphological, biochemical and molecular characterizations (16S rRNA gene sequence analysis). Optimized culture conditions for the production of protease were pH 8.5, 30°C, sucrose (2%) and yeast extract (0.2%) during 36h of incubation. Furthermore, the protease was partially purified by ammonium sulphate precipitation (80%) and again by Superdex 200 10/300 GL and Superdex 75 10/300 GL column chromatography, which resulted in 5.0 fold purification and a yield of 23%. The molecular mass of the protease was estimated to be 48kDa by SDS-PAGE. The purified enzyme was further characterized and found to be most active at pH 9.0 and 60°C. The activity of the purified protease was enhanced by CaCl2 and CoCl2, but inhibited by PMSF, which indicated it was a serine type protease. Moreover, the protease was moderately stable in surfactants and 81% stable in H2O2. Finally, the enzyme was more active and stable (94–126.5%) in various hydrophilic organic solvents. Considering the stability of protease towards the alkaline pH, high temperature and organic solvents (50%), the enzyme from B. koreensis can be used as an alternative biocatalyst for several industrial applications mainly for peptide synthesis in nonaqueous solvents.

Efficient enzymatic synthesis of mangiferin glycosides in hydrophilic organic solvents

β-Fructofuranosidase from Arthrobacter arilaitensis NJEM01 showed high activity and stability in 15–25% (v/v) methanol, 15–20% (v/v) DMSO, and 15–20% (v/v) ethanol with 91.2–99.7% total molar yields of mangiferin glycosides. The addition of hydrophilic organic solvents not only greatly promoted the solubility of the substrate, but also revealed a regulatory effect on mangiferin glycoside formation with increasing solvent concentration. Extraordinarily high concentrations and high yields of mangiferin glycosides were attained in the 20% DMSO solvent system from 47.6 g L−1 mangiferin with β-fructofuranosidase using sucrose as the donor, which greatly simplified the preparation process. β-Fructofuranosidase showed an exquisite regioselectivity for C-glucosylflavone.

An efficient novel glycosylation of flavonoid by β-fructosidase resistant to hydrophilic organic solvents

An effective approach was successfully developed to isolate glycosidase with resistance of hydrophilic organic solvent, simultaneously with acceptor specificity of the target substrate. By this approach, an efficient solvent tolerant glycosidase producing bacterium Arthrobacter nicotianae XM6 was obtained. The β-fructosidase from strain XM6 shows high activity and stability in 10–25% DMSO and 10–20% methanol with 90–99% yields of puerarin glycosides. The addition of hydrophilic solvents not only greatly promoted the solubility of puerarin, but also regulated main products from multifructosyl puerarin to monofructosyl puerarin with increasing solvent concentration. Extraordinary highly efficient synthesis of puerarin glycosides (111.3g/L of monofructosyl puerarin and 35.6g/L of difructosyl puerarin) was attained in 25% DMSO solvent system from 110.4g/L puerarin, which resulted a great facility for purification in large-scale process. The most novelty was that the β-fructosidase did not hydrolyze almost the newly formed glycosides using simply sucrose as donor.

Prediction of the solvent affecting site and the computational design of stable Candida antarctica lipase B in a hydrophilic organic solvent

Enzyme reactions in organic solvent such as for organic synthesis have great industrial potential. However, enzymes lose their stability in hydrophilic organic solvents due to the deformation of the enzyme by the solvent. It is thus important to enhance the stability of enzymes in hydrophilic organic solvents. Previous approaches have not considered on the interaction between enzymes and solvents due to the lack of information. In this study, the structural motions of the enzyme in methanol cosolvent and the interaction between the enzyme surface and the solvent molecule were investigated using molecular dynamics simulation (MD). By analyzing the MD simulation results, the surface residues of Candida antarctica lipase B (CalB) with higher root mean square deviation (RMSD) in a methanol solvent were considered as methanol affecting site and selected for site-directed mutagenesis. The methanol affecting site was computationally redesigned by lowering the RMSD. Among the candidate mutants, the A8T, A92E, N97Q and T245S mutants showed higher organic solvent stability at various methanol concentrations. The rational approach developed in this study could be applied to the stabilization of other industrial enzymes used in organic solvents.

Determination of Binding Constants for Inclusion Complexes of Cyclodextrins with Organic Solvents, Ethylene Glycol, and Its Related Compounds by Means of 1H NMR Spectroscopy

Abstract The binding constants (Ka’s) for the complexation of α- and β-cyclodextrins (α- and β-CDs) with some hydrophilic organic solvents such as methanol, dimethyl sulfoxide, acetone, etc., were successfully determined in D2O by following changes in the 1H NMR chemical shift of the C(3)–H signal of CD, using the C(1)–H signal as an internal reference. The Ka value obtained for a β-CD–methanol complex was 0.088 mol−1 dm3 at 298 K, which is, as we know, the smallest among Ka’s measured thus far for CD complexes. The same method was also applied to the determination of Ka’s for the complexation of α-CD with ethylene glycol and some related compounds. It was revealed that these molecules were certainly included within the cavity of α-CD, though the Ka values are fairly small.

Stabilization of Candida antarctica lipase B in hydrophilic organic solvent by rational design of hydrogen bond

Enzymatic reactions conducted in organic solvents have many advantages. However, organic solvent molecules may replace water molecules at the protein surface and penetrate into the enzyme, which could lead to the denaturation of the enzyme or changes in its reaction kinetics and substrate specificity. Thus, it is important to enhance the stability of enzymes in organic solvents. To date, there has been no efficient rational approach developed to enhance enzyme stability in hydrophilic solvents. We developed a rational approach to enzyme design. The design rules were established by investigating stable mutants from previous studies of directed evolution. Candida antarctica lipase B (CalB) was used as a target enzyme due to its versatile applications in organic solvents. The N97Q, N264Q, and D265E mutants of CalB showed higher organic solvent stability than the wild type.

A simple preparation of betulinic acid from sycamore bark

Betulinic acid is a biologically active compound, which has been well-known for anti-cancerous properties especially for skin. Recently, it has been drawn attention as a compound for combating AIDS. It can be chemically derived from betulin or by solvent extraction from bark. However, bark extracts are complex mixtures that always contain compounds with a wide spectrum of polarity. In this study, we have found a simple method to get high purity betulinic acid from Sycamore outer bark using hot water pre-treatment followed by hydrophilic organic solvent extraction with a yield of 5–6% (w/w).

Characterization of Acetonitrile-Tolerant Marine Bacterium <i>Exiguobacterium</i> sp. SBH81 and Its Tolerance Mechanism

A Gram-positive marine bacterium, Exiguobacterium sp. SBH81, was isolated as a hydrophilic organic-solvent tolerant bacterium, and exhibited high tolerance to various types of toxic hydrophilic organic solvents, including acetonitrile, at relatively high concentrations (up to 6% [v/v]) under the growing conditions. Investigation of its tolerance mechanisms illustrated that it does not rely on solvent inactivation processes or modification of cell surface characteristics, but rather, increase of the cell size lowers solvent partitioning into cells and the extrusion of solvents through the efflux system. A test using efflux pump inhibitors suggested that secondary transporters, i.e. resistance nodulation cell division (RND) and the multidrug and toxic compound extrusion (MATE) family, are involved in acetonitrile tolerance in this strain. In addition, its acetonitrile tolerance ability could be stably and significantly enhanced by repetitive growth in the presence of toxic acetonitrile. The marked acetonitrile tolerance of Exiguobacterium sp. SBH81 indicates its potential use as a host for biotechnological fermentation processes as well as bioremediation.

Improvement in the photovoltaic efficiency of polymer solar cells by treating the poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) buffer layer with co-solvents of hydrophilic organic solvents and hydrophobic 1,2-dichlorobenzene

Less conductive poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS, Clevios P VP Al 4083) is usually used as a buffer layer in polymer solar cells. The conductivity of PEDOT:PSS can be enhanced from 10 −3 to 10 0 S cm −1 through a treatment with a cosolvent of hydrophilic methanol and hydrophobic 1,2-dichlorobenzene (DCB). The conductivity enhancement is attributed to the preferential solvation of hydrophobic PEDOT and hydrophilic PSS chains with the two components of the cosolvent. It induces PSSH reduction of the PEDOT:PSS films and morphological change of the polymer chains. Moreover, the cosolvent treatment of the PEDOT:PSS buffer layer can remarkably improve the photovoltaic efficiency of polymer solar cells. The treatment of the PEDOT:PSS buffer layer with a cosolvent of methanol and DCB can improve the photovoltaic efficiency of polymer solar cells based on poly(3-hexylthiophene) (P3HT) and [6,6]-phenyl-C 61-butyric acid methyl ester (PCBM) from 3.98% to 4.31%. The PCE improvement is attributed to the high conductivity and high surface area of the cosolvent-treated PEDOT:PSS buffer layer.

Improvement of the biocatalytic properties of one phenylacetone monooxygenase mutant in hydrophilic organic solvents

The presence of different hydrophilic organic solvents or a water soluble polymer such as PEG 4000 led to an enhancement in the enzymatic activity of the M446G mutant of phenylacetone monooxygenase when it is employed in enantioselective sulfoxidations and Baeyer–Villiger reactions. By solvent engineering new substrates were found to be effectively converted by this Baeyer–Villiger monooxygenase. The use of 5% methanol together with the weak anion exchange resin Lewatit MP62 also allows the dynamic kinetic resolution of a set of racemic benzylketones. By this approach ( S)-benzylesters could be obtained with high yields and optical purities.

Role of α-Helical Structure in Organic Solvent-Activated Homodimer of Elastase Strain K

Recombinant elastase strain K overexpressed from E. coli KRX/pCon2(3) was purified to homogeneity by a combination of hydrophobic interaction chromatography and ion exchange chromatography, with a final yield of 48% and a 25-fold increase in specific activity. The purified protein had exhibited a first ever reported homodimer size of 65 kDa by SDS-PAGE and MALDI-TOF, a size which is totally distinct from that of typically reported 33 kDa monomer from P. aeruginosa. The organic solvent stability experiment had demonstrated a stability pattern which completely opposed the rules laid out in previous reports in which activity stability and enhancement were observed in hydrophilic organic solvents such as DMSO, methanol, ethanol and 1-propanol. The high stability and enhancement of the enzyme in hydrophilic solvents were explained from the view of alteration in secondary structures. Elastinolytic activation and stability were observed in 25 and 50% of methanol, respectively, despite slight reduction in α-helical structure caused upon the addition of the solvent. Further characterization experiments had postulated great stability and enhancement of elastase strain K in broad range of temperatures, pHs, metal ions, surfactants, denaturing agents and substrate specificity, indicating its potential application in detergent formulation.

Immobilization strategy of accessible transmission for trypsin to catalyze synthesis of dipeptide in mesoporous support

Immobilized trypsin in mesoporous silica foams was used to catalyze dipeptide synthesis in hydrophilic organic solvent instead of soluble form. The area surface of nano support was measured. The catalytic activity, coupled yield and kinetic characterization of immobilized trypsin were examined. Bz-Arg-OEt was chosen as the acyl donor with Lys-OH as the nucleophile. The trypsin-catalyzed synthesis condition was optimized, such as catalytic temperature, pH, reaction time, physical properties and content of organic solvents, together with the added enzyme amount. The immobilized trypsin showed 112.8% of residual activity with 91.9% of coupled yield, and the kinetic parameters exhibited accessibility for transmission. The product yield of 5.8% was reached at the optimum conditions for enzymatic synthesis of dipeptide: 800 mg of wet immobilized trypsin (200 mg/g support) was used in Tris-HCl buffer (0.1 mol/L, pH 8.0) containing 80% (v/v) ethanol solvents for 6 h of reaction time at 35 °C. This attempt of immobilized strategy for trypsin in nanopores renders the possibility of wide application of inorganic nano-sized support in catalytic synthesis process, which can avoid usage of large amounts of organic solvents in washing steps by chemical methods and reduce the tedious purification process of its soluble form.

Using ionic liquid cosolvents to improve enzymatic synthesis of arylalkyl β-d-glucopyranosides

► Ethylene glycol diacetate, a green and nontoxic organic solvent, proved to be the best for enzymatic synthesis of glycosides. ► The ionic liquid [BMIm]I was applied as the solvent in biocatalysis for the first time. ► The yields were enhanced between 0.2-fold and 0.5-fold by adding 10% (v/v) [BMIm]I. Enzymatic synthesis of various arylalkyl β- d -glucopyranosides catalyzed by prune ( Prunus domestica ) seed meal via reverse hydrolysis in the mixture of organic solvent, ionic liquid (IL) and phosphate buffer was described. Among four hydrophilic organic solvents tested, ethylene glycol diacetate (EGDA) was found to be the most suitable for enzymatic synthesis of salidroside, a bioactive compound of commercial interest, from d -glucose and tyrosol. The effects of the nature of ionic liquids and their contents on the enzymatic glucosylation were studied. The addition of a suitable amount of ILs including denaturing ones was favorable to shift the reaction equilibrium toward the synthesis, thus improving the yields. Among the examined ILs, the novel IL [BMIm]I proved to be the best. And this IL was applied as the solvent in biocatalysis for the first time. The yields were found to be enhanced between 0.2-fold and 0.5-fold after the addition of 10% (v/v) [BMIm]I. In 10% (v/v) [BMIm]I-containing system, the desired arylalkyl β- d -glucopyranosides were synthesized with 15–28% yields, among which salidroside was obtained with a yield of 22%.