Enantioselective synthesis of (S)‐naproxen using immobilized lipase on chitosan beads

S‐naproxen by enantioselective hydrolysis of racemic naproxen methyl ester was produced using immobilized lipase. The lipase enzyme was immobilized on chitosan beads, activated chitosan beads by glutaraldehyde, and Amberlite XAD7. In order to find an appropriate support for the hydrolysis reaction of racemic naproxen methyl ester, the conversion and enantioselectivity for all carriers were compared. In addition, effects of the volumetric ratio of two phases in different organic solvents, addition of cosolvent and surfactant, optimum pH and temperature, reusability, and inhibitory effect of methanol were investigated. The optimum volumetric ratio of two phases was defined as 3:2 of aqueous phase to organic phase. Various water miscible and water immiscible solvents were examined. Finally, isooctane was chosen as an organic solvent, while 2‐ethoxyethanol was added as a cosolvent in the organic phase of the reaction mixture. The optimum reaction conditions were determined to be 35 °C, pH 7, and 24 h. Addition of Tween‐80 in the organic phase increased the accessibility of immobilized enzyme to the reactant. The optimum organic phase compositions using a volumetric ratio of 2‐ethoxyethanol, isooctane and Tween‐80 were 3:7 and 0.1% (v /v/v), respectively. The best conversion and enantioselectivity of immobilized enzyme using chitosan beads activated by glutaraldehyde were 0.45 and 185, respectively.     

Synthetic activity enhancement of membrane-bound lipase from <Emphasis Type="Italic">Rhizopus chinensis</Emphasis> by pretreatment with isooctane

The cell-bound lipase from Rhizopus chinensis CCTCC M201021 with high catalysis ability for ester synthesis was located as a membrane-bound lipase by the treatments of Yatalase™ firstly. In order to improve its synthetic activity in non-aqueous phase, the pretreatments of this enzyme with various organic solvents were investigated. The pretreatment with isooctane improved evidently the lipase synthetic activity, resulting in about 139% in relative synthetic activity and 115% in activity recovery. The morphological changes of mycelia caused by organic solvent pretreatments could influence the exposure of the membrane-bound enzyme from mycelia and the exhibition of the lipase activity. The pretreatment conditions with isooctane and acetone were further investigated, and the optimum effect was obtained by the isooctane pretreatment at 4°C for 1 h, resulting in 156% in relative synthetic activity and 126% in activity recovery. When the pretreated lipases were employed as catalysts for the esterification production of ethyl hexanoate in heptane, higher initial reaction rate and higher final molar conversion were obtained using the lipase pretreated with isooctane, compared with the untreated lyophilized one. This result suggested that the pretreatment of the membrane-bound lipase with isooctane could be an effective method to substitute the lyophilization for preparing biocatalysts used in non-aqueous phase reactions.     

Effect of solvents on hydrolysis of olive oil by immobilized lipase in reverse phase system

Summary Lipase fromCandida rugosa was immobilized by adsorption on three supports which could contain water available for the hydrolysis of olive oil in a reverse phase system. To select the most suitable solvent for this system, the effect of organic solvents on the stability and catalytic activity of immobilized lipase for the hydrolysis reaction has been examined. The results revealed that isooctane was superior to any other solvents tested in this study for enzymatic fat splitting in a reverse phase system. Also the effect of the solvent polarity on the hydrolysis of olive oil has been examined in detail using various organic solvents mixed with an equivolume of isooctane. It was found that the hydrolysis of olive oil by immobilized lipase was markedly affected by the polarity of reaction solvents.     

Lipase-catalyzed transesterification in several reaction systems: An application of room temperature lonic liquids for bi-phasic production ofn-butyl acetate

Organic solvents are widely used in biotransformation systems. There are many efforts to reduce the consumption of organic solvents because of their toxicity to the environment and human health. In recent years, several groups have started to explore novel organic solvents called room temperature ionic liquids in order to substitute conventional organic solvents. In this work, lipase-catalyzed transesterification in several uni-and bi-phasic systems was studied. Two representative hydrophobic ionic liquids based on 1-butyl-3-methylimidazolum coupled with hexafluorophosphate ([BMIM][PF₆]) and bis[(trifluoromethylsulfonyl) imide] ([BMIM] [Tf₂N]) were employed as reaction media for the transesterification ofn-butanol. The commercial lipase, Novozym 435, was used for the transesterification reaction with vinyl acetate as an acyl donor, The conversion yield was increased around 10% in a water/[BMIM][Tf₂N], bi-phasic system compared with that in a water/hexane system. A higher distribution of substrates into the water phase is believed to enhance the conversion yield in a water/[BMIM][Tf₂N] system. Partion coefficients of the substrates in the water/[BMIM][Tf₂N] bi-phasic system were higher than three times that found in the water/hexane system, while n-butyl acetate showed a similar distribution in both systems. Thus, RTILs appear to be a promising substitute of organic solvents in some biotransformation systems.     

Selection of organic solvents for in situ extraction of fermentation products fromClostridium thermohydrosulfuricum cultures

Summary Fifteen organic solvents were examined to determine their biocompatibility for in situ extraction of fermentation products from cultures of the thermophilic anaerobeClostridium thermohydrosul furicum. Five solvents (hexadecane, isooctane, kerosene, oleyl alcohol, Shellsol TD) were found to be non-toxic toClostridium thermohydrosul furicum. Interfacial tensions, phase separation and partition coefficients for ethanol of the biocompatible solvents were compared. With the exception of kerosene, these solvents showed good separation from the aqueous phase. Oleyl alcohol had the highest partition coefficient for ethanol (K_D=0.34 at 65°C) and appears to be suitable for extractive ethanol fermentation.     

Optimization of enantioselective resolution of racemic ibuprofen by native lipase from Aspergillus niger

Resolution of (R,S)-ibuprofen (2-(4-isobutylphenyl)propionic acid) enantiomers by esterification reaction with 1-propanol in different organic solvents was studied using native Aspergillus niger lipase. The main variables controlling the process (enzyme concentration and 1-propanol:ibuprofen molar ratio) have been optimized using response surface methodology based on a five-level, two-variable central composite rotatable design, in which the selected objective function was enantioselectivity. This enzyme preparation showed preferentially catalyzes the esterification of R(−)-ibuprofen, and under optimum conditions (7% w/v of enzyme and molar ratio of 2.41:1) the enantiomeric excess of active S(+)-ibuprofen and total conversion values were 79.1 and 48.0%, respectively, and the E-value was 32, after 168 h of reaction in isooctane.     

Enzymatic esterification in organic media: role of water and organic solvent in kinetics and yield of butyl butyrate synthesis

Summary The respective roles of organic solvent and of water in butyl butyrate synthesis from n-butanol and n-butyric acid in n-hexane by Mucor miehei lipase have been investigated by analysis of the kinetics and the reaction balances. Esterificaton was found to take place in both low water systems containing solid enzyme in hexane and in biphasic aqueous enzyme solution/hexane systems. In the solid enzyme system, the enzyme adsorbed the water produced, thus delaying the appearance of a discrete aqueous phase. As expected, the presence of some water was indispensable for this system, as its removal or exclusion by various means (adsorption, distillation) affected enzyme activity. However, water removal had little effect on the final yield of esterification. Reaction velocities were quite similar for the solid enzyme/hexane system and for the biphasic aqueous enzyme solution/hexane system. In the latter case, the butyl butyrate formed was almost exclusively found in the organic phase. Ethyl butyrate, a more polar compound, was synthesized with a lower yield. These results allow the conclusion that the reaction took place in a phase consisting of either solid hydrated enzyme with no discrete aqueous phase or of an aqueous enzyme solution by basically similar mechanisms according to the amount of water available to the system, the esterification being driven to completion by transfer of the ester into the organic phase because of a favourable partition coefficient. Offprint requests to: F. Monot     

TADH,the thermostable alcohol dehydrogenase from <Emphasis Type="Italic">Thermus</Emphasis> sp. ATN1: a versatile new biocatalyst for organic synthesis

The alcohol dehydrogenase from Thermus sp. ATN1 (TADH) was characterized biochemically with respect to its potential as a biocatalyst for organic synthesis. TADH is a NAD(H)-dependent enzyme and shows a very broad substrate spectrum producing exclusively the (S)-enantiomer in high enantiomeric excess (>99%) during asymmetric reduction of ketones. TADH is active in the presence of 10% (v/v) water-miscible solvents like 2-propanol or acetone, which permits the use of these solvents as sacrificial substrates in substrate-coupled cofactor regeneration approaches. Furthermore, the presence of a second phase of a water-insoluble solvent like hexane or octane had only minor effects on the enzyme, which retained 80% of its activity, allowing the use of these solvents in aqueous/organic mixtures to increase the availability of low-water soluble substrates. A further activity of TADH, the production of carboxylic acids by dismutation of aldehydes, was investigated. This reaction usually proceeds without net change of the NAD⁺/NADH concentration, leading to equimolar amounts of alcohol and carboxylic acid. When applying cofactor regeneration at high pH, however, the ratio of acid to alcohol could be changed, and full conversion to the carboxylic acid was achieved.     

Immobilization of Pseudomonas cepacia lipase onto electrospun polyacrylonitrile fibers through physical adsorption and application to transesterification in nonaqueous solvent

The lipase of Pseudomonas cepacia was immobilized onto electrospun polyacrylonitrile (PAN) fibers and used for the conversion of (S)-glycidol with vinyl n-butyrate to glycidyl n-butyrate in isooctane. The rate of reaction with the adsorbed lipase was 23-fold higher than the initial material. After 10 recyclings, the initial reaction rate was 80% of the original rate. This system of enzyme immobilization is therefore suitable for carrying out transesterification reactions in nonaqueous solvents.     

Alleviation of Toxicity of Poisonous Organic Compounds on Hydrophilic Carrier/Hydrophobic Organic Solvent Interface

Growth tests of type culture strains were done on interfaces between nutrient agar and hydrophobic organic solvents. All strains examined were able to grow against n-paraffin (C₁₁-C₁₅), and many could grow against isooctane, and pentyl- and heptylbenzene. The interface cultivation method was also successful even when the solvent layer contained compounds toxic for microorganism.     

Optimisation of n-octyl oleate enzymatic synthesis over Rhizomucor miehei lipase

Octyl oleate is a useful organic compound with several applications in cosmetic, lubricant and pharmaceutical industry. At first, the enzymatic synthesis of n-octyl oleate by direct lipase-catalysed esterification of oleic acid and 1-octanol was investigated in a stirred batch reactor in solvent-free system. A systematic screening and optimisation of the reaction parameters were performed to gain insight into the kinetics mechanism. Particularly, enzyme concentration, reaction temperature, stirrer speed, water content, substrates concentration and molar ratio were optimised with respect to the final product concentration and reaction rate. The kinetics mechanism of the reaction was investigated. Finally, a comparison of the experimental results obtained in a solvent free-system with those using two different solvents, supercritical carbon dioxide (SC-CO₂) and n-hexane, was proposed. It resulted that in SC-CO₂ higher concentration of the desired product was attained, requiring lower enzyme concentrations to achieve comparable conversion of free fatty acid into fatty acid ester.     

Microbial Stereoselective Oxidation of 2-methyl-1,3-propanediol to (R)-β-hydroxyisobutyric Acid in Aqueous/organic Biphasic Systems

The aim of this work was to evaluate (R)-&#103-hydroxyisobutyric acid (HIBA) production by microbial stereoselective oxidation of 2-methyl-1,3 propanediol under different conditions, and to compare the performance of this bioconversion in traditional aqueous media and aqueous/organic biphasic media. The oxidation is a two-step reaction with hydroxyisobutanal as an intermediate. Among the operational factors tested, pH and aeration were those, which most significantly affected the biocatalytic activity. Enantiomeric excesses higher than 95% were consistently obtained. For substrate concentrations above 50 mg ml^?1 a slight substrate inhibition was observed. Product inhibition was much stronger, and together with the decrease of the pH during the bioconversion was the most important limiting factor in long-term bioconversions. Kinetic parameters were determined for different pH values. A compromise pH value of 4 was determined to be the optimum for HIBA production and simultaneous extraction with an organic phase of trioctyl phosphine oxide (TOPO) in isooctane.     

Magnetic lipase adsorbed to a magnetic fluid

A magnetic fluid was synthesized by oxidation of ferrous ions (Fe^2&+) in the presence of a synthetic alternating copolymer of polyethylene glycol (PEG) and maleic acid (MA), poly(PEG-MA). The magnetic fluid dispersed stably both in aqueous solution and in organic solvents. Its particle size was approximately 10 nm. The magnetic fluid was mixed with lipase in water, followed by lyophilization. Although the enzyme and the magnetic fluid were dissociated in aqueous solution, they remained associated in organic solvents such as benzene. The magnetic fluid-adsorbed lipase dispersed in benzene and exerted high enzymic activity (2.9 μmol min⁻¹ mg⁻¹ lyophilized powder) for lauryl laurate synthesis from lauric acid and lauryl alcohol, and was readily recovered from the reaction mixture in a magnetic field (6000 Oe) without loss of enzymic activity.     

Stimulation of n-alkane conversion to dicarboxylic acid by organic-solvent- and detergent-treated microbes

Summary A wild-type strain of Cryptococcus neoformans and Pseudomonas aeruginosa were used to convert n-pentadecane to the corresponding dioic acid, tridecane 1,13-dicarboxylic acid (DC-15). Altering the cell permeability by treating C. neoformans with 1% (v/v) toluene or 7% (v/v) Triton X-100 stimulated production of DC-15 by 1.5-fold and fourfold, respectively. Furthermore, DC-15 productivity was increased from 2.5 mg/l per hour to 18 or 30 mg/l per hour, respectively. If 10% (v/v) hexane was used to treat the yeast culture, stimulation of DC-15 production could reach 200% and more viable cells remained compared to the toluene-treated culture. Data from the organic solvent treatment experiment indicated that the solvent with a higher polarity showed a more adverse effect on DC-15 production. P. aeruginosa was vulnerable to most organic solvents; however, Tween 80 could greatly stimulate the conversion of n-pentadecane to DC-15. Although organic solvents and non-ionic detergents could enhance DC-15 formation by microbial conversion, it was inhibited by elevated levels of DC-15.Offprint requests to: E.-C. Chan     

Control with Organic Solvents of Efficiency of Persolvent Cholesterol Fermentation by Pseudomonas sp. Strain ST-200

Pseudomonas sp. strain ST-200 oxidizes cholesterol dissolved in an organic solvent overlying the medium. Major conversion products are cholest-4-en-3-one (C4EO), 6β-hydroxycholest-4-en-3-one (HCEO), and cholest-4-ene-3,6-dione (CEDO). Productivity of each conversion product was altered by changing organic solvents used to dissolve the cholesterol. Generally, HCEO was predominant among the products. HCEO was produced even by cells grown without cholesterol and then killed with harmful organic solvents. The yield of the most oxidized product, CEDO, was improved when the cells were grown in the presence of cholesterol dissolved in a less toxic solvent, cyclooctane.     

Improvement in lipase-catalyzed methanolysis of triacylglycerols for biodiesel production using a solvent engineering method

A solvent engineering strategy was applied to the lipase-catalyzed methanolysis of triacylglycerols for biodiesel production. The effect of different pure organic solvents and co-solvent mixtures on the methanolysis was compared. The substrate conversions in the co-solvent mixtures were all higher than those of the corresponding pure organic solvents. Further study showed that addition of co-solvent decreased the values of |log P_interface − log P_substrate| and thus led to a faster reaction. The more the values of |log P_interface − log P_substrate| decreased, the faster the reaction proceeded and the higher the conversion attained. Different co-solvent ratio was further investigated. The co-solvent mixture of 25% t-pentanol:75% isooctane (v/v) was optimal, with which both the negative effects caused by excessive methanol and by-product glycerol could be eliminated. There was no obvious loss in lipase activity even after being repeatedly used for 60 cycles (720 h) with this co-solvent mixture as reaction medium. Other lipases and lipase combinations can also catalyze methanolysis in this co-solvent mixture. Furthermore, other vegetable oils were also explored for biodiesel production in this co-solvent mixture and it had been found that this co-solvent mixture media has extensive applicability.     

Lipase-catalyzed ammonolysis of trimethylsilylmethyl acetate in organic solvent*

Lipase could catalyze the ammonolysis of trimethylsilylmethyl acetate in organic solvents and Novozym 435 was the best biocatalyst for the reaction. The influences of some factors on the reaction were investigated. Cyclohexane, n-hexane and heptane were found to be suitable reaction media and ammonium carbamate was the best ammonium source. The optimal initial water activity, temperature and pH value were 0.55–0.75, 35°C and 6.5 respectively, under which a substrate conversion of 97.6% could be achieved after reaction for 140 h.     

Parameters influencing asymmetric synthesis of (R)-mandelonitrile by a novel (R)-hydroxynitrile lyase from Eriobotrya japonica

(R)-Mandelonitrile was successfully synthesized by an enzymatic transcyanation reaction of benzaldehyde and acetone cyanohydrin catalyzed by a hydroxynitrile lyase from Eriobotrya japonica (EjHNL) in an aqueous-organic biphasic system. The effects of pH, temperature, organic solvent, substrate concentration and enzyme concentration on the initial activity and enantioselectivity of the enzyme were studied. Both pH and temperature had a large effect on the initial velocity and enantiomeric excess (e.e.) of the product, (R)-mandelonitrile. High enantiomeric purity of the product was observed at low pH and temperature because the non-enzymatic reaction producing racemates of mandelonitrile was almost suppressed. The optimum pH and temperature to obtain high e.e. were pH 4.0 and 10 °C, respectively. Surprisingly, the organic solvents had a significant influence on the initial velocity of the reaction but less influence on the enantiomeric purity of product. The EjHNL was very stable in ethyl acetate, diethyl ether, methyl-t-butyl ether, diisopropyl ether, dibutyl ether and hexane for 12 h. The best solvent for the highest initial velocity and e.e. was diethyl ether with an optimum aqueous phase content of 50% (v/v). The initial reaction rate increase as the aqueous phase content rose, but when the content was more than 50%, a reduction of e.e. was observed. Increasing the concentration of the substrates accelerated the initial velocity, but caused a slight decrease in the e.e. of the product. Under the optimized conditions, the conversion and e.e. of (R)-mandelonitrile for 3 h were 40 and 99%, respectively. The aqueous phase containing the enzyme also showed considerably efficient reusability for 4 batch reactions.     

Synthesis of (R)-2-trimethylsilyl-2-hydroxyl-propionitrile catalyzed by (R)-hydroxynitrile lyase from Prunus japonica seed meal

Synthesis of (R)-2-trimethylsilyl-2-hydroxyl-propionitrile via asymmetric transcyanation of acetyltrimethylsilane with acetone cyanohydrin in an aqueous/organic biphasic system catalyzed by (R)-hydroxynitrile lyase from Prunus japonica seed meal was successfully carried out for the first time. The optimal volume ratio of aqueous to organic phase, buffer pH value and reaction temperature were 15% (v/v), 5.0 and 30°C, respectively, under which both substrate conversion and product enantiomeric excess (ee) were 99%. Silicon atom in the substrate showed great effect on the reaction. Acetyltrimethylsilane was a much better substrate for (R)-hydroxynitrile lyase from Prunus japonica than its carbon analogue.     

Modelling and experimental studies on lipase-catalyzed isoamyl acetate synthesis in a microreactor

A lipase-catalyzed synthesis of isoamyl acetate was studied in a continuously operated pressure-driven microreactor. The esterification of isoamyl alcohol and acetic acid occurred at the interface between n-hexane and an aqueous phase with dissolved lipase B from Candida antarctica. By adjusting flow rates of both phases, a parallel laminar flow with liquid–liquid boundary in the middle of the microchannel could be reestablished and a separation of phases was achieved at the y-shaped exit of the microreactor. Since product remained in the organic phase, this also enabled its continuous separation from the aqueous phase with the enzyme. A three-dimensional mathematical model was developed, considering the velocity profile developed for steady-state conditions between two immiscible fluids. The model contained convection, diffusion, and enzyme reaction terms, where esterification rate was described with a Ping-Pong Bi-Bi mechanism and inhibition by both substrates. Experimental data, which were in good agreement with model simulations, have demonstrated 35% conversion at residence time 36.5 s at 45 °C and at 0.5 M acetic acid and isoamyl alcohol inlet concentrations, which is much faster as in any literature reported so far. According to model simulations, obtained by non-equidistant finite differences numerical solutions of complex non-linear equations system, further microreactor design and process optimization are feasible.