Effect of different reaction parameters on the lipase-catalyzed selective acylation of polyhydroxylated natural compounds in ionic liquids

The effect of various reaction parameters on the enzymatic acylation of plant polyhydroxylated compounds, including phenolic and flavonoid glucosides (salicin, helicin, esculin and naringin), was investigated in imidazolium-based ionic liquids (1-butyl-3-methylimidazolium tetrafluoroborate [bmim]BF₄ and 1-butyl-3-methylimidazolium hexafluorophosphate [bmim]PF₆), using immobilized lipase B from Candida antarctica. The conversion yield, the regioselectivity and the reaction rate of the biocatalytic process strongly depended on the ionic liquid used, their water content, the incubation temperature, as well as the solubility and the concentration of substrates. For most glucosides tested, one major product (monoacylated derivative) was detected as a result of the acylation of the primary hydroxyl group of glucose moiety. The acylation rate and the regioselectivity of the process are higher in [bmim]BF₄, where the solubility of all glucosides is significantly higher than in [bmim]PF₆ or acetone. Response surface methodology (RSM) based on a five level-three variable central composite circumscribed design, was employed to evaluate the interactive effect of the molar ratio of substrates (MR), the initial concentration of glucoside (N) and the reaction time (RT), as well as for their optimization in [bmim]BF₄. At the optimal reaction conditions the maximum acylation yield was 87%. The amount of monoacylated derivatives produced in a single-step biocatalytic process reached values up to 31.6 g/l which is considerably higher than those reported for organic media.     

Alpha-chymotrypsin catalysis in imidazolium-based ionic liquids

The transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with 1-propanol catalyzed by alpha-chymotrypsin was examined in the ionic liquids 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF(6)]) and 1-octyl-3-methylimidazolium hexafluorophosphate ([omim][PF(6)]), and in combination with supercritical carbon dioxide (SC-CO(2)). The activity of alpha-chymotrypsin was studied to determine whether trends in solvent polarity, water activity, and enzyme support properties, observed with this enzyme in conventional organic solvents, hold for the novel environment provided by ionic liquids. alpha-Chymotrypsin freeze-dried with K(2)HPO(4), KCl, or poly(ethylene glycol) demonstrated no activity in [bmim][PF(6)] or [omim][PF(6)] at very low water concentrations, but moderate transesterification rates were observed with the ionic liquids containing 0.25% water (v/v) and higher. However, the physical complexation of the enzyme with poly(ethylene glycol) or KCl did not substantially stimulate activity in the ionic liquids, unlike that observed in hexane or isooctane. Activities were considerably higher in [omim][PF(6)] than [bmim][PF(6)]. Added water was not necessary for enzyme activity when ionic liquids were combined with SC-CO(2). These results indicate that [bmim][PF(6)] and [omim][PF(6)] provide a relatively polar environment, which can be modified with nonpolar SC-CO(2) to optimize enzyme activity.     

Enzymatic selective acylation of glycosides in ionic liquids: significantly enhanced reactivity and regioselectivity

The enzymatic selective acylations of carbohydrates in ionic liquids were explored in both organic solvents and ionic liquids to see any significant differences in terms of reactivity and regioselectivity between two different classes of reaction media. Monoprotected glycosides (methyl-6-O-trityl-glucosides and galactosides) were chosen as the substrates with Candida rugosa lipase as an acylation enzyme. Two organic solvents, THF and chloroform, and two ionic liquids, [BMIM]⁺PF₆⁻ ([BMIM]⁺ = 1-butyl-3-methylimidazolium) and [MOEMIM]⁺PF₆⁻ ([MOEMIM]⁺ = 1-methoxyethyl-3-methylimidazolium), were employed as reaction media. The enzymatic reactions were performed in the presence of vinyl acetate at room temperature. It was observed that the reactions in ionic liquids took place more rapidly and more selectively than those in conventional organic solvents.     

Optimization of (R,S)-1-phenylethanol kinetic resolution over Candida antarctica lipase B in ionic liquids

The kinetic resolution of racemates constitutes one major route to manufacture optically pure compounds. The enzymatic kinetic resolution of (R,S)-1-phenylethanol over Candida antarctica lipase B (CALB) by using vinyl acetate as the acyl donor in the acylation reaction was chosen as model reaction. A systematic screening and optimization of the reaction parameters, such as enzyme, ionic liquid and substrates concentrations with respect to the final product concentration, were performed. The enantioselectivity of immobilized CALB commercial preparation, Novozym 435, was assayed in several ionic liquids as reaction media. In particular, three different ionic liquids: (i) 1-butyl-3-methylimidazolium hexafluorophosphate [bmim][PF₆], (ii) 1-butyl-3-methylimidazolium tetrafluoroborate [bmim][BF₄] and (iii) 1-ethyl-3-methylimidazolium triflimide [emim][NTf₂] were tested. At 6.6% (w/w) of Novozym 435, dispersed in 9.520 M of [bmim][PF₆] at 313.15 K, using an equimolar ratio of vinyl acetate/(R,S)-1-phenylethanol after 3 h of bioconversion, the highest possible conversion (50%) was reached with enantiomeric excess for substrate higher than 99%.     

Pseudomonas cepacia lipase-catalysed resolution of racemic alcohols in ionic liquid using succinic anhydride: role of triethylamine in enhancement of catalytic activity

Racemic secondary alcohols were resolved via enantioselective acylation using succinic anhydride as acyl donor catalysed by lipase from Pseudomonas cepacia supported on celite (PS-C) in ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate [bmim]PF₆. Organic base, namely triethylamine as an additive in ionic liquid has been found to enhance the rate of the reaction.     

Obtaining high transesterification activity for subtilisin in ionic liquids

It is known that subtilisin shows poor transesterification activity in ionic liquids (ILs). The present work, taking subtilisin as the system, explores approaches for biocatalyst preparations, which are capable of yielding higher/adequate transesterification activity in these solvents. Of all the approaches tried, enzyme precipitated and rinsed with n-propanol (EPRP) gave the best results (about 10,000 times increase in initial rates in 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF(6)]) over what is obtained with pH tuned lyophilized powders). In case of water soluble ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF(4)]), pH tuned lyophilized subtilisin did not show any transesterification activity. EPRP, however, gave an initial rate (for transesterification) of 2.78 mmol mg(-1) h(-1).     

Enzymatic hydrolysis of penicillin in mixed ionic liquids/water two-phase system

In this paper, an integrated process involving the mixed ionic liquids/water two-phase system (MILWS) is proposed to improve the efficiency for enzymatic hydrolysis of penicillin G. First, hydrophilic [C4mim]BF4 (1-butyl-3-methylimidazolium tetrafluoraborate) and NaH2PO4 salt form an ionic liquids aqueous two-phase system (ILATPS), which could extract penicillin from its fermentation broth efficiently. Second, a hydrophobic [C4mim]PF6 (1-butyl-3-methylimidazolium hexafluoraphosphate) is introduced into the ionic liquids-rich phase of ILATPS containing penicillin and converses it into MILWS. Penicillin is hydrolyzed by penicillin acylase in the water phase of MILWS at pH 5. The byproduct phenylacetic acid (PAA) is partitioned into the ionic liquids mixture phase, while the intended product 6-aminopenicillanic acid (6-APA) is precipitated at this pH. In comparison with a similar butyl acetate/water system (BAWS) at pH 4, MILWS exhibits two advantages. (1) The selectivity between PAA and penicillin is greatly optimized at pH 5 by varying the mole ratio of [C4mim]PF6/[C4mim]BF4 in MILWS, whereas in BAWS the unalterable nature of the organic solvent restricts the optimized pH for maximum selectivity between PAA and penicillin at pH 4. (2) The pH for 6-APA precipitation in BAWS is 4, whereas it shifts to pH 5 in MILWS due to the complexation between negatively charged 6-APA and the cationic surface of the ionic liquids micelle. As a result, the removal of the two products from the enzyme sphere at relatively high pH is permitted in MILWS, which is beneficial for enzymatic activity and stability in comparison with the acidic pH 4 environment in BAWS.     

Environmentally friendly, efficient resolution of racemic secondary alcohols by lipase-catalyzed enantioselective transesterification in ionic liquids in the presence of organic bases

The lipase-catalyzed enantioselective transesterification of racemic secondary alcohols was studied using vinyl acetate as acyl donor in two imidazolium-based ionic liquids vs. hexane (Scheme), both in the absence and presence of catalytic amounts of organic bases such as triethylamine (Et(3)N) or pyridine. The organic bases generally enhanced both the rate and enantioselectivity of the reaction. Further, the system 1-butyl-3-methyl-1H-imidazolium hexafluorophosphate/Candida antarctica lipase B ([bmim][PF(6)]/CALB) could be readily recycled four times without significant loss in activity or enantioselectivity.     

Regioselective acylation of flavonoids catalyzed by immobilized Candida antarctica lipase under reduced pressure

A single-step acylation of rutin and naringin, catalyzed by immobilized Candida antarctica lipase B in 2-methyl-2-butanol, occurred preferentially on the primary hydroxyl group. Using palmitic methyl ester as acyl donor, the acylation rate of naringin was 10-fold higher than that of rutin. Under optimal conditions, i.e. a molar ratio acyl donor/naringin of 7:1 and 200 mbar, 92% naringin was acylated.     

Synthesis of benzylidenated hexopyranosides in ionic liquids

The synthesis of 4,6-O-benzylidenated monosaccharides and disaccharides has been studied using ionic liquids as a unique solvent alternative. An examination of several imidazolium ionic liquids indicates that the benzylidenation of hexopyranosides in 3-butyl-1-methylimidazolium tetrafluoroborate, [bmim]BF₄, gives the highest yields for most of the substrates, and that this solvent system could be readily recycled.     

Tyrosinase activity in ionic liquids

Activity of mushroom tyrosinase was studied in three ionic liquids, 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIm][PF₆]), 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF₄]) and 1-butyl-3-methylimidazolium methylsulfate ([BMIm][MeSO₄]), and was compared to that in chloroform. Kinetic parameters of the enzyme were determined and the results indicate that the enzyme in ionic liquids basically follows the same catalytic mechanism as in water, and that the ionic liquids may affect the enzyme activity by direct interacting with the enzyme and thus hindering the E–S binding due to their high hydrophilicity and polarity.     

Properties of an ionic liquid-tolerant <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">amyloliquefaciens</Emphasis> CMW1 and its extracellular protease

An ionic liquid-tolerant bacterium, Bacillus amyloliquefaciens CMW1, was isolated from a Japanese fermented soybean paste. Strain CMW1 grew in the presence of 10 % (v/v) 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), a commonly used ionic liquid. Additionally, strain CMW1 grew adequately in the presence of the hydrophilic ionic liquids 10 % (v/v) 1-ethyl-3-methylimidazolium trifluoromethanesulfonate ([EMIM]CF₃SO₃) or 2.5 % (v/v) 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([BMIM]CF₃SO₃). Strain CMW1 produced an extracellular protease (BapIL) in the culture medium. BapIL was stable in the presence of 80 % (v/v) ionic liquids, [EMIM]CF₃SO₃, [BMIM]Cl, [BMIM]CF₃SO₃, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate, and 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, and functioned in 10 % (v/v) these ionic liquids. BapIL was stable at pH 4.0–12.6 or in 4004 mM NaCl solution, and exhibited activity in the presence of 50 % (v/v) hydrophilic or hydrophobic organic solvents. BapIL was completely inhibited by 1 mM PMSF and partially by 5 mM EDTA. BapIL belongs to the true subtilisins according to analysis of the deduced amino acid sequence. We showed that BapIL from the ionic liquid-tolerant B. amyloliquefaciens CMW1 exhibited tolerance to ionic liquid and halo, alkaline, and organic solvents.     

Biocatalytic preparation of acylated derivatives of flavonoid glycosides enhances their antioxidant and antimicrobial activity

Enzymatic synthesis of acylated derivatives of a monosaccharidic flavonoid chrysoeriol-7-O-beta-D-(3'-E-p-coumaroyl)-glucopyranoside as well as of a disaccharidic flavonoid chrysoeriol-7-[6'-O-acetyl-beta-D-allosyl-(1-->2)-beta-D-glucopyranoside], isolated from Greek endemic plants, was performed using an immobilized Candida antarctica lipase in non-toxic organic solvents. The influence of the reaction parameters such as the molar ratio of acyl donor to flavonoid, as well as the nature of the acyl donor, on the performance of the biocatalytic process was pointed out using the acylation of naringin as a model reaction. With vinyl laurate as acyl donor, the highest conversion was observed at relatively high molar ratio (>or=10), using acetone as solvent. Lipase exhibits specificity towards primary alcohol of the glucose moiety of both flavonoid glycosides. The introduction of an acyl group into glucosylated flavonoids significantly improved their antioxidant activity towards both LDL and serum model in vitro. Furthermore, the acylated derivative of disaccharidic flavonoid increased its antimicrobial activity against two Gram-positive bacteria.     

Retention factors and resolutions of amino benzoic acid isomers with some lonic liquids

Ionic liquids in the form of organic salts are being widely used as new solvent media. In this paper three positional isomers,o-amino benzoic acid,m-amino benzoic acid, andp-amino benzoic acids were separated with four different ionic liquids as mobile phase additives using high performance liquid chromatography (HPLC). The following ionic liquids were used: 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF₄]), 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIm][BF₄]), 1-ethyl-3-methylimidazolium methylsulfate ([EMIm][MS]), and 1-octyl-3-methylimidazolium methylsulfate ([OMIm][MS]). The effects of the alkyl group length on the imidazolium ring and its counterion, and the concentrations of the ionic liquids on the retention factors and resolutions of amino benzoic acid isomers were tested. The results of the separations with ionic liquids as the eluents were better than those without ionic liquids. Excellent separations of the three isomers were achieved using 2.0≈8.0 mM/L [OMIm][MS] and 1.0≈8.0 mM/L [EMIm][MS] as the eluent modifiers.     

Lipase-catalyzed naproxen methyl ester hydrolysis in water-saturated ionic liquid: significantly enhanced enantioselectivity and stability

The lipase selective hydrolysis of Naproxen methyl ester was explored in both water-saturated isooctane and water-saturated ionic liquid 1-butyl-3-methylimidazolium hexafluoro-phoshate ([bmim]PF₆) to see any significant differences in terms of enantioselectivity and stability between two different classes of reaction media. It is shown that polar and hydrophobic of [bmim]PF₆ made it an unearthly reaction medium for hydrolysis of Naproxen methyl ester. It not only decreases the equilibrium constant (K) and enhances the enantiomeric ratio (E), consequently improves the equilibrium conversion (C_Eq) of the hydrolysis reaction and enantiomeric excess of product (ee_p), but also maintains the lipase activity. Because the lipase would not dissolve in the 1-butyl-3-methylimidazolium hexafluoro-phoshate, it can be filtrated up from 1-butyl-3-methylimidazolium hexafluoro-phoshate and recycled for several runs. The stability of lipase was improved due to the higher solubility of methanol in 1-butyl-3-methylimidazolium hexafluoro-phoshate than in isooctane.     

Biocatalysis in ionic liquids: the stereoconvergent hydrolysis of trans-β-methylstyrene oxide catalyzed by soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) was shown to catalyze hydrolysis of epoxides using the ionic liquids (ILs) [bmim][PF₆], [bmim][N(Tf)₂], and [bmim][BF₄] (where bmim=1-butyl-3-methylimidazolium, PF₆=hexafluorophosphate, N(Tf)₂=bis(trifluoromethylsulfonyl)imide, and BF₄=tetrafluoroborate) as reaction medium. Reaction rates were generally comparable with those observed in buffer solution, and when the cress enzyme was used the hydrolysis of trans-β-methylstyrene oxide gave, through a stereoconvergent process, the corresponding optically active (1S,2R)-erythro-1-phenylpropane-1,2-diol.     

The study of factors affecting the enzymatic hydrolysis of cellulose after ionic liquid pretreatment

Cellulose resource has got much attention as a promising replacement of fossil fuel. The hydrolysis of cellulose is the key step to chemical product and liquid transportation fuel. In this paper a serials of chloride, acetate, and formate based ionic liquids were used as solvents to dissolve cellulose. The cellulose regenerated from ILs was characterized by FTIR and X-ray powder diffraction. From the characterization and analysis, it was found that the original close and compact structure has changed a lot. After enzymatic hydrolysis, different kinds of ionic liquids (ILs) have different yields of the reducing sugar (TRS). They are 100%, 90.72%, and 88.92% from 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]), 1-butyl-3-methylimidazolium formate ([BMIM][HCOO]) respectively after enzymatic hydrolysis at 50 °C for 5 h. The results indicated that the yields and the hydrolysis rates were improved apparently after ILs pretreatment comparing with the untreated substrates.     

Lipase-catalyzed esterification of rutin and naringin with fatty acids of medium carbon chain

Flavonoids rutin and naringin were acylated with fatty acids of medium carbon chain (with 8–12 carbon atoms on their molecule) in a reaction catalyzed by immobilized lipase from Candida antarctica (Novozyme) in various solvent systems. The reaction parameters affecting the acylation rate and the conversion of the enzymatic process, such as the nature of the organic solvent and acyl donor used, the water activity (a_w) of the system, as well as the kinetic of the reaction have been investigated. In all cases studied, only flavonoid monoester is identified as the product, which indicates that this lipase-catalyzed esterification is regioselective. The enzymatic acylation of flavonoids seems to follow Michaelis–Menten kinetics.     

Penicillin acylase catalysis in the presence of ionic liquids

Several ionic liquids were used as reaction media for penicillin G acylase catalysis. In all the assayed ionic liquids, [bmim]PF6 proved good media for PGA-catalyzed hydrolysis. A novel [bmim]PF6/water two-phase system is provided for 6-aminopenicillanic acid (APA) production, which will be more benefical than aquous batch systems used widely in industrial production of APA.     

Identification of suitable ionic liquids for application in the enzymatic hydrolysis of rutin by an automated screening

An automated method in milliliter scale was developed for the screening of process parameters concerning the hydrolysis of the flavonoid rutin catalyzed by the rhamnosidase activity of naringinase from Penicillium decumbens. Besides the effect of additives such as ionic liquids and low molecular salts, the productivity in a multiple phase system as well as the recyclability of the enzyme in repetitive batches were studied. The hydrophobic ionic liquid (IL) trihexyl(tetradecyl)phosphonium bis(trifluormethylsulfonyl)imide [P(h₃)t][Tf₂N] was identified to combine the most favorable characteristics out of 23 investigated ILs with regard to enzyme compatibility, substrate solubility and enzyme partition coefficient. Also, for the corresponding cations 1-ethyl-3-methylimidazolium [EMIM], 1-butyl-3-methylimidazolium [BMIM], 1-butyl-1-methylpyrrolidinium [BMPL] and 1-octyl-3-methylimidazolium [OMIM], the entity with the [Tf₂N] anion was best tolerated by the naringinase. With increasing IL content, higher space time yields with up to 1.5 g/(L h) for 80% (v/v) [P(h₃)t][Tf₂N] were achieved. Enhanced specific enzyme activity was observed in the presence of Ca²⁺ ions. By addition of [P(h₃)t][Tf₂N] and calcium chloride, the reactive aqueous phase was successfully used in three repetitive batches with full conversion.