Enantioselective enzymatic resolution of racemic alcohols by lipases in green organic solvents

The effects of two eco-friendly solvents, 2-methyltetrahydrofuran (MeTHF) and cyclopentyl methyl ether (CPME), on the enzyme activity and enantioselectivity of Novozym 435, Candida rugosa lipase (CRL), Porcine pancreas lipase (PPL), Lipase AK, Lipase PS, and Lipozyme, a series of commercial lipases, in the enantioselective transesterfications of racemic menthol, racemic sulcatol and racemic α-cyclogeraniol were studied. Vinyl acetate was chosen as the acyl donor and the reactions were carried out at water activity 0.06. The activity of lipases in CPME was similar to that observed in other largely employed organic solvents [toluene and tert-butyl methyl ether (MTBE)], and was slightly lower in MeTHF. However, for most of the lipases tested, the enantioselectivity was higher in the eco-friendly solvents. Lipase AK exhibited a high enantioselectivity (E = 232) for the resolution of racemic menthol but the reaction rate was low. Lipase formulation (the enzyme was frozen and lyophilized in potassium phosphate buffer without and with 5% (w/v) of sucrose, d-mannitol, or methoxy poly(ethylene glycol)) was tested with this lipase in order to improve its activity, which increased up to 4.5 times, compared to the untreated enzyme. CALB was found to be a useful biocatalyst for the resolution of racemic sulcatol, where high activity and enantioselectivity were obtained (E ≥ 1000). For the resolution of the racemic primary alcohol α-cyclogeraniol, most of the lipases tested were active but not enantioselective, except lipase PS which displayed a moderate enantioselectivity (E = 19). The effect of the presence of a low percentage of two ionic liquids (ILs) 1-Butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([BMIM][TFSI]) (5% (v/v)) and 1-Butyl-3-methylimidazoliumtetrafluoroborate ([BMIM][BF4]) (1% (v/v)) in the medium was also investigated. Only in the case of CRL the ILs slightly increased the enantioselectivity from E = 91 to E = 103 and E = 120 for [BMIM][TFSI] and [BMIM][BF4], respectively. However, in all cases ILs caused a decrease of enzyme activity.     

Production and Characterization of a Halo-, Solvent-, Thermo-tolerant Alkaline Lipase by Staphylococcus arlettae JPBW-1, Isolated from Rock Salt Mine

Studies on lipase production and characterization were carried out with a bacterial strain Staphylococcus arlettae JPBW-1 isolated from rock salt mine, Darang, HP, India. Higher lipase activity has been obtained using 10 % inoculum with 5 % of soybean oil as carbon source utilizing a pH 8.0 in 3 h at 35 °C and 100 rpm through submerged fermentation. Partially purified S. arlettae lipase has been found to be active over a broad range of temperature (30–90 °C), pH (7.0–12.0) and NaCl concentration (0–20 %). It has shown extreme stability with solvents such as benzene, xylene, n-hexane, methanol, ethanol and toluene up to 30 % (v/v). The lipase activity has been found to be inhibited by metal ions of K⁺, Co²⁺ and Fe ²⁺ and stimulated by Mn²⁺, Ca²⁺ and Hg²⁺. Lipase activity has been diminished with denaturants, but enhanced effect has been observed with surfactants, such as Tween 80, Tween 40 and chelator EDTA. The K _m and V _max values were found to be 7.05 mM and 2.67 mmol/min, respectively. Thus, the lipase from S. arlettae may have considerable potential for industrial application from the perspectives of its tolerance towards industrial extreme conditions of pH, temperature, salt and solvent.     

Solubility and Thermal Degradation of Quercetin in CO2-Expanded Liquids

The solubility of quercetin and its thermal degradation was studied in CO₂-expanded ethanol and ethyl lactate. An equipment setup was constructed that enabled the separation of the products of degradation while quantifying the solubility of quercetin. Three different conditions of temperature were analyzed (308, 323, and 343 K) at 10 MPa. Higher solubility and thermal degradation of quercetin were observed for CO₂-expanded ethyl lactate in comparison with CO₂-expanded ethanol. At the same time, as the amount of CO₂ was increased in the CO₂-expanded liquids mixtures, the thermal degradation of quercetin decreased for almost all the conditions of temperature considered in this work. The importance of considering thermal degradation while performing solubility measurements of compounds that are thermally unstable such as quercetin was highlighted.     

Perfluoromethacrylate-styrene statistical copolymers synthesized in CO2-expanded monomers

Statistical copolymers of perfluoroalkyl ethyl methacrylate (Zonyl-TM) and styrene (S) were synthesized in CO₂-expanded monomer medium at a low initial pressure of 25 MPa. Different Zonyl-TM/S feed ratios were used during copolymerizations, and it was determined that the increase in the Zonyl-TM content and decrease of the CO₂ amount in the comonomer feed resulted in a decrease of the molecular weights of copolymers due to earlier precipitation of copolymers giving shorter chains. The cloudy CO₂-expanded liquid monomer phase was found to be the main loci of copolymerization. In addition, the increase in the Zonyl-TM feed ratio resulted in an increase in the critical degree of the polymerization time (J _crit) as the time when the copolymer chains start to precipitate. The higher the Zonyl-TM content used in the feed, the higher the J _crit time and the lower the weight-average molecular weight (M _w) of the copolymer obtained. Thermal analysis results of the copolymer indicated that the copolymers are stable up to 387–403 °C.     

Extraction of aroma compounds in blackcurrant buds by alternative solvents: Theoretical and experimental solubility study

This study was designed to evaluate the performance of nine alternative solvents (α-pinene, MeTHF, ethyl acetate, methyl acetate, ethyl lactate, butanol, isopropanol, ethanol and CO₂ supercritical fluid) for extracting aromas from blackcurrant buds (Ribes nigrum L) compared to that of n-hexane, commonly used. This study has been performed via experimentation and simulation using Hansen solubility methodology for the comprehension of the dissolving mechanism. Experimentally, the extracts were analysed to compare the solvents performance in terms of aroma compositions. The results indicated that an alternative solvent, i.e. MeTHF, could be the most promising one for n-hexane substitution with good yield and selectivity of aromas.     

Dispersion Property of CO2 in Oil. Part 3: Aggregation of CO2 Molecule in Organic Liquid at Near Critical and Supercritical Condition of CO2

To study the dispersion property of CO₂ in organic liquids, the solubility of CO₂, the volume of CO₂ + organic liquids under different pressure at 318.15 K were measured and the radial distribution function of CO₂ molecule aggregate in the organic liquids was calculated by molecule dynamic simulation. The results show that the aggregate of CO₂ molecules in the organic liquids is formed obviously; the aggregation and disperse property of CO₂ molecule in the organic liquids is affected by the structure and polarity of the organic molecule at near critical and supercritical condition of CO₂ dominantly.     

Efficient Regioselective Synthesis of the Crotonyl Polydatin Prodrug by <Emphasis Type="Italic">Thermomyces lanuginosus</Emphasis> Lipase: a Kinetics Study in Eco-friendly 2-Methyltetrahydrofuran

Bio-based solvents have recently been discussed as sustainable green and promising alternatives to conventional organic media for enzymatic processes. In this paper, highly regioselective synthesis of the 6″-O-crotonyl-polydatin catalyzed by Thermomyces lanuginosus lipase (TLL) in biomass-derived 2-methyltetrahydrofuran (2-MeTHF) was successfully performed for the first time. The results indicated that TLL lipase displayed significantly improved catalytic performance in 2-MeTHF than in other traditional solvents. Under the optimal conditions, the initial reaction rate, 6″-regioselectivity, and maximum substrate conversion were as high as 12.38 mM h^?1, 100 %, and 100 %, respectively. Moreover, further investigations on the operational stability, kinetic parameters like V _max, K _m, V _max/K _m, and E _a revealed that 2-MeTHF exhibited excellent biocompatibility and rendered the greener process of the enzymatic acylation.     

Production and Biotechnological Application of Extracellular Alkalophilic Lipase from Marine Macroalga-Associated <Emphasis Type="Italic">Shewanella algae</Emphasis> to Produce Enriched C<Subscript>20-22</Subscript><Emphasis Type="Italic">n</Emphasis>-3 Polyunsaturated Fatty Acid Concentrate

An extracellular alkalophilic lipase was partially purified from heterotrophic Shewanella algae (KX 272637) associated with marine macroalgae Padina gymnospora. The enzyme possessed a molecular mass of 20 kD, and was purified 60-fold with a specific activity of 36.33 U/mg. The enzyme exhibited V_max and K_m of 1000 mM/mg/min and 157 mM, respectively, with an optimum activity at 55 °C and pH 10.0. The catalytic activity of the enzyme was improved by Ca²⁺ and Mg²⁺ ions, and the enzyme showed a good tolerance towards organic solvents, such as methanol, isopropanol, and ethanol. The purified lipase hydrolyzed the refined liver oil from leafscale gulper shark Centrophorus squamosus, yielding a total C_20-22 n-3 PUFA concentration of 34.99% with EPA + DHA accounting the major share (34% TFA), after 3 h of hydrolysis. This study recognized the industrial applicability of the thermostable and alkalophilic lipase from marine macroalga-associated bacterium Shewanella algae to produce enriched C_20-22 n-3 polyunsaturated fatty acid concentrate.     

A Calcium-Ion-Stabilized Lipase from <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> ZS04 and its Application in Resolution of Chiral Aryl Alcohols

An extracellular organic solvent-tolerant lipase-producing bacterium was isolated from oil-contaminated soil samples and was identified taxonomically as Pseudomonas stutzeri, from which the lipase was purified and exhibited maximal activity at temperature of 50 °C and pH of 9.0. Meanwhile, the lipase was stable below or at 30 °C and over an alkaline pH range (7.5–11.0). Ca²⁺ could significantly improve the lipase thermal stability which prompts a promising application in biocatalysis through convenient medium engineering. The lipase demonstrated striking features such as distinct stability to the most tested hydrophilic and hydrophobic solvents (25 %, v/v), and DMSO could activate the lipase dramatically. In the enzyme-catalyzed resolution, lipase ZS04 manifested excellent enantioselective esterification toward the (R)-1-(4-methoxyphenyl)-ethanol (MOPE), a crucial chiral intermediate in pharmaceuticals as well as in other analogs with strict substrate specificity and theoretical highest conversion yield. This strong advantage over other related schemes made lipase ZS04 a promising biocatalyst in organic synthesis and pharmaceutical applications.     

Electrochemistry of nickel(II) and copper(II) N,N′-ethylenebis(acetylacetoniminato) complexes and their electrocatalytic activity for reduction of carbon dioxide and carboxylic acid protons

The effect of the metal center of [ML] complexes [M = Ni(II), Cu(II); L = N,N′-ethylenebis(acetylacetoniminato)] on their electrochemistry and electrocatalytic activity for the reduction of CO₂ and protons has been studied using cyclic voltammetry and bulk electrolysis. The two complexes exhibit different electrochemistries, which are not significantly dependent on the nature of the solvent. The electrocatalytic activity of [NiL] is significantly higher than that of [CuL] for CO₂ reduction, due to the higher stability of the electrochemically generated [Ni(I)L] complex, relative to the Cu(I) analog. The diffusion coefficient of [NiL] calculated from the steady-state diffusion limiting current is 3.0 × 10^?6 cm² s^?1. The catalytic efficiency of [NiL] in non-aqueous solvents in terms of i _p(CO₂)/i _p(N₂) per nickel center is smaller than that of [Ni(cyclam)]²⁺, but greater than those of sterically hindered mononuclear [Ni(1,3,6,8,10, 13,15-heptaazatricyclo(11.3.1.1) octadecane)]²⁺ or multinuclear [Ni₃ (X)]⁶⁺ where X = 8,8′,8″-{2,2′,2″(-nitrilotriethyl)-tris(1,3,6,8,10,13,15-heptaazatricyclo(11.3.1.1) octadecane}. Both [NiL] and [CuL] are also electrocatalysts for the reduction of carboxylic acid protons, with the catalytic pathway being different for acetic and trifluoroacetic acids in MeCN.     

Near-infrared spectroscopic measurements of volume expansion and composition of CO2-expanded ethyl acetate,acetone, tetrahydrofuran,acetonitrile, methanol-OD,and dimethyl sulfoxide

CO₂-expanded liquid (CXL) is a mixture of organic solvent with high-pressure CO₂ whose volume is increased by CO₂ dissolved in it. CXLs have attracted attention as tunable solvents, because the solvent properties can be widely controlled by the pressure. The volume expansion and the solubility of CO₂ were measured by near-infrared spectroscopy for 6 CXLs at various pressures up to 55 bar and 40 °C. The molarity of organic solvent was determined from the absorbance of the 3ν and 2ν + δ bands, and that of CO₂ was obtained from the area of the 3ν₃ band, whose peak shifted to higher frequency with increasing pressure due to a decrease in the molecular interaction around CO₂. The expansion coefficient was shown to be an increasing function of the pressure with larger slope at higher pressure, and the mole fraction of CO₂ in the liquid phase was an almost linearly increasing function of the pressure. The results were in quantitative agreement with the literature data measured by conventional sampling method, indicating the validity of the spectroscopic method.     

Transesterification of Waste Cooking Oil by an Organic Solvent-Tolerant Alkaline Lipase from Streptomyces sp. CS273

The aim of this present study was to produce a microbial enzyme that can potentially be utilized for the enzymatic transesterification of waste cooking oil. To that end, an extracellular lipase was isolated and purified from the culture broth of Streptomyces sp. CS273. The molecular mass of purified lipase was estimated to be 36.55 kDa by SDS PAGE. The optimum lipolytic activity was obtained at alkaline pH 8.0 to 8.5 and temperature 40 °C, while the enzyme was stable in the pH range 7.0?～?9.0 and at temperature ≤40 °C. The lipase showed highest hydrolytic activity towards p-nitrophenyl myristate (C14). The lipase activity was enhanced by several salts and detergents including NaCl, MnSo₄, and deoxy cholic acid, while phenylmethylsulfonyl fluoride at concentration 10 mM inhibited the activity. The lipase showed tolerance towards different organic solvents including ethanol and methanol which are commonly used in transesterification reactions to displace alcohol from triglycerides (ester) contained in renewable resources to yield fatty acid alkyl esters known as biodiesel. Applicability of the lipase in transesterification of waste cooking oil was confirmed by gas chromatography mass spectrometry analysis.     

Sonochemical Effect on Activity and Conformation of Commercial Lipases

The enzyme under lower-intensity ultrasonic irradiation leads to favourable conformational changes, thereby enhancing its activity. The augmentation of activity of ultrasound-treated enzyme is strongly dependent on ultrasound intensity, duty cycle and exposure time, which was investigated for commercial lipases. Thermomyces lanuginosus (TL) lipase showed a 1.3-fold enhanced activity after irradiating at 22 kHz and 11.38 W cm^?2 with 50 % duty cycle for 25-min ultrasonic treatment and 1.5-fold enhanced activity was observed for lipozyme (candida antarctica lipase B (CALB)) lipase, at 22 kHz and 15.48 W cm^?2 with 66.67 % duty cycle for 20-min ultrasonic treatment. After sonication, thermodynamic parameters viz. E _a, ΔH, ΔS and ΔG were evaluated and values were found to be significantly lower for both lipases. In addition, the changes in secondary structure due to sonication were investigated by using Fourier transform infrared (FT-IR), which revealed increase in a certain number of random coiled structure, loss of β-sheets, β-turns and α-helix content in TL lipase and CALB lipase. Also, fluorescence spectroscopy exhibited the increased number of tryptophan on surface of both lipases. Moreover, particle size distribution after sonication also helped to improve surface area and enhanced mass transfer, which contributed to improvement in lipase activity.     

Synthesis of mesoporous Ca-MCM catalysts and their use in suitable multicomponent synthesis of polyfunctionalized pyrans

Mesoporous Ca-MCM catalysts were prepared using a mixture of CaCO₃, TEOS, cetyl trimethyl ammonium bromide (CTAB), triethanolamine (TEA), ethanol and H₂O. Three catalysts with different CaCO₃ contents, M-Ca 1 %, M-Ca 5 % and M-Ca 10 %, respectively, were prepared and characterized by N₂ physisorption, XRD, FT-IR, XPS, TPD-CO₂ and Pyridine-FTIR. A green and simple protocol was developed for the synthesis of 6-amino-4H-pyran scaffolds through a multicomponent coupling reaction of an aldehyde, malononitrile, and 1,3-dicarbonyl compound in different reaction solvents. This procedure was performed at room temperature (20 °C), obtaining good and excellent yields of 4H-pyran derivatives. The mesoporous materials are insoluble in polar media, which allows easy removal of the reaction products without affecting their catalytic activity. The leaching test showed an excellent stability, and Ca-MCM catalysts can be used three times without appreciable loss of their catalytic activity.     

Characterization of a Novel Organic Solvent Tolerant Protease from a Moderately Halophilic Bacterium and Its Behavior in Ionic Liquids

An extracellular protease was purified from a novel moderately halophilic bacterium Salinivibrio sp. strain MS-7 by the combination of an acetone precipitation (40–80 %) step and a DEAE-cellulose anion exchange column chromatography. Kinetic parameters of the enzyme exhibited V _max and K _m of 130 U/mg and 1.14 mg/ml, respectively, using casein as a substrate. The biochemical properties of the enzyme revealed that the 21-kDa protease had a temperature and pH optimum of 50 °C and 8.0, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, Pefabloc SC, chymostatin, and also EDTA, indicating that it belongs to the class of serine metalloproteases. Interestingly, Ba²⁺ and Ca²⁺ (2 mM) strongly enhanced the enzyme activity, while Fe²⁺ and Mg²⁺ activated moderately and Zn²⁺, Ni²⁺, and Hg²⁺ decreased the enzyme activity. The effect of organic solvents with different logP on the purified protease revealed complete stability in toluene, ethyl acetate, chloroform, and n-hexane at 10 and 50 % (v/v) and moderate stability even in 50 % of DMSO and ethanol. The behavior of the MS-7 protease in three imidazolium-based ionic liquids exhibited suitable activity in these green solvent systems, especially in 1-hexyl-3-methylimidazolium hexafluorophosphate ([C₆MIM][PF₆]). Comparison of the purified protease with other previously reported proteases suggests that strain MS-7 secrets a novel organic solvent-tolerant protease with outstanding activity in organic solvents and imidazolium-based ionic liquids, which could be applied in low water synthetic section of industrial biotechnology.     

Development in the green synthesis of cyclic carbonate from carbon dioxide using ionic liquids

This brief review presents the recent development in the synthesis of cyclic carbonate from carbon dioxide (CO₂) using ionic liquids as catalyst and/or reaction medium. The synthesis of cyclic carbonate includes three aspects: catalytic reaction of CO₂ and epoxide, electrochemical reaction of CO₂ and epoxide, and oxidative carboxylation of olefin. Some ionic liquids are suitable catalysts and/or solvents to the CO₂ fixation to produce cyclic carbonate. The activity of ionic liquid is greatly enhanced by the addition of Lewis acidic compounds of metal halides or metal complexes that have no or low activity by themselves. Using ionic liquids for the electrochemical synthesis of the cyclic carbonate can avoid harmful organic solvents, supporting electrolytes and catalysts, which are necessary for conventional electrochemical reaction systems. Although the ionic liquid is better for the oxidative carboxylation of olefin than the ordinary catalysts reported previously, this reaction system is at a preliminary stage. Using the ionic liquids, the synthesis process will become greener and simpler because of easy product separation and catalyst recycling and unnecessary use of volatile and harmful organic solvents.     

Production, Characterization, and Application of an Organic Solvent-Tolerant Lipase Present in Active Inclusion Bodies

An organic solvent-tolerant lipase from Serratia marcescens ECU1010 (rSML) was overproduced in Escherichia coli in an insoluble form. High concentrations of both biomass (50 g cell wet weight/L culture broth) and inclusion bodies (10.5 g/L) were obtained by applying a high-cell-density cultivation procedure. Activity assays indicated that the enzymatic activity of rSML reached 600 U/L. After treatment with isopropyl ether for 12 h, the maximum lipase activity reached 6,000 U/L. Scanning electron microscopy and Fourier transform infrared microspectroscopy revealed the activation mechanism of rSML in the presence of organic solvents. rSML was stable in broad ranges of temperatures and pH values, as well as in a series of organic solvents. Besides, rSML showed the best enantioselectivity for the kinetic resolution of (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester. These features render the S. marcescens ECU1010 lipase attractive for biotechnological applications in the field of organic synthesis and pharmaceutical industry.     

Basic ionic liquids. A short review

Basic ionic liquids as environmental-friendly solvents and catalysts with high activity and selectivity and easily recovered materials were used to replace traditional bases such as KOH, NaOH, K₂CO₃, NaHCO₃, NaOAc, triethylamine, or tetrabutylammonium acetate. Using the traditional bases generally suffered from disadvantages such as waste production, corrosion and environmental problems. Basic ionic liquids offering a new possibility for developing environmentally friendly basic catalysts due to the combination of the advantages of inorganic bases and ionic liquids. They are flexible, nonvolatile,noncorrosive, and immiscible with many organic solvents. Basic ionic liquids (BILs) have been used in base-catalyzed processes such as Michael addition, Markovnikov addition, Knoevenagel condensation, Henry reaction, Mannich reaction, oximation, Feist-Benary reaction and etc. In this short review, we wish to present an overview of the types, properties, synthesis and applications of basic ionic liquids.     

Experimental investigations of thermal stability of some morpholinecarbamic acid complexes of copper(II) and zinc(II)

Some new carbamates, viz. M(MorphcbmH)₂X₂ (MorphcbmH = morpholinecarbamic acid, M = Cu, X = Cl, ClO₄,NO₃; M = Zn, X = Cl, ClO₄, NO₃, CH₃COO and X₂ = SO₄), have been synthesized and investigated. Compounds were characterized by elemental analysis, molar conductance, FT infrared, fluorescence, NMR (¹H and ¹³C) and solution electronic absorption spectral studies. Room temperature field-dependent magnetic susceptibility measurements, PXRD spectral and cyclic voltametric studies were also conducted. Chelating bidentate mode of coordination of ligand, MorphcbmH with four coordination around metal ion has been proposed. Ligand and its compounds have also been studied using non-isothermal thermogravimetric analysis and differential scanning calorimetric analytical techniques which inferred formation of metal oxide/MCO₃ as final thermal decomposition products. Compounds were screened against the lipase enzyme for assaying their enzyme activity and were found to retard the lipase activity from 48.16 to 0.044 µmol mL^?1 min^?1.     

Diastereoselective and enantioselective alkaline-hydrolysis of 2-aryl-1-cyclohexyl acetate: a CAL-B catalyzed deacylation/acylation tandem process

Candida antarctica lipase proved to be a particularly efficient lipase for the resolution of racemic 2-arylcyclohexyl acetate in hydrolysis reaction with Na₂CO₃ in an organic medium. The (1R,2S)-trans-2-arylcyclohexanols 2a–2d were obtained with high ee values (up to >99%) and the selectivity reached E > 200. The influence of the enol ester and the solvent on (±)-trans-2-arylcyclohexanol in the CAL-B catalyzed acylation was also studied and compared with the deacylation. The CAL-B exhibits a better affinity for the alkaline hydrolysis reaction compared with acylation with the enol esters in the same organic solvents. The best conditions were applied to resolve a stereoisomeric mixture cis/trans-2-phenyl-1-cyclohexanol and its corresponding acetate by acylation and deacylation. The obtained results show a highly enantio- and diastereoselectivity of the CAL-B during the acylation and the deacylation in favor of the trans-(R)-enantiomer product. The resolution of a mixture of cis/trans-2-arylcyclohexanols was an easy, convenient approach to provide only one stereoisomer of a mixture of four with high enantiomeric excess.