Lipase-immobilized biocatalytic membranes for enzymatic esterification: Comparison of various approaches to membrane preparation

In this paper, lipase-immobilized membranes were prepared by both non-covalent and covalent immobilization methods using (i) lipase adsorption on membranes, (ii) inclusion of enzyme in membrane structure by filtration and (iii) covalent attachment of lipase to membrane. The catalytic properties of these membranes have been studied in reaction of butyloleate synthesis through esterification of oleic acid with n-butanol in isooctane. Ultrafiltration membranes made of regenerated cellulose (C030F) and polyethersulphone (PM30) were used for lipase immobilization. It was found that the lipase inclusion in the wide porous supporting layer of membrane was the most efficient method in preparing highly effective biocatalytic membranes. The degree of oleic acid conversion using these membranes was about 70–72% with a reaction time of 8 h. It was shown that the distribution profile of the lipase in the membrane was important for the effective enzyme utilization.The profile imaging atomic force microscopy (AFM) technique was used to visualize surfaces of lipase-immobilized biocatalytic membranes. AFM has also been used to directly quantify interactions between lipase-coated tip and membrane surfaces. It was concluded that the direct measurements of the interaction force between the enzyme-coated tip and the membrane surface would be a useful and practical approach for the choice of membranes as porous polymeric support for lipase immobilization through adsorption.     

膜反应器中萘普生甲酯的动态拆分

在碱催化连续原位消旋条件下,利用CRL脂肪酶（Candida rugosa lipase）催化的萘普生甲酯立体选择性水解反应。动态拆分制备（S）－普生。使用硫水硅橡胶膜隔离生物催化拆分反应和碱催化消旋反应,解决了常规动态拆分反应中生物催化剂难以承受原位化学消旋苛刻反应条件的难题。为了利于从水－有机溶剂乳化体系中分离产物和克服产物抑制,将亲水半透膜引入搅拌罐反应器,在该膜反应器中进行动态拆分反应。当转化率超过60％时,产物（S）－萘普生的对映体过量值（eep)仍在96％以上,在反应过程中还发现CRL脂肪酶同工酶的转化。     

Esterification of (RS)-Ibuprofen by native and commercial lipases in a two-phase system containing ionic liquids

Four commercially available lipases and two native lipases from Aspergillus niger AC-54 and Aspergillus terreus AC-430 were used for the resolution of (RS)-Ibuprofen in systems containing the ionic liquids [BMIM][PF₆] and [BMIM][BF₄]. The lipases showed higher conversion in a two-phase system using [BMIM][PF₆] and isooctane compared to that in pure isooctane. Although the best enzyme was a commercially available lipase from Candida rugosa (E = 8.5), another native lipase, produced in our laboratory, from A. niger gave better enantioselectivity (E = 4.6) than the other lipases tested (E = 1.9–3.3.). After thorough optimization of several reaction conditions (type and ratios of isooctane/ionic liquid, amount of enzyme, and reaction time), the E-value of A. niger lipase (15% w/v) could be duplicated (E = 9.2) in a solvent system composed of [BMIM][PF₆] and isooctane (1:1) after 96 h of reaction.     

Study of immobilized candida rugosa lipase for biodiesel fuel production from palm oil by flow microcalorimetry

Enzymatic transesterification of palm oil with methanol and ethanol was studied. Of the four lipases that were tested in the initial screening, lipase Candida Rugosa (CR) resulted in the highest yield of mono alkyl esters. Lipase CR was further investigated in immobilized form within an activated carbon as support. The activated carbon was prepared by activation physical. Using the immobilized lipase CR, the effects of water and alcohol concentration, enzyme loading and enzyme thermal stability in the transesterification reaction were investigated. The optimal conditions for processing 50 g of palm oil were: 37 °C, 1:14.5 oil/methanol molar ratio, 1.0 g water and 500 mg lipase for the reactions with methanol, 35 °C, 1:15.0 oil/ethanol molar ratio, 1.0 g water, 500 mg lipase for the reactions with ethanol, and 35 °C, 1:10.0 oil/n-butanol molar ratio, 1.0 g water, 500 mg lipase for the reactions with ethanol. Subject to the optimal conditions, methyl and ethyl esters formation of 70 and 85 mol% in 1 h of reaction were obtained for the immobilized enzyme reactions. The flow microcalorimetry is an important and novel techniques is used in evaluation of biodiesel production.     

Stability and performance of porous silica–zirconia composite membranes for pervaporation of aqueous organic solutions

Porous silica–zirconia membranes were fabricated by the sol–gel techniques to study their stability against water and the pervaporation performance of aqueous solutions of organic solvents. Zirconia (10–70 mol%) was added to silica to obtain silica–zirconia composite membranes by firing at 400–500 °C for pervaporation tests with organic solvent/water mixtures, such as iso-propyl alcohol (IPA)/water and tetrahydrofuran (THF)/water mixtures at their normal boiling points.The membrane coatings have been done effectively by the hot-coating methods proposed previously. Boiling water treatments introduced in the coating processes have made the membranes quite stable even in the high water concentration region of aqueous organic solutions at their normal boiling points. Zirconia contents larger than about 40 mol% have made the silica–zirconia membranes quite stable. The membranes of zirconia contents less than about 30 mol% were found not stable in a dilute aqueous solution of IPA. The membranes fabricated by the conventional dip-coating methods with slow drying were not stable against water because of the probable segregation of silica and/or silica-rich phases during drying.The membranes fired at lower temperature (400 °C) gave a higher water flux of around 500 mol m⁻² h⁻¹ (9 kg m⁻² h⁻¹) with a separation factor larger than 1500 at 10 wt.% of water in the boiling feed of IPA/water mixture, for example.     

Mechanistic aspects of aldehyde and imine electro-reduction in a liquid–liquid carbon nanofiber membrane microreactor

A simple and electrolyte-free ion-transfer electrosynthesis micro-reactor system (volume 100 μL, up to 10 mg batches) for processes at liquid–liquid interfaces is developed and demonstrated for the reduction of aldehydes and imines. These cathodic reactions occur at an amphiphilic carbon nanofiber membrane accompanied by proton cation transfer from an aqueous phase into an organic phase.     

A green route to enantioenriched (S)-arylalkyl carbinols by deracemization via combined lipase alkaline-hydrolysis/Mitsunobu esterification

Herein we report results of the chemoenzymatic deracemization of a range of secondary benzylic acetates 1a–9a via a sequence of hydrolysis with CAL-B lipase in non-conventional media, combined with esterification of the recovered alcohol according to the Mitsunobu protocol following an enzymatic kinetic resolution (KR). The KR of racemic acetates 1a–9a via an enzymatic hydrolysis, with CAL-B lipase and Na₂CO₃, in non-aqueous media was optimized and gave high selectivities (E ? 200) at good conversions (C >49%) for all of the substrates studied. This method competes well with the traditional one performed in a phosphate buffer solution. The deracemization using Mitsunobu inversion gave the (S)-acetates in moderate to excellent enantiomeric excess 75% < ee < 99%, in acceptable isolated yields 70% < yield < 89%, and with some variations according to the acetate structure.     

On the influence of some strong electrolytes on the partitioning of acetic acid to aqueous/organic two-phase systems in the presence of tri-n-octylamine: Part I: Methyl isobutyl ketone as organic solvent

The recovery of carboxylic acids from aqueous phases is often achieved by reactive extraction with water-insoluble amines which are dissolved in an organic solvent. The basic design of such downstream processes requires a thermodynamic framework for the encountered liquid–liquid equilibrium. The thermodynamic framework should be able to describe the rather uncommon and surprising effects that comparatively small amounts of strong electrolytes might have. Such strong electrolytes can either reduce or increase the affinity of a carboxylic acid for the organic phase in particular at low aqueous phase concentrations of the carboxylic acids. That behavior was explained in previous investigations with citric acid as a model compound for a carboxylic acid and modeled by combining the dissociation/protonation equilibrium in the aqueous phase with the formation of organic phase complexes of (amine + acid(s) + water). In the present investigation this work is extended to acetic acid as another example for a carboxylic acid. New experimental results are reported for the influence of sodium chloride, sodium nitrate, sodium sulfate, sodium citrate and hydrochloric acid on the partitioning of acetic acid to coexisting aqueous/organic liquid phases of the system (water + methyl isobutyl ketone (organic solvent) + tri-n-octylamine (chemical extractant)) at 25 °C. The phase behavior is described by an extension of the previously published thermodynamic framework that is able to describe/predict the influence of a strong electrolyte on the partitioning of acetic acid.     

Application of hollow fiber liquid phase microextraction for pinic acid and pinonic acid analysis from organic aerosols

A method based on hollow fiber liquid phase microextraction (HF-LPME) for analysis of pinic acid and pinonic acid was developed and for the first time successfully applied to ambient aerosol samples. In this method, the aerosol samples were dissolved in 0.05 M H₂SO₄ and the solution was extracted using three-phase HF-LPME where donor phase was 0.1 M (NH₄)₂CO₃. Different parameters like type of organic solvent for membrane phase, extraction time and stirring speed etc. were optimized. Optimum extraction time was 4.5 h and optimum-stirring speed was found to be 900 rpm. We used 6-undecanone as organic phase along with tri-n-octylphosphine oxide (optimum TOPO contents was 15% w/v), which gave an enormous enrichment for both pinic and pinonic acid. Enrichment factors of 28,050 and 27,400 times were obtained for pinonic acid and pinic acid, respectively, that are the highest ever published. The extraction efficiency for pinic acid and pinonic acid were 68.5% and 70.1%, respectively. Very low limits of detection were obtained. Values of 1.0 ng L^?1 and 0.5 ng L^?1 in aqueous solutions, corresponding to 24 pg m^?3 and 12 pg m^?3 in aerosol samples were the limits of detections for pinonic acid and pinic acid, respectively. Both pinonic acid and pinic acid were found in all aerosol samples analyzed.     

Partial oxidation of ethane to syngas in an oxygen-permeable membrane reactor

A perovskite-type oxide of Ba_0.5Sr_0.5Co_0.8Fe_0.2O_3−δ (BSCFO) with mixed electronic and oxygen ionic conductivity at high temperatures was used as an oxygen-permeable membrane. A tubular membrane of BSCFO made by extrusion method has been used in the membrane reactor to exclusively transport oxygen for the partial oxidation of ethane (POE) to syngas with catalyst of LiLaNiO/γ-Al₂O₃ at temperatures of 800–900 °C. After only 30 min POE reaction in the membrane reactor, the oxygen permeation flux reached at 8.2 ml cm⁻² min⁻¹. After that, the oxygen permeation flux increased slowly and it took 12 h to reach at 11.0 ml cm⁻² min⁻¹. SEM and EDS analysis showed that Sr and Ba segregations occurred on the used membrane surface exposed to air while Co slightly enriched on the membrane surface exposed to ethane. The oxygen permeation flux increased with increasing of concentration of C₂H₆, which was attributed to increasing of the driving force resulting from the more reducing conditions produced with an increase of concentration of C₂H₆ in the feed gas. The tubular membrane reactor was successfully operated for POE reaction at 875 °C for more than 100 h without failure, with ethane conversion of ∼100%, CO selectivity of >91% and oxygen permeation fluxes of 10–11 ml cm⁻² min⁻¹.     

Non-dispersive extraction separation of metals using hydrophilic microporous and cation exchange membranes

Non-dispersive extraction of Zn(II) and Cu(II) from single and binary solutions across a flat-sheet membrane to an organic solution containing di(2-ethylhexyl)phosphoric acid (D2EHPA) was studied. Hydrophilic microporous and cation exchange membranes were used. Experiments were performed as a function of the pH (2–6), metal concentration (0.31–3.13 mol/m³), and D2EHPA concentration (50–500 mol/m³). It was shown that the presence of one metal retarded the transport of the other. Compared to the hydrophilic microporous membrane, the cation exchange membrane gave a low extraction rate of both metals in either single or binary systems, but gave a higher selectivity of Zn(II) over Cu(II) in binary systems at high D2EHPA concentrations.     

Oxygen reduction at the silver/hydroxide-exchange membrane interface

A solid-state cell is used to study the electrocatalysis of oxygen reduction at the silver/hydroxide-exchange membrane interface. The catalyst/membrane interface exhibits improved performance in comparison to a catalyst/aqueous sodium hydroxide interface. Surprisingly, the half-wave potential for oxygen reduction is shown to shift 185 mV higher at the silver/hydroxide-exchange membrane interface than for the silver/aqueous hydroxide solution interface, and the exchange current density is significantly higher at 1.02 × 10⁻⁶ A m⁻². On a cost per performance basis, silver electrocatalysts in a hydroxide-exchange membrane fuel cell may provide better performance than platinum in a proton-exchange membrane fuel cell.     

Efficient resolution of profen ethyl ester racemates by engineered Yarrowia lipolytica Lip2p lipase

Enzyme-catalyzed enantiomer discrimination is still a great challenge for the development of industrial pharmaceutical processes. For the resolution of ibuprofen, naproxen and ketoprofen racemates, three major anti-inflammatory drugs, only lipases from Candida rugosa present a high selectivity if solvent and surfactant use is discarded. However, their catalytic activities are too low. In the present work, we demonstrate that the lipase Lip2p from the yeast Yarrowia lipolytica has a higher catalytic activity than C. rugosa lipases to hydrolyze the ethyl esters of ibuprofen, naproxen and ketoprofen, but its selectivity is not sufficient [E = 52 (S); 11 (S) and 1.5 (R) respectively]. The enantioselectivity was further improved by site-directed mutagenesis, targeted at the substrate binding site and guided by molecular modelling studies. By investigating the binding modes of the (R)- and (S)-enantiomers in the active site, two amino acid residues located in the hydrophobic substrate binding site of the lipase, namely residues 232 and 235, were identified as crucial for enantiomer discrimination and enzyme activity. The (S) enantioselectivity of Lip2p towards ethyl ibuprofen esters was rendered infinite (E ? 300) by replacing V232 by an A or C residue. Substitution of V235 by C, M, S, or T amino acids led to a great increase in the (S)-enantioselectivity (E ? 300) towards naproxen ethyl ester. Finally, the variant V232F enabled the efficient kinetic resolution of ethyl ketoprofen ester enantiomers [(R)-enantiopreference; E ? 300]. In addition to the increase in selectivity, a remarkable increase in velocity by 2.6, 2.7 and 2.5 times, respectively, was found for ibuprofen, naproxen and ketoprofen ethyl esters.     

Experimental investigation of incipient equilibrium conditions for the formation of semi-clathrate hydrates from quaternary mixtures of (CO2 + N2 + TBAB + H2O)

The three-phase (vapour + liquid + solid) equilibrium conditions for semi-clathrates formed from three mixtures of (CO₂ + N₂), in aqueous solutions of tetra-butyl ammonium bromide (TBAB), were measured in an isochoric reactor. The experiments were conducted at temperatures between (281 and 290) K, at pressures between (1.9 and 5.9) MPa and in aqueous TBAB solutions of w_TBAB = (0.05, 0.10, and 0.20). The experimental results obtained in this study were compared with previously obtained results for gas hydrates, formed from the same three mixtures of (CO₂ + N₂) and it was observed that semi-clathrates formed at a substantially lower pressure than did gas hydrates.     

Square wave voltammetry assay of organophosphorus inhibition on cholinesterase in two phases of isooctane/water

A novel method for the detection of pesticides by inhibition of cholinesterase (ChE) from earthworm was developed. Two immiscible phases are employed where the organic phase isooctane contains substrate and the aqueous phase does enzyme. Water insoluble indophenol acetate was hydrolyzed by ChE at the interface of two phases to produce water soluble indophenol. The latter spontaneously penetrates into aqueous solution and gives the change of electrochemical signal. Organophosphorus compounds methyl parathion dissolved in isooctane phase could inhibit the activity of ChE at the interface of two phases and a corresponding inhibition relationship is given in the concentration range of 50 ng/ml–100 μg/ml.     

Assessing ion-exchange properties and purity of lipophilic electrolytes by potentiometry and spectrophotometry

Many ionic salts synthesized using metathesis are often found to contain significant amounts of impurities, despite careful control of the weighing of starting materials. In this work, a potentiometric method is devised to monitor ion-exchange properties (or ‘purity’) of an organic solvent containing a lipophilic electrolyte. Its permselective behaviour is monitored by treating the solvent as a liquid membrane and contacting it with two aqueous solutions with different electrolyte activities. This electrolyte mismatch results in a drastic potential change when excess lipophilic cation-exchanger is titrated with anion-exchanger, altering the membrane from being cation to anion responsive. Here, the cation-exchanger potassium tetrakis(4-chlorophenyl)borate (KTpClPB) dissolved in nitrobenzene was titrated with tetradodecylammonium chloride (TDDACl), in contact with Ag/AgCl electrodes placed in aqueous 1 M and 10^?2 M KCl, respectively. The predicted potential change of ?214 mV was observed at the equivalence point, forming the inert lipophilic electrolyte ETH 500, in a very small concentration range of added anion-exchanger (0.8% for ±10 mV), suggesting good precision. The approach was confirmed by monitoring absorbance and fluorescence intensity changes of the chromoionophore Nile Blue. This method may be applied for the synthesis of a range of highly lipophilic salts for which established metathesis protocols are not suitable.     

Esterification for butyl butyrate formation using Candida cylindracea lipase produced from palm oil mill effluent supplemented medium

The ability of Candida cylindracea lipase produced using palm oil mill effluent (POME) as a basal medium to catalyze the esterification reaction for butyl butyrate formation was investigated. Butyric acid and n-butanol were used as substrates at different molar ratios. Different conversion yields were observed according to the affinity of the produced lipase toward the substrates. The n-butanol to butyric acid molar ratio of 8 and lipase concentration of 75 U/mg gave the highest butyl butyrate formation of 63.33% based on the statistical optimization using face centered central composite design (FCCCD) after 12 h reaction. The esterification potential of the POME based lipase when compared with the commercial lipase from the same strain using the optimum levels was found to show a similar pattern. It can be concluded therefore that the produced lipase possesses appropriate characteristics to be used as a biocatalyst in the esterification reactions for butyl butyrate formation.     

Adsorption and photocatalytic degradation of 1,4-dioxane on TiO2

1,4-Dioxane in hexane as a solvent was adsorbed on TiO₂ due to an electrostatic interaction. The porous TiO₂ pellets (SG) prepared by sol–gel method were superior adsorbent to ST-B21 and Degussa P-25. Effects of firing temperature of the pellets and the initial concentrations of 1,4-dioxane on the adsorption percents were examined. Photocatalytic degradation of aqueous 1,4-dioxane gave 1,2-ethanediol diformate and formic acid as intermediates. Analysis of total organic carbon indicated that 1,4-dioxane was mineralized effectively in the following order: P-25 > ST-B21 > SG. The photocatalytic degradation of 1,4-dioxane adsorbed on the TiO₂ pellets in air showed that ST-B21 had a higher activity than SG. These facts indicate that SG pellet acts as a good adsorbent because of its high specific surface area but the internal region of the pores is not illuminated and acts only as a support.     

Changes in Secondary Metabolite Contents Following Crude Drug Preparation

Crude drug is commonly prepared by directly drying of freshly harvested plant organ or after slicing. These processes may decrease or maintain the content of the desired metabolites presence in the crude drug. Plant rhizome can be directly sliced followed by drying or after storage at certain period. Certain rhizome can maintain the secondary metabolites after being stored for three months (12 weeks), while others decreased just after being stored for two weeks. Drying process can be performed under the sun or in an air circulated oven with temperature not higher than 60 °C. Phenolic content of crude drugs on the other hand is the lowest if it is dried at 60 °C. Drying at 40 °C, 80 °C and 100 °C produce crude drug with higher phenolic content compared to those dried at 60 °C. This may associated with the activity of peroxidase that has optimal activity at 60° C. At above 60 °C, the activity of peroxidase may decrease due to the degradation of the enzyme. Moist treatment of fresh material may increase the content of the secondary metabolites. Boiling of Cosmos caudatus leaves increased the content of the flavonoid glycoside. However, part of the flavonoid was presence in the aliquot that hamper further step of crude drug preparation. Steaming of potato peels increased the chlorogenic acid content. From these observations, steaming can be considered as one of pre-treatment steps in the preparation of crude drugs prior drying process. The increase of flavonoid glycoside in Cosmos caudatus leaves upon boiling has been confirmed not due to the increase the extractability of the flavonoid. The increase of key enzyme activity that involved in the biosynthetic pathway upon moist-heat treatment need to be further studied     

Tetrakis(4-sulfonatophenyl)porphyrin fluorescence as reporter of human serum albumin structural changes induced by guanidine hydrochloride

The interaction of the drug carrier protein human serum albumin (HSA) with the ionic, free base porphyrin tetrakis(4-sulfonatophenyl)porphyrin (TSPP) was investigated under chemical denaturation conditions using guanidine hydrochloride (Gdn-HCl) in aqueous solution at pH 7 and 2.5. Protein stability was studied by fluorescence spectroscopy using intrinsic tryptophan fluorescence, whereas far-UV circular dichroism gave information regarding conformational changes. Steady-state and time-resolved fluorescence as well as extinction and induced visible CD of TSPP were also monitored in the presence of the denaturant.The addition of 1.0 M Gdn-HCl inhibited the FRET process between the sole tryptophan residue of HSA and the porphyrin as inferred by an increase in the intrinsic fluorescence of the former together with a drop in the fluorescence of the latter. Simultaneously, an induced bisignate CD band was detected in the Soret region of TSPP extinction following the changes in the monomer ? aggregate equilibrium of TSPP. The features in the extinction spectra pointed to the formation of J-aggregates at pH = 2.5 and were confirmed by fluorescence lifetime measurements. At pH = 7, no TSPP dimers were detected in the absence of the protein or in the presence of native HSA. However, H-dimers or higher aggregates of TSPP associated to HSA were induced at concentrations of Gdn-HCl below 2 M.The main unfolding transition probed by HSA intrinsic fluorescence took place between 2 and 3 M Gdn-HCl at pH = 7, whereas at pH = 2.5 it was detected only above 2.8 M Gdn-HCl, coinciding with TSPP release into solution which occurs at high denaturant concentration for both pH studied. The results suggest that the chemical unfolding of HSA is a multistep process. The free base porphyrin contributes to an increase in the protein stability, particularly important under acidic conditions, where the protein is known to be in an expanded form in the absence of TSPP.The analysis of TSPP fluorescence fluctuations in the autocorrelation functions obtained using fluorescence correlation spectroscopy (FCS) in the presence of HSA at different denaturant concentrations showed that the porphyrin only interacts with the native form of the protein.Both fluorescence and circular dichroism data confirmed that in the noncovalent complex HSA–TSPP the free base porphyrin can act as a reporter of the protein structural changes induced either by pH or chemical denaturation.