Biosynthesis of glycyrrhetic acid 3-O-mono-β-d-glucuronide catalyzed by β-d-glucuronidase with enhanced bond selectivity in an ionic liquid/buffer biphasic system

The bond selective hydrolysis of glycyrrhizin (GL) to glycyrrhetic acid 3-O-mono-β-d-glucuronide (GAMG) catalyzed by recombinant β-d-glucuronidase from Escherichia coli BL21 (PGUS-E) was successfully performed in an ionic liquid (IL)/buffer biphasic system. Five ILs were analyzed, however, a hydrophobic IL 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF₆) showed the best biocompatibility with PGUS-E. An obvious enhancement in the initial reaction rate, substrate conversion, GAMG yield and chemical bond selectivity (S_cb) was observed using 40% (v/v) [BMIM]PF₆/buffer as the reaction medium when compared to the acetate buffer medium. Under the optimized conditions (pH 6.0, temperature 50 °C, substrate concentration 6 mM and shaking speed 200 rpm), the initial reaction rate, the GAMG yield and the S_cb reached 3.15 mM h⁻¹, 74.36% and 98.12%, respectively. The recyclability of [BMIM]PF₆ was also studied and found to be reusable for five batches with high recovery percentage (≥92%). Furthermore, the desired product and byproduct were easily separated since they were distributed in different phases. Additionally, higher V_max (3.14 versus 2.24 mM h⁻¹), lower apparent K_m (1.21 versus 1.80 mM) and E_a (25.97 versus 32.60 kJ mol⁻¹) were achieved in [BMIM]PF₆/buffer biphasic system than that in monophasic buffer system.     

Asymmetric reduction of (4S)-(+)-carvone catalyzed by baker's yeast: A green method for monitoring the conversion based on liquid–liquid–liquid microextraction with polypropylene hollow fiber membranes

The α,β-unsaturated carbonyl compound (4S)-(+)-carvone was selectively reduced to (1R,2R,4S)-iso-dihydrocarveol using baker's yeasts. The conversion of the bioreduction reaction was monitored using a green hollow-fiber liquid–liquid–liquid microextraction (HF-LLLME) technique. Several parameters which may affect the bioreduction of (4S)-(+)-carvone, such as temperature, time, substrate/enzyme ratio, pH and buffer concentration, were evaluated. The effect of some additives, such as trehalose, DMSO and the ionic liquid [BMIm][PF₆], was also studied. The (1R,2R,4S)-iso-dihydrocarveol was recovered with 52.7% conversion and diastereoisomeric excess >99% after 48 h of reaction at 40 °C in an aqueous monophasic system, with 0.1 mol L^?1 buffer concentration (pH 7.5) and a substrate/yeast cell mass ratio of 8.0 mg g^?1. The HF-LLLME microextraction technique allowed the optimization of the reaction with a reduction of over 99.5% in relation to the use of organic solvents.     

Lipase-catalyzed acylation of lily polysaccharide in ionic liquid-containing systems

A comparative study was made of immobilized Burkholderia cepacia lipase (PSL-C)-catalyzed acylation of lily polysaccharide (LP) with vinyl acetate in organic solvents, ionic liquids (ILs) and IL-containing systems. The degree of substitution (DS) of the modified LP was used to evaluate the extent of acylation and thus enzymatic activity. In this manner, an eco-friendly solvent, 2-methyltetrahydrofuran (MeTHF), was found to be the most suitable organic reaction medium. However, compared to MeTHF, enhanced enzyme activity was observed when 1-butyl-3-methylimidazolium tetrafluorobrate ([C₄MIm][BF₄]) was used as the solvent. To further enhance the DS of the modified LP product, co-solvent mixtures of [C₄MIm][BF₄] and MeTHF were investigated. Among the various MeTHF–[C₄MIm][BF₄] systems examined, 20% (v/v) MeTHF–[C₄MIm][BF₄] produced the highest DS. In this reaction medium, the optimal water activity, reaction temperature and time were 0.33, 55 °C and 18 h, respectively, producing a product DS as high as 0.67. The PSL-C enzyme exhibited a much higher stability in the IL-containing system. Additionally, PSL-C-catalyzed acylation of LP was highly regioselective, causing acylation of only C6OH.     

Enzymatic hydrolysis of racemic ibuprofen esters using Rhizomucor miehei lipase immobilized on different supports

This research describes the immobilization of Rhizomucor miehei lipase (RML) and chemically aminated RML (NH₂-RML) on different supports including octyl-sepharose (octyl-RML), activated sepharose with cyanogen bromide (CNBr-RML and CNBr-NH₂-RML), glyoxyl sepharose (Gx-RML and Gx-NH₂-RML) and glyoxyl sepharose dithiothreitol (Gx-DTT-RML and Gx-DTT-NH₂-RML). The highest immobilization yield was achieved for octyl-RML (>98%) followed by CNBr-RML (88%). Octyl-RML had the most specific activity (13.6) among all derivatives. The other preparations had moderate activities likely because of chemical reaction during covalent attachment of the enzyme. The catalytic behavior of lipase immobilized in hydrolysis reactions was investigated using methyl, ethyl, propyl, butyl and isobutyl-ibuprofen esters and the influence of the alkyl chain and the alcoholic residue of the ester were studied. Butyl ester was the most interesting ester for carrying out hydrolysis. The highest enantioselectivity of enzyme (E = 8.8) was obtained with isooctane/sodium phosphate buffer pH 7.0 at temperature of 40 °C. Increasing temperature from 40 to 50 °C caused decreasing in enantioselectivities and conversions. Also esterification of ibuprofen was carried out in solvent systems containing isooctane and two ionic liquids (ILs); [BMIM][PF₆] and [BMIM][BF₄]. Poor conversions and enantioselectivities were observed during esterification in all solvents.     

Improvements of enzyme activity and enantioselectivity in lipase-catalyzed alcoholysis of (R,S)-azolides

With Candida antarctica lipase B (CALB)-catalyzed alcoholysis of (R,S)-naproxenyl 1,2,4-triazolide at the optimal conditions (i.e. anhydrous MTBE as the solvent, and methanol as the acyl acceptor at 45 °C) as the model system, the enzyme enantioselectivity in terms of V_R/V_S = 105.8 and specific activity for the fast-reacting (R)-azolide V_R/(E_t) = 0.979 mmol/(h g) were greatly improved in comparison with V_R/V_S = 8.0 and V_R/(E_t) = 0.113 mmol/(h g) of using (R,S)-naproxenyl 2,2,2-trifluoroethyl ester as the substrate. The resolution strategy was successfully extended to other (R,S)-profenyl 1,2,4-triazolides and lipases from Candida rugosa (Lipase MY) and Carica papaya (CPL) having opposite enantioselectivity to CALB. Moreover, the kinetic constants were estimated, compared with those obtained via hydrolysis, and employed for modeling time-course conversions of (R,S)-naproxenyl 1,2,4-triazolide in anhydrous MTBE. The advantages of easy substrate preparation, high enzyme reactivity and enantioselectivity, as well as easy product separation from the remaining substrate via reactive extraction demonstrate merits of using (R,S)-azolides but not the corresponding esters for the alcoholytic resolution.     

Methyl-palladium(II) complexes of pyridine-bridged bis(nucleophilic heterocyclic carbene) ligands: Substituent effects on structure,stability, and catalytic performance

A series of discrete, mononuclear palladium(II)–methyl complexes, together with several palladium(II)–chloro analogues, of pyridine-functionalised bis-NHC ligands have been prepared via ligand transmetallation from the silver(I)-NHC complexes. The reported complexes comprise examples with both the methylene-bridged 2,6-bis[(3-R-imidazolin-2-yliden-1-yl)methyl]pyridine (_RC^∧N^∧C; R = Mes, dipp, ^tBu) and planar 2,6-bis(3-R-imidazolin-2-yliden-1-yl)pyridine (_RCNC; R = Mes, dipp) ligands and, when combined with the previously reported _MeC^∧N^∧C/_MeCNC examples, cover a broad spectrum of ligand substituent steric and electronic properties, including the bulky Mes and dipp groups frequently used in catalytic applications. The palladium(II) complexes have been characterised by a variety of methods, including single crystal X-ray crystallography, with the shielding of the Pd–Me groups in the proton NMR spectra of some of the N-aryl substituted examples correlated with the proximity of the aryl rings to the methyl group in the solid state structures. The [PdMe(_RC^∧N^∧C/_RCNC)]⁺ complexes undergo thermal degradation via reductive methyl-NHC coupling to give 2-methyl-3-R-imidazolium-1-yl species with relative stabilities in the order of [PdMe(_MesC^∧N^∧C)]BF₄ > [PdMe(_MeC^∧N^∧C)]BF₄ ≈ [PdMe(_MesCNC)]BF₄ > [PdMe(_MeCNC)]BF₄ > [PdMe(_tBuC^∧N^∧C)]BF₄ ≫ [PdMe(_tBuCNC)]BF₄ (not isolable). A comparison of the activity of the complexes as precatalysts in a model Heck coupling reaction shows greatest activity in those species bearing bulkier N-substituents, with complexes bearing _RC^∧N^∧C ligands generally more efficient precatalysts than those bearing _RCNC ligands.     

Enantioselective reduction of aryl ketones using immobilized cells of Candida viswanathii

Enantioselective reduction of 1-acetonapthone to S(−)-1-(1-naphthyl) ethanol, a key intermediate for the synthesis of HMG Co-A reductase inhibitor, was successfully carried out using immobilized cells of a newly isolated carbonyl reductase producing yeast strain Candida viswanathii MTCC 5158. Calcium alginate (1.5%, w/v) gave the best immobilization efficiency. Among different organic solvents and ionic liquids tried as reaction media, isopropanol gave the best enantioselectivity with moderate conversion. The immobilized cells (100 mg/ml in 50 mM Tris buffer pH 9) showed best results at a substrate concentration of 0.2 mg/ml at 30 °C. After twelve cycles of reaction, no significant decrease in bioreduction efficiency of the immobilized cells was observed as compared to the free cells.     

Biocatalytic synthesis of ethyl (R)-2-hydroxy-4-phenylbutyrate with Candida krusei SW2026: A practical process for high enantiopurity and product titer

Ethyl (R)-2-hydroxy-4-phenylbutyrate ((R)-HPBE), a key intermediate in the production of angiotensin-converting enzyme (ACE) inhibitors, was prepared by the microbial reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE). Among 63 microorganisms tested, Candida krusei SW2026, for the first time, was proven to be a highly effective biocatalyst in this reduction process, leading to the (R)-enantiomer in 99.7% ee and 95.1% yield at 2.5 g/L of OPBE (under optimal conditions of 30 °C, pH 6.6, and in the presence of 5% glucose as co-substrate). In order to achieve higher product concentration with desired enantiopurity and yield for application in large-scale production, strategies such as substrate fed-batch and aqueous/organic biphasic system were successfully conducted in the biotransformation reaction. At 20 g/L of OPBE, the enantiomeric excess (ee), yield, and product concentration were enhanced to 97.4%, 82.0%, and 16.6 g/L, respectively, in water/dibutyl phthalate biphasic system, compared with 87.5%, 45.8%, and 9.2 g/L in aqueous medium. This study provides an attractive process of (R)-HPBE production for potential green chemistry applications.     

Use of an ionic liquid to improve asymmetric reduction of 4′-methoxyacetophenone catalyzed by immobilized Rhodotorula sp. AS2.2241 cells

Rhodotorula sp. AS2.2241, a newly isolated strain, was used as biocatalyst for asymmetric reduction of 4′-methoxyacetophenone (MOAP) to enantiopure (S)-1-(4-methoxyphenyl)ethanol {(S)-MOPE}. Despite the improved efficiency of the reaction with immobilized cells compared to free cells, the inhibition of the reaction by substrate and product in monophasic aqueous system proved to be big problem. For high efficient biotransformation, several water-immiscible ionic liquids (ILs) were employed as green solvents to construct ionic liquid-involving biphasic systems. Of the six ILs tested, C₄MIM·PF₆ exhibited the best biocompatibility with the cells, and consequently the biocatalytic reduction proceeded with the fastest initial reaction rate and the highest maximum substrate conversion in the C₄MIM·PF₆-based biphasic system. To better understand the bioreduction conducted in the C₄MIM·PF₆-based biphasic system, various variables that influenced the performance of the reaction were examined. The optimal buffer pH, reaction temperature, volume ratio of buffer to C₄MIM·PF₆ and substrate concentration were 7.5, 25 °C, 4/1 and 40 mM, respectively. Under the optimal conditions, the initial reaction rate, maximum substrate conversion and product e.e. were 1.6 μmol/h, 95.5% and >99%, respectively. Additionally, the cells still remained above 90% of their original activity in the C₄MIM·PF₆-based biphasic system, which was much higher than that in the monophasic buffer system (about 25% of their original activity), after being repeatedly used for 8 batches (50 h per batch), indicating that C₄MIM·PF₆ markedly enhanced the operational stability of the cells.     

Effect of ionic liquid [BMIM][PF<Subscript>6</Subscript>] on asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The effect of ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF₆]) on the asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (EOPB) to synthesize optical active ethyl 2-hydroxy-4-phenylbutyrate (EHPB) catalyzed by Saccharomyces cerevisiae was investigated. (R)-EHPB [70.4%, e.e.(R)] is obtained using ethyl ether or benzene as the solvent. The main product is (S)-EHPB [27.7%, e.e.(S)] in [BMIM][PF₆]. However, in ionic liquid-water (10:1, v/v) biphasic system, the enantioselectivity of the reduction is shifted towards (R)-side, and e.e.(R) is increased from 6.6 to 82.5% with the addition of ethanol (1%, v/v). The effect of the use of [BMIM][PF₆] as an additive in relatively small amounts on the reduction was also studied. We find that there is a decline in the enantioselectivity of the reduction in benzene. In addition, a decrease in the conversion of EOPB and the yield of EHPB with increasing [BMIM][PF₆] concentrations occurs in either organic solvent–water biphasic systems or benzene.     

Efficient production of (R)-(−)-mandelic acid with highly substrate/product tolerant and enantioselective nitrilase of recombinant Alcaligenes sp.

For efficient production of (R)-(−)-mandelic acid, a nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli. After simple optimization of the culture conditions, the biocatalyst production was greatly increased from 500 to 7000 U/l. The recombinant E. coli whole cells showed strong tolerance against a high substrate concentration of up to 200 mM, and the concentration of (R)-(−)-mandelic acid after only 4 h of transformation reached 197 mM with an enantiomeric excess (ee_p) of 99%. In a fed-batch reaction with 600 mM mandelonitrile as the substrate, the cumulative production of (R)-(−)-mandelic acid after 17.5 h of conversion reached 520 mM. The recombinant E. coli cells could also be repeatedly used in the biotransformation, retaining 40% of the initial activity after 10 batches of reaction. The highly substrate/product tolerable and enantioselective nature of this recombinant nitrilase suggests that it is of great potential for the practical production of optically pure (R)-(−)-mandelic acid.     

A novel control of enzymatic enantioselectivity through the racemic temperature influenced by reaction media

The influence of reaction media on the racemic temperature (T_r) in the lipase-catalyzed resolution of ketoprofen vinyl ester was investigated. An effective approach to the control of the enzymatic enantioselectivity and the prediction of the increasing tendency was developed based on the T_r influenced by reaction media. The T_r for the resolution catalyzed by Candida rugosa lipase (CRL) was found at 29 °C in aqueous and S-ketoprofen was obtained predominantly at 40 °C. However, CRL showed R-selectivity at 40 °C in diisopropyl ether because the T_r was changed to 56 °C. CRL, lipase from AYS Amano^® and Mucor javanicus lipase were further applied for the investigation of the enzymatic enantioselectivity in dioxane, DIPE, isooctane and their mixed media with water. The effects of the reaction medium on T_r could be related to the solvent hydrophobicity, the lipase conformational flexibility and the interaction between the enantiomers and the lipase.     

Structural comparison and enhanced enzymatic hydrolysis of eucalyptus cellulose via pretreatment with different ionic liquids and catalysts

Eucalyptus was fractionated with mild alkaline process, and the obtained cellulose fraction was pretreated with various ionic liquids (ILs) to enhance the enzymatic saccharification. The results showed that the ILs used was efficient for the hydrolysis of cellulose, with the maximum total reducing sugars (TRS) yield over 80% at 50 °C. The regenerated cellulose substrate exhibited a significant improvement about 4.4–6.4 folds enhancement on saccharification rate during the first 4 h reaction. The crystallinity index (CrI) of cellulose via 1-ally-3-methylimidazolium ([AMIM]Cl) pretreatment was significantly decreased from 70.2% to 31.2%, resulting in structural change from cellulose I to cellulose II, which enabled the cellulase enzymes easier access to hydrolyze cellulose. However, 1-butyl-3methylimidazolium acesulfamate ([BMIM]Ace) pretreatment had no large effect on the CrI although a high conversion yield in glucose was obtained. The surface morphologies of the regenerated substrate which was pretreated via 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) and 1-ethyl-3-methylimidazolium acetate ([EMIM]Ac) showed more porous and incompact network of cellulose when compared with the untreated substrate. This result indicated a better accessibility by cellulases to the cellulose surface. Besides, a certain amount of catalysts such as MgCl₂ and H₂SO₄ could improve the rate of enzymatic saccharification.     

Biosynthesis of ethyl caproate and other short ethyl esters catalyzed by cutinase in organic solvent

The main objective of this work was to study the enzymatic synthesis of short chain ethyl esters, a group of relevant aroma molecules, by Fusarium solani pisi cutinase in an organic solvent media (iso-octane), and to assess the influence of different parameters on the reaction yield.Cutinase displayed high initial esterification rates in iso-octane, which amounted to 1.15 μmol min⁻¹ mg⁻¹ for ethyl butyrate (C₄ acid chain) and 1.06 μmol min⁻¹ mg⁻¹ for ethyl valerate (C₅ acid chain). High product yields, 84% for ethyl butyrate and 96% for ethyl valerate, were observed after 6 h of reaction, for an initial equimolar concentration of substrates (0.1 M).The highest product yield (97%) was observed for ethyl caproate (C₆) synthesis, a compound which is a part of natural apple and pineapple flavour, for an alcohol:acid molar ratio of 2 (0.2 M ethanol concentration).Cutinase affinity for short chain length carboxylic acids (C₄–C₆) in ester synthesis in iso-octane confirmed previous observations in reversed micellar system.     

Trispyrazolylmethane piano stool complexes of iron(II) and cobalt(II)

A series of cationic trispyrazolylmethane complexes of the general form [Tm^RM(CH₃CN)₃]²⁺ (Tm = tris(pyrazolyl)methane, 1, R = 3,5-Me₂, M = Fe(II); 2, R = 3-Ph, M = Fe(II); 3, R = 3,5-Me₂, M = Co(II); 4, R = 3-Ph, M = Co(II)) with ‘piano-stool’ structures was prepared by the reaction of the N₃tripodal ligands (Tm^R)with [(CH₃CN)₆M](BF₄)₂ in a 1:1 stoichiometric ratio. Magnetic susceptibility measurements indicate that all four complexes with BF₄⁻ counter anions are paramagnetic, high-spin systems in the solid state with μ_eff at high temperatures of 5.2 (1, S = 2), 5.4 (2, S = 2), 4.9 (3, S = 3/2) and 4.6 (4, S = 3/2) BM, respectively. Comparisons of bond lengths from the metal centre to the Tm^R nitrogen donors, and from the metal centre to the acetonitrile nitrogen donors indicate that the neutral tripodal ligands appear to be more weakly coordinated to the metal centre than are the acetonitrile ligands. Reactions of these tripodal complexes with bidentate phosphine ligands, such as 1,2-diphosphinoethane or 1,2-bis(diallylphosphino)ethane leads to displacement of the tripodal ligand, or to the formation of more thermally stable bis-ligand complexes M(Tm^R)₂ (R = 3,5-dimethyl).     

Impressive effect of immobilization conditions on the catalytic activity and enantioselectivity of Candida rugosa lipase toward S-Naproxen production

Candida rugosa lipase (CRL) was immobilized on Amberlite XAD 7 and the advantage of immobilization under the best reaction conditions in achieving high activity and enantioselectivity was shown for the hydrolysis of racemic Naproxen methyl ester. The performance of CRL was found to be better when the enzyme was immobilized at the temperature and pH values where higher conversion and enantioselectivity were obtained. The effects of immobilized lipase load, temperature, pH and substrate concentration on the conversion and enantioselectivity toward S-Naproxen production in aqueous phase/isooctane biphasic batch system were also evaluated. The increase in immobilized lipase load in 320–800 U/mL range increased the conversion of the substrate and enantioselectivity for S-Naproxen. The kinetic resolution of racemic Naproxen methyl ester conducted at the temperatures of 40, 45 and 50 °C and at the pH values of 4, 6, 7.5 and 9 resulted in the highest conversion and enantioselectivity at 45 °C and pH 6. Higher concentration of racemic Naproxen methyl ester than 10 mg/mL decreased both the conversion and enantioselectivity. CRL, which was immobilized at the temperature and pH values where the enzyme was more enantioselective, was successfully used in three successive batch runs each of 180 h. The highest enantiomeric ratio achieved in the S-Naproxen production was 174.2 with the conversion of 49%.     

Synthesis of methyl (R)-3-(4-fluorophenyl)glutarate via enzymatic desymmetrization of a prochiral diester

An efficient procedure for enzymatic desymmetrization of the prochiral dimethyl 3-(4-fluorophenyl)glutarate (3-DFG) in an aqueous–organic phase was successfully developed to prepare methyl (R)-3-(4-fluorophenyl)glutarate ((R)-3-MFG). Novozym 435 was selected as a highly efficient biocatalyst through lipase screening. The effects of various parameters in terms of co-solvent and its concentration, buffer pH, ionic strength and reaction temperature, on the reaction were investigated. It was found that 0.2 M phosphate buffer (pH 8.0) containing 20% MTBE (v/v) was the optimum reaction medium, and the optimum reaction temperature was 30 °C. Under the optimized reaction conditions, (R)-3-MFG was obtained in 95.6% ee value and 92.6% yield after 64 h when the concentration of 3-DFG and Novozym 435 were 200 mmol/l and 20 g/l respectively. Furthermore, Novozym 435 showed an excellent operational stability, retaining above 95% of the initial activity and enantioselectivity after 10 cycles of reaction. The developed method has a potential to be used for efficient enzymatic production of (R)-3-MFG.     

Substrate effects on the mechanism of enantioselective hydrogenation using ruthenium bis(phosphine) complexes as catalyst: A mechanistic investigation of the hydrogenation of α,β-unsaturated acids and esters based on deuterium labeling studies

The mechanism of ruthenium-bis(phosphine) catalyzed enantioselective hydrogenation of olefins was examined using [Ru((R)-BINAP)(H)(MeCN)_n(sol)_3 − n]BF₄ (n = 0–3, sol = solvent used in reaction) as catalyst. Tiglic and angelic acids were used as standard α,β-unsaturated acid substrates; (Z)-methyl α-acetamidocinnamate and dimethyl itaconate were used as standard α,β-unsaturated ester substrates. Isotopic labeling studies (deuterium scrambling) indicate that two distinct mechanisms are in operation for α,β-unsaturated acids versus α,β-unsaturated esters. In each case, 5-membered metallocycle intermediates are formed via olefin-hydride insertion. The mechanisms, however, deviate primarily in the activation of dihydrogen, which is strongly affected by the nature of the substrate. Hydrogenation of α,β-unsaturated acids proceed via heterolytic cleavage of dihydrogen, whereas hydrogenation of α,β-unsaturated esters proceed via homolytic cleavage of dihydrogen. A full discussion of the mechanisms is presented.     

Efficient production of glycyrrhetic acid 3-O-mono-β-d-glucuronide by whole-cell biocatalysis in an ionic liquid/buffer biphasic system

Hydrolysis of glycyrrhizin (GL) to glycyrrhetic acid 3-O-mono-β-d-glucuronide (GAMG) by whole-cell biocatalysts in a system containing non-conventional solvents was performed. Three whole-cell biocatalysts were used, including wild-type Penicillium purpurogenum Li-3 (w-PGUS) and recombinant strains Escherichia coli BL21 and Pichia pastoris GS115. The biotransformation of GL to GAMG by w-PGUS in a 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim]PF₆)/buffer biphasic system was the main focus of this study because w-PGUS showed a higher GAMG yield and a higher relative activity in this system than the other two whole-cell biocatalysts. Using the optimized reaction conditions determined as a pH 5.2 buffer, a 6.0 mM substrate concentration, a reaction temperature of 30 °C, and a 60 g/L (1.23 U/g) cell concentration, a GAMG yield of 87.63% was achieved after 60 h. After eight reaction cycles, [Bmim]PF₆ retained a high recovery percentage (85.48%)_[0], indicating the reusability of this IL. The biotransformation activity of w-PGUS was not significantly affected, even after two batch reaction cycles. Furthermore, the product GAMG and the byproduct glycyrrhetinic acid were spontaneously separated in the biphasic system. In conclusion, the combination of whole cells and ionic liquid is a promising approach for economical and industrial-scale production of GAMG.     

Microwave-assisted enzymatic resolution of (R,S)-2-octanol in ionic liquid

Combination use of microwave irradiation (MW) as heating mode and ionic liquid (IL) as reaction medium in enzymatic resolution of (R,S)-2-octanol with vinyl acetate as the acyl donor through transesterification by Novozym 435 was investigated. A synergistic effect of MW and IL [EMIM][NTf2], which was screened as the best reaction medium for this reaction, on improving enzyme activity and enantioselectivity was observed. The activity and enantioselectivity of Novozym 435 in [EMIM][NTf2] under MW were much higher than that in solvent free system under conventional heating, in solvent free system under MW, and in [EMIM][NTf2] under conventional heating, respectively. A systematic screening and optimization of the reaction parameters in [EMIM][NTf2] under MW were performed. Under the optimum conditions, 50% yield of (S)-2-octanol with 99% enantiomeric excess was obtained in 6 h. Furthermore, increased thermal stability and reusability of Novozym 435 under the combination use of MW and IL condition were also observed.