Lipase-catalyzed synthesis of geranyl acetate in n-hexane with membrane-mediated water removal.

The esterification of geraniol with acetic acid in n-hexane was investigated. A commercial lipase preparation from Candida antarctica was used as catalyst. The equilibrium conversion (no water removal) was found to be 94% for the reaction of 0.1 M alcohol and 0.1 M acid in n-hexane at 30 degrees C. This was shown by both hydrolysis and esterification reactions. The activation energy of reaction over the temperature range 10 degrees to 50 degrees C was found to be 16 kJ/mol. The standard heat of reaction was -28 kJ/mol. Membrane pervaporation using a cellulose acetate/ceramic composite membrane was then employed for selective removal of water from the reaction mixture. The membrane was highly effective at removing water while retaining all reaction components. Negligible transport of the solvent n-hexane was observed. Water removal by pervaporation increased the reaction rate by approximately 150% and increased steady-state conversion to 100%.     

Investigating Pervaporation for In Situ Acetone Removal as Process Intensification Tool in ω-Transaminase Catalyzed Chiral Amine Synthesis

Hydrophobic pervaporation (PV), allowing for the separation of an organic component from an aqueous stream, was investigated for in situ acetone removal from a transamination reaction. A poly(dimethylsiloxane) membrane was applied in a coupled enzymatic process at 5 L scale. Among the four components, there was no loss of donor and product amines through PV which was highly desirable. However, in addition to removal of acetone, there was also an unwanted loss of acetophenone (substrate ketone) because of PV. The coupled enzyme-PV process resulted in 13% more product formation compared to the control process (where no PV was applied) after 9 h. Results from a qualitative simulation study (based on partial vapor pressures and a vapor–liquid equilibrium of the feed solution) indicated that PV might have an advantage over direct distillation strategy for selective removal of acetone from the reaction medium. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2731, 2019     

Immobilization of proteases to porous chitosan beads and their catalysis for ester and peptide synthesis in organic solvents.

alpha-Chymotrypsin (CT), subtilisin BPN' (STB), and subtilisin Carlsberg (STC) were immobilized by adsorption to porous chitosan beads (Chitopearl, CP). The immobilized enzymes showed higher catalytic activities than free enzymes for amino acid esterification in many hydrophilic organic solvents except for methanol and DMF. In ethanol, the initial rate of the esterification increased with water content, whereas in ethyl acetate, the maximum rate was obtained at 2%-3% water. CP-immobilized CT also catalysed transesterification of Ac-Tyr-OMe in ethanol and peptide synthesis in acetonitrile from Ac-Tyr-OH or its ethyl ester and amino acid amides. The immobilized enzymes are highly stable in organic solutions, and can easily be separated from the reaction solutions. Repeated esterifications of Ac-Tyr-OH in acetonitrile by a CP-immobilized CT gave almost constant yields of the ester for more than 3 weeks.     

Pervaporation Aided Esterification of Carboxylic Acids with Ethanol Catalyzed by Porcine Pancreatic Lipase

The results of pervaporation-coupled esterification of various carboxylic acids with ethanol catalyzed by Porcine pancreatic lipase are reported. The effect of lipase and substrate concentrations has been studied and the advantage of pervaporation on the equilibrium conversion has been deduced. The kinetics of reaction were analyzed with a three-parameter model which coupled the effect of pervaporation. The intrinsic kinetic constants for all the reactions were estimated and correlated with the carbon number, an indicator of hydrophobicity of the acids. It was found that the rate constant increases with decrease in carbon number. The experimental concentration profiles were simulated from the model for all the reactions and the model prediction was found to be reasonably good. The water permeability was also correlated well with acid hydrophobicity. The pervaporation coupled reaction efficiency, as represented by the reaction time for equilibrium conversion, was found to bear a profound relation to membrane surface area per unit volume of the reaction mixture (A/V). The time for equilibrium conversion was found to decrease with an increase in A/V value, reaching a minimum and then increasing with a further increase of A/V. A probable explanation has been postulated for such an observation.     

Pervaporation Aided Esterification of Carboxylic Acids with Ethanol Catalyzed by Porcine pancreatic Lipase

The results of pervaporation-coupled esterification of various carboxylic acids with ethanol catalyzed by Porcine pancreatic lipase are reported. The effect of lipase and substrate concentrations has been studied and the advantage of pervaporation on the equilibrium conversion has been deduced. The kinetics of reaction were analyzed with a three-parameter model which coupled the effect of pervaporation. The intrinsic kinetic constants for all the reactions were estimated and correlated with the carbon number, an indicator of hydrophobicity of the acids. It was found that the rate constant increases with decrease in carbon number. The experimental concentration profiles were simulated from the model for all the reactions and the model prediction was found to be reasonably good. The water permeability was also correlated well with acid hydrophobicity. The pervaporation coupled reaction efficiency, as represented by the reaction time for equilibrium conversion, was found to bear a profound relation to membrane surface area per unit volume of the reaction mixture (A/V). The time for equilibrium conversion was found to decrease with an increase in A/V value, reaching a minimum and then increasing with a further increase of A/V. A probable explanation has been postulated for such an observation.     

Microbial lipases: at the interface of aqueous and non-aqueous media. A review

In recent times, biotechnological applications of microbial lipases in synthesis of many organic molecules have rapidly increased in non-aqueous media. Microbial lipases are the 'working horses' in biocatalysis and have been extensively studied when their exceptionally high stability in non-aqueous media has been discovered. Stability of lipases in organic solvents makes them commercially feasibile in the enzymatic esterification reactions. Their stability is affected by temperature, reaction medium, water concentration and by the biocatalyst's preparation. An optimization process for ester synthesis from pilot scale to industrial scale in the reaction medium is discussed. The water released during the esterification process can be controlled over a wide range and has a profound effect on the activity of the lipases. Approaches to lipase catalysis like protein engineering, directed evolution and metagenome approach were studied. This review reports the recent development in the field ofnon-aqueous microbial lipase catalysis and factors controlling the esterification/transesterification processes in organic media.     

pH-optima in lipase-catalysed esterification

Though lipases are frequently applied in ester synthesis, fundamental information on optimal pH or substrate concentration, can almost only be found for the reverse reaction - hydrolysis. This study demonstrates that the pH-optima of lipase-catalysed esterifications differ significantly from the optima of the hydrolysis reaction. In the esterification of n-butanol and propionic acid with lipases of Candida rugosa (CRL) and Thermomyces lanuginosa (TLL) pH-optima of 3.5 and 4.25, respectively, were found. This is about 3-4 units (CRL) and 7 units (TLL) in pH lower than optimum for hydrolysis. Enzyme activity increased with increasing concentrations of protonated acid indicating that the protonated acid rather than the deprotonated form is substrate for esterification. The rate of esterification can be drastically increased by ensuring acid concentrations up to 1000 mmol L^-1 for CRL and 600 mmol L^-1 for TLL in the reaction system.     

Ethanol production in an integrated process of fermentation and ethanol recovery by pervaporation

In ethanol fermentations inhibition of the microorganism by ethanol limits the amount of substrate in the feed that can be converted. In a process high feed concentrations are desirable to minimize the flows. Such high feed concentrations can be realized in integrated processes in which ethanol is recovered from the fermentation broth as it is formed. In this study ethanol recovery by pervaporation was coupled to glucose fermentations by baker's yeast. Pervaporation was carried out with commercial silicone based hollow-fibre membrane modules with relatively high fluxes. Three different types of process configurations with pervaporation were investigated. Two of these configurations also included cell retention by microfiltration, in order to optimize the productivity. In the systems with pervaporation a feed containing 360 kg/m³ glucose could be converted almost completely. This feed concentration is a factor three higher than in a process without ethanol recovery. The productivity was 14 kg/m³ h in a system with pervaporation only, and could be increased to 43 kg/m³ h in the system with all recycle by microfiltration. The kinetic data suggest that accumulation of inhibitory compounds occurs in the integrated system. The integrated process was relatively easy in operation.     

Continuous acetone-butanol-ethanol (ABE) fermentation using immobilized cells of Clostridium acetobutylicum in a packed bed reactor and integration with product removal by pervaporation

An integrated solvent (ABE) fermentation and product removal process was investigated. A stable solvent productivity of 3.5 g/L h was achieved by using cells of Clostridium acetobutylicum immobilized onto a packed bed of bonechar, coupled with continuous product removal by pervaporation. Using a concentrated feed solution containing lactose at 130g/L, a lactose value of 97.9% was observed. The integrated fermentation and product removal system, with recycling of the treated fermentor effluent containing only low amount of solvents (/but lactose and acids), leads to only low acid losses. Therefore, most of the acids are converted to solvents, and this results in a high solvent yield of 0.39 g solvents/g lactose utilized. The pervaporation system provided a high product removal rate even at low solvent concentrations. A solvent membrane flux of 7.1 g/m(2) h with a selectivity of 5 was achieved during these investigations. The system proved to be very reliable.     

Production of highly concentrated ethanol in a coupled fermentation/pervaporation process using silicalite membranes

The fermentation performance of a coupled fermentation/pervaporation process using silicalite membranes, which are ethanol permselective for an ethanol/water solution, was studied. The process exhibited about a 20% increase in an average glucose consumption rate as compared with that without the pervaporation unit. A strong correlation was observed between the membrane flux and the consumption rate. Ethanol concentrations in the permeates reached a maximum of 85% (v/v).     

pH-optima in lipase-catalysed esterification

Though lipases are frequently applied in ester synthesis, fundamental information on optimal pH or substrate concentration, can almost only be found for the reverse reaction – hydrolysis. This study demonstrates that the pH-optima of lipase-catalysed esterifications differ significantly from the optima of the hydrolysis reaction. In the esterification of n-butanol and propionic acid with lipases of Candida rugosa (CRL) and Thermomyces lanuginosa (TLL) pH-optima of 3.5 and 4.25, respectively, were found. This is about 3–4 units (CRL) and 7 units (TLL) in pH lower than optimum for hydrolysis. Enzyme activity increased with increasing concentrations of protonated acid indicating that the protonated acid rather than the deprotonated form is substrate for esterification. The rate of esterification can be drastically increased by ensuring acid concentrations up to 1000 mmol L^?1 for CRL and 600 mmol L^?1 for TLL in the reaction system.     

Recovery of 6-pentyl-alpha-pyrone fromTrichoderma viride culture medium by perevaporation

Summary Cultivation ofTrichoderma viride yields the volatile lactone 6-pentyl-alpha-pyrone (6-PP) as principal metabolite in concentration of up to 1 g/l. Separation and enrichment of 6-PP from the culture medium is effected by pervaporation through hydrophobic membranes. Virtually complete removal of 6-PP from inactive culture medium is achieved by batch pervaporation. Unlike steam distillation, pervaporation may be directly coupled to the active fermenter.     

Computer-aided real-time estimation of reaction conversion for lipase-catalyzed esterification in solvent-free systems

Real-time conversion estimation through macroscopic balancing was investigated for enzymatic esterification reactions in a solvent-free system. In principle, the conversion of ester synthesis can be determined from the amount of water produced by the reaction because water is formed as a by-product in the same molar ratio as the product. In this study, we show that the water production rate, and thereby the reaction conversion, can be estimated on-line from measurements of the relative humidity of the inlet and outlet air and the material balances of water in the system. In order to test the performance of the real-time conversion estimation method, the lipase-catalyzed esterification reaction of n-capric acid and n-decyl alcohol in solvent-free media was conducted while controlling the water activity at various values. When the reaction conversions estimated on-line were compared with those analyzed off-line by gas chromatography, good agreement was obtained: the average mean absolute error was +/- 2.4% of the reaction conversion despite the simplicity of the method. The on-line estimation method presented here requires no expensive or complicated analytical instruments and no sampling of reaction medium. It can be used for monitoring nonaqueous enzymatic reactions where water is produced or consumed during reaction.     

Candida Rugosa lipase reactions in nonionic w/o-microemulsion with a technical surfactant

In enzyme catalysis there is a great interest in finding suitable organic media for less water soluble substrates in order to increase the substrate concentration and therefore the reaction rates. These requirements are fulfilled by using w/o-microemulsions as reaction media. The influences of pH, temperature, water concentration and the kinetic parameters of Candida Rugosa Lipase in a nonionic w/o-microemulsion with a surfactant of technical grade, Marlipal O13-60, are presented. In an example the enantiospecific esterification of racemic menthol with propionic anhydride using this nonionic microemulsion likely to be affordable in large scale applications is shown. For a continuous process an ultrafiltration unit is attached to a reactor within a loop. In this way, the reverse micelles containing the enzymes can be separated from the oil, containing the product, and reused afterwards.     

Preparation of new lipases derivatives with high activity–stability in anhydrous media: adsorption on hydrophobic supports plus hydrophilization with polyethylenimine

A novel method to prepare immobilized lipases derivatives is hereby proposed. Lipases are firstly adsorbed on supports having large internal surfaces covered by hydrophobic groups (e.g. polyacrylic resins covered by C18 moieties). Then, immobilized lipases are incubated in the presence of polyethyleneimine (PEI) at a pH value over the isoelectric point of the enzyme in order to cover the lipase surface with this polymer. In this way, we try to minimize all possible direct interactions between immobilized lipase and organic solvents when using these derivatives in anhydrous media.

Lipases from Rhizomucor miehie (RML) and Candida rugosa (CRL) were immobilized according to the proposed protocol. These derivatives were very active and very stable when catalyzing esterifications and transesterifications in anhydrous media. For example, RML derivatives exhibited a very high synthetic activity (more than 1000 Units/g immobilized biocatalyst) even when catalyzing the esterification of lauric acid with octanol at water activity values very close to zero. On the contrary, covalently immobilized derivatives exhibited a much lower synthetic activity under similar conditions (less than 10 Units/g of immobilized biocatalyst). Moreover, these new RML derivatives preserve 100% activity after incubation for 3 days in anhydrous butanone in the presence of molecular sieves. Under the same conditions, commercial immobilized RML lost more than 90% of activity in less than 10 min.     

Production of 2-phenylethanol and 2-phenylethylacetate from L-phenylalanine by coupling whole-cell biocatalysis with organophilic pervaporation

An integrated bioprocess for the production of the natural rose-like aroma compounds, 2-phenylethanol (2-PE) and 2-phenylethylacetate (2-PEAc), from L-phenylalanine (L-phe) with yeasts was investigated. The hydrophobicity of the products leads to product inhibition, which can be compensated by in situ product removal (ISPR). An organophilic pervaporation unit, equipped with a polyoctylmethylsiloxane (POMS) membrane, was coupled via a bypass to a bioreactor and proved to be a suitable technique for the in situ removal of high-boiling products from culture broth. With batch cultures of the thermotolerant yeast Kluyveromyces marxianus CBS 600 in a standard medium at 35 degrees C, the use of pervaporation resulted in a double 2-PE concentration (2.2 g/L) and 1.3 g/L 2-PEAc, which only accumulated transiently in low concentrations during cultivation without ISPR. Using a previously optimized medium, the variation of the temperature from 30 degrees C to 40 degrees C caused an increase in the total conversion yield from 63% to 79%, corresponding to total product concentrations of 5.23 and 5.85 g/L, respectively. In the 40 degrees C batch experiment, the volumetric productivity (2-PE + 2-PEAc) during the exponential phase was 5.2 mmol/L h. While for 2-PE, there is still potential for further optimization, the more hydrophobic 2-PEAc was nearly completely removed from the aqueous culture broth (enrichment factor >400), resulting in highly aroma-enriched permeates. Due to the temperature-correlated performance of the pervaporation, the bioconversion was still efficient even at 45 degrees C (conversion yield: 69%). Surprisingly, at 45 degrees C, the molar ratio of the two products inverted and 2-PEAc turned out to be the main product (4.0 g/L), which opens easy control of the reaction's selectivity by external means. Retrofitting the process with interim heating and cooling equipment to use different temperature levels for cultivation and pervaporation resulted in a decreased yield and product concentration caused by multiple stress factors. The medium composition affected the pervaporation efficiency with molasses acting detrimental.     

Esterification of side-chain oxysterols by lysosomal phospholipase A2

Side-chain oxysterols produced from cholesterol either enzymatically or non-enzymatically show various bioactivities. Lecithin-cholesterol acyltransferase (LCAT) esterifies the C3-hydroxyl group of these sterols as well as cholesterol. Lysosomal phospholipase A2 (LPLA2) is related to LCAT but does not catalyze esterification of cholesterol. First, esterification of side-chain oxysterols by LPLA2 was investigated using recombinant mouse LPLA2 and dioleoyl-PC/sulfatide/oxysterol liposomes under acidic conditions. TLC and LC-MS/MS showed that the C3 and C27-hydroxyl groups of 27-hydroxycholesterol could be individually esterified by LPLA2 to form a monoester with the C27-hydroxyl preference. Cholesterol did not inhibit this reaction. Also, LPLA2 esterified other side-chain oxysterols. Their esterifications by mouse serum containing LCAT supported the idea that their esterifications by LPLA2 occur at the C3-hydroxyl group. N-acetylsphingosine (NAS) acting as an acyl acceptor in LPLA2 transacylation inhibited the side-chain oxysterol esterification by LPLA2. This suggests a competition between hydroxycholesterol and NAS on the acyl-LPLA2 intermediate formed during the reaction. Raising cationic amphiphilic drug concentration or ionic strength in the reaction mixture evoked a reduction of the side-chain oxysterol esterification by LPLA2. This indicates that the esterification could progress via an interfacial interaction of LPLA2 with the lipid membrane surface through an electrostatic interaction. The docking model of acyl-LPLA2 intermediate and side-chain oxysterol provided new insight to elucidate the transacylation mechanism of sterols by LPLA2. Finally, exogenous 25-hydroxycholesterol esterification within alveolar macrophages prepared from wild-type mice was significantly higher than that from LPLA2 deficient mice. This suggests that there is an esterification pathway of side-chain oxysterols via LPLA2.     

Removal of water produced from lipase-catalyzed esterification in organic solvent by pervaporation

Esterification of oleic acid with n-butanol in the presence of Lipozyme(R) was carried out at 25 degrees C in isooctane with various initial water activities. Initial reaction rate as well as equilibrium conversion decreased at high initial water activity. Therefore, removal of water present in the reaction mixtures was essential. A pervaporation process was applied to the lipase-catalyzed synthesis of n-butyloleate to remove water. Pervaporation selectively separated water from the reaction mixture using a nonporous polymeric membrane, cellulose acetate. Therefore, pervaporation is potentially applicable to remove the water produced from various enzymatic processes, such as synthesis of various esters, peptides, and glycosides in a solvent system as well as in a solvent-free system. (c) 1995 John Wiley & Sons, Inc.     

Application of ion exchange to purify acarbose from fermentation broths

Acarbose is conventionally used to reduce the insuline consumption of the diabetic patients. This compound is an oligosaccharide with the general formulae C25H43NO18 and obtained from fermentation processes by certain strains of Actinoplanes Utahensis. After the fermentation process, the acarbose has to be isolated from the fermentation broth where is accompanied of a large amount of substances, such as substrates, intermediate metabolites, proteins and different salts.

Four strong acid resins considering geliform and macroporous matrix types in aqueous and organic media have been tested in order to reach an easy and selective separation process. According to the experimental data, the Finex CS10GC (a gel strong cationic ion exchanger) presented the maximum acarbosa uptake and also the highest rate of ion exchange in water. The best behavior in non-aqueous media was observed with the Purolite CT151 (macroporous ion exchanger) but its maximum capacity of ion exchange was really lower than that exhibited by the Finex CS10GC resin in aqueous media. These results suggest that the acarbose removal from fermentation broths must be carried out in aqueous media to ensure the maximum usage of the resin uptake capacity. The results obtained provide a significant insight into the main equilibrium phenomena that takes place depending on the characteristics of the liquid phase. Finally, the elution of acarbose from the resin can be accomplished of a selectivity way by using a solution of 2.25N of HCl. The proposed separation method seems to be technically and economically feasible.     

Study of vitamin ester synthesis by lipase-catalyzed transesterification in organic media

Immobilized lipase from Candida antarctica (Novozym 435) was used in organic media to catalyze esterifications of vitamins (ascorbic acid and retinol) from hydroxy acid. We described the synthesis of retinyl L-lactate by transesterification between retinol and L-methyl lactate with yield reaching 90% and the synthesis of ascorbyl L-lactate by transesterification between ascorbic acid and L-methyl lactate with yield reaching 80%. The kinetic study of the esterification of vitamins with L-methyl lactate in organic media has been carried out and agrees with ping-pong-ordered Bi-Bi when the initial vitamin concentration is low. When initial vitamin concentration is high, the kinetic is similar to a hybrid ping-pong-ordered Bi Bi or hybrid ping-pong-random Bi Bi mechanism. However, with high initial substrate concentration, change of the kinetic by other phenomena, such as interaction of substrates with molecular sieves, adsorption of the methanol formed, and decreases of substrate diffusion, could be considered. It is obvious that in these conditions, classical enzymology (i.e., Michaelian enzymology) cannot be used for the interpretation of results.