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.     

Enhanced esterification activity through interfacial activation and cross‐linked immobilization mechanism of Rhizopus oryzae lipase in a nonaqueous medium

Interfacial activation via surfactant (Tween 80, Triton X‐100) treatment was conducted to improve the esterification activity of Rhizopus oryzae lipase that had undergone immobilization through cross‐linked enzyme aggregates (CLEA®) technique. Surfactant pretreated immobilized enzymes exhibited better esterification activity compared to free and non‐pretreated immobilized enzyme (Control CLEAs) since higher conversion rates were obtained within shorter times. The superiority of surfactant pretreated CLEAs, especially Tween 80 pretreated CLEAs (T 80 PT CLEAs), were clearly pronounced when longer alcohols were used as substrates. Conversion values exceeded 90% for octyl octanoate, oleyl octanoate and oleyl oleate synthesis with T 80 PT CLEAs whereas Control CLEAs and free enzyme showed no activity. Maximum conversions were achieved in the case equal molars of the substrates or in the case excess of the alcohol to acid in cyclohexane. In solvent free medium containing equal molars of substrates the conversion rates were 85% and 87% with T 80 PT CLEAs respectively for octyl octanoate and oleyl oleate within 2 hours. T 80 PT CLEAs showed 59% of its original activity after 7 consecutive usage for oleyl oleate synthesis. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:899–904, 2016     

Oleyl Oleate and Homologous Wax Esters Synthesized Coordinately from Oleic Acid by Acinetobacter and Coryneform Strains

Newly isolated Acinetobacter (NRRL B-14920, B-14921, B-14923) and coryneform (NRRL B-14922) strains accumulated oleyl oleate and homologous liquid wax esters (C_30:2–C_36:2) in culture broths. Diunsaturated oleyl oleate preponderated in 75 mg liquid wax esters (280 mg lipid extract) recovered from 100-ml cultures of Acinetobacter B-14920 supplemented with 810 mg oleic acid–oleyl alcohol. With soybean oil instead of oleic acid, wax esters (260 mg) were increased to approximately 50% of the lipid extract. Production of wax esters by cultures supplemented with combined fatty (C₈–C₁₈) alcohols and acids suggests a coordinated synthesis whereby the exogenous alcohol remains unaltered, and the fatty acid is partially oxidized with removal of C₂ units before esterification. Consequently, C₈–C₁₈ primary alcohols control chain lengths of the wax esters. Exogenous fatty acids are presumed to enter an intracellular oxidation pool from which is produced a homologous series of liquid wax esters.     

Esterification of fatty acids using nylon-immobilized lipase in n-hexane: kinetic parameters and chain-length effects.

The esterification of long-chain fatty acids in n-hexane catalyzed by nylon-immobilized lipase from Candida rugosa has been investigated. Butyl oleate (22 carbon atoms), oleyl butyrate (22 carbon atoms) and oleyl oleate (36 carbon atoms) were produced at maximum reaction rates of approximately equal to 60 mmol h(-1) g(-1) immobilized enzyme when the substrates were present in equimolar proportions at an initial concentration of 0.6 mol l(-1). The observed kinetic behavior of all the esterification reactions is found to follow a ping-pong bi-bi mechanism with competitive inhibition by both substrates. The effect of the chain-length of the fatty acids and the alcohols could be correlated to some mechanistic models, in accordance with the calculated kinetic parameters.     

Acid Carboxypeptidase-like Activity Contaminating Proctase B

Ester synthesis by the purified lipase from Pseudomonas fragi 22.39 B was investigated. The lipase could synthesize esters from oleic acid and primary or secondary alcohols, but it did not react with tertiary alcohols. Also, the enzyme could use the fatty acids with straight carbon chains as substrates. The activity was enhanced by increasing the carbon number of the fatty acid, but this is not the case for alcohol. The lipase synthesized glycerides from glycerol and oleic acid. 1(3)-Monoolein and 1,3-diolein were the main products and triolein was minor. Synthesis of monoester such as butyl oleate was scarcely affected by the water content in the reaction mixture, while that of glyceride of oleic acid was much affected.     

Exploiting the pressure effect on lipase-catalyzed wax ester synthesis in dense carbon dioxide

The present work focuses on the thermodynamic interpretation of the lauryl oleate biosynthesis in high-pressure carbon dioxide. Lipase-catalyzed lauryl oleate production by oleic acid esterification with 1-dodecanol over immobilized lipase from Rhizomucor miehei (Lipozyme RM IM) was successfully performed in a sapphire window batch stirred tank reactor (BSTR) using dense CO(2) as reaction medium. The experiments were planned to elucidate the pressure effect on the reaction performance. With increasing the pressure up to 10 MPa, the catalytic efficiency of the studied enzyme improved rising up to a maximum and decreased at higher pressure values. Kinetic observations, exhibiting that dense CO(2) expanded reaction mixture in subcritical conditions led to higher performance than when diluted in a single supercritical phase, were elucidated by phase-equilibrium arguments. The experimental results were justified with emphasis on thermodynamic interpretation of the studied system. Particularly, the different reaction performances obtained were related to the position of the operating point with respect to the location of liquid-vapor phase boundaries of the reactant fatty acid/alcohol/CO(2) ternary system. The outlook for exploitation of CO(2) expanded phase at lower pressure compared to supercritical phase, with heterogeneous system in which the solid catalyst particles are exposed to dense CO(2) expanded reaction mixture, in developing new biotransformation schemes is promising.     

Synthesis of N-Octyl Oleate with Lipase from Mucor miehei Immobilized onto Polyethylene Based Graft Copolymers

Lpase from Mucor miehei was immobilized onto partially hydrolyzed poly(ethylene)-g.co-hydroxyethyl methacrylate (PE/HEMA) via spacer arms of 1,6-diaminohexane and glutaraldehyde-. The PE/HEMA-lipase system was used for the enzymatic esterification of n-octanol with oleic acid in the absence of organic solvents. The influence of lipase' concentration, in the attachment solution, on the ester production profile and initial reaction rate was studied. It was found that very small amounts of lipase gave preparations which reached good degrees of conversion. The effect of the initial oleic acid concentration on that pseudo-first order reaction, as well as the presence of water in the reactional medium and the influence of temperature were evaluated. It was found that initial oleic acid concentrations lesser than 1.2 M did not inhibit the immobilized lipase activity; the presence of small amount of water (10–30μ) solubilized in the reaction mixture (6.5 cm³) increased the lipase activity and a maximum of activity of the immobilized lipase preparation was found at 55d`C. The operational stability of the preparation was determined at 37d`C in a BSTR type reactor and a half-life time of three days for the immobilized lipase was obtained.     

Enzymatic synthesis of short‐chain esters in n‐hexane and supercritical carbon dioxide: Effect of the acid chain length

Enzymatic synthesis is the preferred way to produce so‐called “natural products.” Hydrolases have been used for short‐chain ester synthesis. These esters present a pleasant flavor and they have a lot of applications in different industries. Novozym 435 from Candida antarctica (EC 3.1.1.3, triacylglycerol lipase) was used for hexyl ester synthesis in n‐hexane and supercritical carbon dioxide (SCCO₂). Direct esterification provided higher yields than transesterification for the synthesis of esters. Several carboxylic acids of different chain lengths were tested for the esterification reactions: acetic, propionic, butyric, caproic and caprylic acids. The reactions were carried out at 40°C and the amount of enzyme used was 13.8 g/mol alcohol. Substrates were added at equimolar concentrations, with sufficient stirring to avoid external diffusion control. Different substrate concentrations up to 1.5 M were used. The working pressure was 14 MPa in the case of SCCO₂ and atmospheric pressure in the case of organic solvent. The results in both solvents show that the reaction rate increases with the chain length of the acid, but the final yields were similar. However, some of the reactions prove to be faster in SCCO₂, except for hexyl acetate and propionate synthesis, in which acetic and propionic acid presented a lower solubility in SCCO₂ due to its high polarity. Moreover, an acetic acid concentration of 1.5 M brought about a strong inhibition of the enzyme activity.     

Enantioselective esterification of ibuprofen in supercritical carbon dioxide by immobilized lipase

The enantioselective esterification of racemic ibuprofen with n-propanol by immobilized Mucor miehel lipase in supercritical carbon dioxide was studied. The enantiomeric excess of the product (ee_p) was 70 % at 15...20 % conversion. The enantioselectivity was faintly affected by temperature and the concentration of ibuprofen and lipase. The optimum temperature was 45 °C. The initial reaction rate increased with pressure, but enantioselectivity was not affected by pressure changes. The reaction rates in supercritical carbon dioxide at optimized conditions and in n-hexane were similar.     

Erratum to: Solvent-free geranyl oleate production by enzymatic esterification

This study reports the maximization of geranyl oleate production by esterification of geraniol and oleic acid in a solvent-free system using a commercial lipase as catalyst. The operating conditions that maximized geranyl oleate production were determined to be 40?°C, geraniol to oleic acid molar ratio of 5:1, 150?rpm and 10?wt% of enzyme, with a resulting reaction conversion of about 93%. After determining the best reaction parameters, a kinetic study was performed and the results obtained in this step allow to conclude that an excess of alcohol (alcohol to acid molar ratio of 5:1), relatively low enzyme concentration (5?wt%) and temperature of 50?°C afforded nearly complete reaction conversion after 1?h of reaction. New experimental data on enzymatic esterification of geraniol and oleic acid for geranyl oleate production are reported in this work, showing a promising perspective of the technique to overcome the inconvenience of the chemical-catalyzed route.     

Enzymatic reaction kinetic: comparison in an organic solvent and in supercritical carbon dioxide

Myristic acid esterification has been performed by an immobilized lipase from Mucor Miehei both in n-hexane and in supercritical carbon dioxide (SCCO(2)). The enzyme is stable in SCCO(2) at 15 MPa and 323 K. The reaction rate is influenced by the concentration of water and by the reaction medium composition. A reaction mechanism is proposed, and kinetic parameters are determined at 12.5 MPa and 313 K. Maxium velocity appears 1.5-fold higher in SCCO(2) than in n-hexane; however, as solubility of myristic acid is greater in n-hexane, it is not yet definitively clear that the supercritical medium is more favorable than the classical organic solvent for this type of enzyme reaction.     

Solvent-free geranyl oleate production by enzymatic esterification

This study reports the maximization of geranyl oleate production by esterification of geraniol and oleic acid in a solvent-free system using a commercial lipase as catalyst. The operating conditions that maximized geranyl oleate production were determined to be 40 °C, geraniol to oleic acid molar ratio of 5:1, 150 rpm and 10 wt% of enzyme, with a resulting reaction conversion of about 93%. After determining the best reaction parameters, a kinetic study was performed and the results obtained in this step allow to conclude that an excess of alcohol (alcohol to acid molar ratio of 5:1), relatively low enzyme concentration (5 wt%) and temperature of 50 °C afforded nearly complete reaction conversion after 1 h of reaction. New experimental data on enzymatic esterification of geraniol and oleic acid for geranyl oleate production are reported in this work, showing a promising perspective of the technique to overcome the inconvenience of the chemical-catalyzed route.     

Enzymatic synthesis of cetyl oleate in a solvent-free medium using microwave irradiation and physicochemical evaluation

Abstract

A cosmetic ester, cetyl oleate was synthesized using microwave irradiated system. The esterification reaction was carried using Candida antarctica lipase B in a solvent-free media. The influence of various reaction parameters was studied, and the efficiency of Fermase CALB^TM10000 was compared with other enzymes. Equilibrium conversion of 97.5% was obtained within 20?min at 60?°C temperature, 1:2 oleic acid to cetyl alcohol molar ratio and 4% w/w dose of lipase. A comparative study showed that microwave irradiation is a much more efficient method than ultrasound irradiation and conventional heating. Fermase CALB^TM10000 was reusable over 6 enzymatic cycles as its stability improved under microwave system. Physicochemical parameters of cetyl oleate were tested in order to analyze its suitability for further cosmetic use.     

有机溶剂中固定化脂肪酶催化硅醇的酯化反应

固定化Mucor miehei脂肪酶可催化有机硅醇和脂肪酸的酯化反应．对固定化酶用量、脂肪酸链长、不同有机硅醇底物、有机溶剂极性和水含量等影响因素进行了初步研究．     

Preparation and properties of highly soluble chitosan–l-glutamic acid aerogel derivative

The objective of this work is to improve the solubility of chitosan at neutral or basic pH using the supercritical carbon dioxide (sc.CO₂). A novel water-soluble chitosan–l-glutamic acid (Cl-GA) aerogel derivative was synthesized by reaction of 85% deacetylated chitosan with l-glutamic acid (l-GA) in aq.AcOH subjected to solvent exchange prior to using sc.CO₂ as a nonsolvent for the polymer. The prepared aerogel derivative and molecular conformation of modified chitosan are characterized by using UV, FTIR, ¹H NMR, and CD techniques. Some physical properties and surface morphology were analyzed by X-ray diffraction, differential scanning calorimetry (DSC), thermogravimetry (TG), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and porosimetry analysis. Overall, the sc.CO₂ assisted chitosan aerogel derivative opens new perspectives in biomedical applications.     

Comparison of lipase-catalysed esterification in supercritical carbon dioxide and in n-hexane

Summary Oleic acid esterification by ethanol has been performed by an immobilized lipase fromMucor miehei in supercritical carbon dioxide and in n-hexane as solvents. In both media, determination of apparent kinetic constants has been achieved and influence of water content has been shown to be different due to various rates of water solubilities. Stability of the lipase has been proved to be correct and similar in both solvents. Inhibition by ethanol excess has been found but is greater in n-hexane. That can explain the higher initial velocities obtained in supercritical carbon dioxide for the highest ethanol concentrations.     

Separation of protein and fatty acids from tuna viscera using supercritical carbon dioxide

Supercritical carbon dioxide extraction was investigated as a method for removing lipids and bad flavor from tuna viscera. To find the optimum conditions, different experimental variables, such as pressure, temperature, flow rate of solvent and sample size, were evaluated for the effective removal of lipids and the undesirable smell. Ethanol was used as the entrainer, with a 3% by vol CO₂ flow rate. By increasing the pressure at constant temperature, the efficiency of the lipid removal was improved and the protein was concentrated without denaturalization. The main fatty acids extracted from the tuna viscera were palmitic acid (16∶0), heptadecanoic acid (17∶1), oleic acid (18∶1) and docosahexaenoic acid (22∶6). The major amino acids in the tuna viscera treated by supercritical carbon dioxide were glutamic acid, leucine and lysine, and the free amino acids werel-proline, taurine andl-α-aminoadipic acid.     

Single step thin-layer chromatographic method for quantitation of enzymatic formation of fatty acid anilides

The activity of the enzyme involved in catalyzing the formation of fatty acid anilides can be measured by quantitating the fatty acid anilides formed. We have shown earlier that oleic acid is the most preferred substrate among other fatty acids studied for the conjugation with aniline. The reaction product (oleyl anilide) could be separated by thin-layer chromatography (TLC) and then quantified by reversed-phase high-performance liquid chromatography (HPLC). Using [1-¹⁴C]oleic acid as substrate, the fatty acid anilide forming activity can be determined in a single step by TLC analysis. The conventional TLC methods used for the separation of the fatty acid esters, however, could not resolve oleyl anilide from the residual [1-¹⁴C]oleic acid. Therefore, a simple and reliable TLC method was developed for the separation of oleyl anilide from oleic acid using a freshly prepared solvent consisting of petroleum ether–ethyl acetate–ammonium hydroxide (80:20:1, v/v). Using this solvent system the relative flow (R_f) values were found to be 0.54 for oleyl anilide and 0.34 for aniline, whereas oleic acid remained at the origin. The TLC procedure developed in the present study could be used to determine the fatty acid anilide forming activity using [1-¹⁴C]oleic or other fatty acids as substrate and was also found suitable for the analysis of fatty acid anilides from the biological samples.     

Effect of moisture on enzymatic reaction in supercritical carbon dioxide

The continuous acidolysis of triolein and stearic acid was carried out by an immobilized lipase to elucidate the characteristics of supercritical carbon dioxide (SC-CO₂) as a reaction medium. At first, an effect of temperature and pressure on the water adsorption to the immobilized lipase in the SC-CO₂ was examined. Then, the continuous interesterification of triolein and stearic acid by the moist immobilized lipase was examined. The amount of water adsorption to the immobilized lipase in the supercritical carbon dioxide measured under the condition of a different temperature and pressure has been expressed by a correlation equation of Freundlich type by using relative water standardized with the solubility of water in each condition. Optimum operating conditions of the interesterification by immobilized lipase in the SC-CO₂ was 323 K, 16.9 MPa and adsorbed-water concentration of 2 wt%. The production rate obtained by enzymatic acidolysis in the SC-CO₂ was found to be about 0.03 mmol/h2g-immobilized enzyme, leaving 74% residual triglyceride at the optimum operating conditions.     

Esterification activity and stability of Talaromyces thermophilus lipase immobilized onto chitosan

The Talaromyces thermophilus lipase (TTL) was immobilized by different methods namely adsorption, ionic binding and covalent coupling, using various carriers. Chitosan, pre-treated with glutaraldehyde, was selected as the most suitable support material preserving the catalytic activity almost intact and offering maximum immobilization capacity (76% and 91%, respectively). The chitosan-immobilized lipase could be reputably used for ten cycles with more than 80% of its initial hydrolytic activity. Shift in the optimal temperature from 50 to 60 °C and in the pH from 9.5 to 10, were observed for the immobilized lipase when compared to the free enzyme.The catalytic esterification of oleic acid with 1-butanol has been carried out using hexane as organic solvent. A high performance synthesis of 1-butyl oleate was obtained (95% of conversion yield) at 60 °C with a molar ratio of 1:1 oleic acid to butanol and using 100 U (0.2 g) of immobilized lipase. The esterification product is analysed by GC/MS to confirm the conversion percentage calculated by titration.