Response surface methodology as a tool to study the lipase‐catalyzed synthesis of betulinic acid ester

BACKGROUND: The synthesis of betulinic acid ester using betulinic acid and oleyl alcohol catalyzed by Novozym 435 (immobilized Candida antarctica lipase) was carried out. Response surface methodology (RSM) based on a five‐level, three‐variable, central composite rotatable design (CCRD) was employed to evaluate the interactive effects of various parameters. The parameters were reaction time (8–16 h), temperature (20–60 °C) and enzyme amount (120–160 mg). RESULTS: Simultaneously increasing reaction time, temperature and amount of enzyme increased the yields of betulinic acid ester produced. CONCLUSION: The optimum conditions derived via RSM for the reaction were reaction time of 10.2 h, temperature of 53.1 °C and enzyme amount of 138 mg. The actual experimental yield was 48.5% under optimum conditions, which compared well with the maximum predicted value of 47.6%. Copyright © 2008 Society of Chemical Industry     

Enzymatic synthesis of monoacylglycerol citrate optimized by response surface methodology

The ability of immobilized lipase B from Candida antarctica (Novozym 435) to catalyze the direct esterification of citric acid (CA) and monoglyceride (MG) for citrate esters of monoacylglycerols (CITREM) preparation was investigated. The effects of substrate concentration, molecular‐sieve amount, substrate molar ratio, reaction temperature, time, and enzyme load on the conversion of CA in the reaction were investigated. Enzyme screening and the effect of solvent on the esterification were also investigated. RSM was used to optimize the effects of the reaction temperature (45–55°C), the enzyme load (6–10%; relative to the weight of total substrates), and the reaction time (24–48 h) on the conversion of CA. Validation of the RSM model was verified by the good agreement between the experimental and the predicted values of CA conversion. The optimum preparation conditions were as follows: CA concentration 0.12 mol/L, molecular‐sieve 120 g/L, molar ratio of MG/CA 2:1, temperature 54.18°C, enzyme load 9.0% (relative to the weight of total substrates), and reaction time 47.5 h. Under the suggested conditions, the conversion of CA was 77.4%. Repeated reaction tests indicated that Novozym 435 could be used eight times under the optimum conditions with 92% of its original catalytic activity still retained.     

Optimization of Enzymatic Synthesis of Cetyl 2-Ethylhexanoate by Novozym<Superscript>®</Superscript> 435

Waxes are esters obtained from long-chain fatty acids and long-chain alcohols which are biodegradable, biocompatible and nontoxic. Seafowl feather oil is a natural wax ester that exists on seafowl feathers. Cetyl 2-ethylhexanoate is the major ingredient of seafowl feather oil. Cetyl 2-ethylhexanoate is widely used in cosmetics as a base oil because of its lubricity, moisture retention and non-toxic properties. An optimal production of cetyl 2-ethylhexanoate by direct esterification of cetyl alcohol with 2-ethylhexanoic acid was developed using an immobilized lipase (Novozym^® 435) as a catalyst in n-hexane. Response surface methodology (RSM) and 5-level-4-factor central composite rotatable design (CCRD) were employed to evaluate the effects of reaction time, reaction temperature, substrate molar ratio, and enzyme amount on the yield of cetyl 2-ethylhexanoate. The results show that reaction time, reaction temperature, substrate molar ratio, and enzyme amount have significant effects on the yield of the esterification reaction. On the basis of ridge-max analysis, the optimum conditions were as follows: a reaction time of 2.65 days, a reaction temperature of 56.18 °C, a substrate molar ratio of 2.55:1, and an enzyme amount of 251.39%. The predicted and experimental values of molar conversion were 91.95 and 89.75 ± 1.06%, respectively.     

Five-factor response surface optimization of the enzymatic synthesis of citronellyl butyrate by lipase IM77 from Mucor miehei

Response surface methodology (RSM) and a five-level-five-factor central composite rotatable design (CCRD) were used to evaluate the effects of synthetic variables, such as reaction time (3 to 27 h), temperature (25 to 65 °C), enzyme amount (10 to 50%), substrate molar ratio of citronellol to butyric acid (1∶1 to 1∶3), and added water amount (0 to 20%) on molar percent yield of citronellyl butyrate by direct esterification, using lipase IM77 from Mucor miehei. Reaction time and temperature were the most important variables. Substrate molar ratio had no effect on percent molar conversion. Based on contour plots, optimal synthetic conditions were these: reaction time 24 h, temperature 60°C, enzyme amount 20%, substrate molar ratio 1∶1.5, and added water 0%. The predicted molar conversion value was 100%. An actual experimental value of 98% molar conversion was obtained.     

Product Selectivity and Optimization of Lipase-Catalyzed 1,3-Propylene Glycol Esters by Mixture Design and RSM

Propylene glycol mono- (PGM) and diesters (PGD) are widely used as emulsifiers in food and pharmaceutical industry. Solvent engineering was applied to determine the optimum solvent mixtures for the lipase-catalyzed synthesis of 1,3-PGM and 1,3-PGD. After 24 h reaction, the results showed that the molar production of 1,3-PGM was 75% under pure 2M2B (2-methyl-2-butanol) system, whereas 1,3-PGD was preferred to produce in binary mixture system (n-hexane: octane 1:1) with 55% of molar production. Furthermore, the reaction parameters that affect esterification of 1,3-PGD using oleic acid as acyl donor in optimum cosolvent environment were evaluated by response surface methodology (RSM). The reaction temperature and reaction time were the most important parameters. Based on a ridge max analysis, the optimum conditions for 1,3-PGD synthesis were predicted to consist of a reaction time of 40.6 min, a temperature of 59 °C, an enzyme amount of 70.4%, a substrate molar ratio (1,3-propylene glycol/oleic acid) of 1:2.7 and an enzyme pretreatment pH of 6.4 on percentage of molar production of 1,3-PGD of 43.3 ± 4.2%.     

Optimized synthesis of lipase‐catalyzed biodiesel by Novozym 435

The ability of immobilized lipase from Candida antarctica (Novozym 435) to catalyze the alcoholysis of canola oil and methanol was investigated. Response surface methodology (RSM) and five–level–five–factor central composite rotatable design (CCRD) were employed to evaluate the effects of synthesis parameters, such as reaction time, temperature, enzyme concentration, substrate molar ratio of methanol to canola oil, and added water content on percentage weight conversion of canola oil methyl ester by alcoholysis. Reaction temperature and enzyme concentration were the most important variables. High temperature and superabundant methanol inhibited the ability of Novozym 435 to catalyze the synthesis of biodiesel. Based on the analysis of ridge max, the optimum synthesis conditions were as follows: reaction time 12.4 h, temperature 38.0 °C, enzyme concentration 42.3%, substrate molar ratio 3.5:1, and added water 7.2%. The predicted value was 99.4% weight conversion, and the actual experimental value was 97.9% weight conversion. Copyright © 2004 Society of Chemical Industry     

Synergistic Effect of Pd/C and Novozyme 435 on the Dynamic Kinetic Resolution of 1,1,1-trifluoroisopropylamine

Response surface methodology (RSM) was successfully applied to study the synergistic effect of Pd/C and Novozyme 435 on the dynamic kinetic resolution of 1,1,1-trifluoroisopropylamine (TFPA). The variables taken into consideration were reaction temperature, substrate concentration, the Pd/C amount, and the Novozyme 435 amount. A statistical model was used to evaluate the influence of the variables on the conversion and enantiomeric excess (ee). It was found that the interaction between the Novozyme 435 and Pd/C was a significant parameter that affected TFPA conversion. The optimum conditions for RSM were: reaction temperature of 35°C, substrate (±) ? 1 concentration of 0.4 mol/L, 60 g/L of Novozyme 435, and 42.4 g/L of Pd/C (3 wt% of Pd on active carbon). The actual experimental conversion was 95.6% under optimum conditions, which was comparable to the maximum predicted value of 95.7%.     

Modeling and optimization of lipase‐catalyzed synthesis of dilauryl adipate ester by response surface methodology

BACKGROUND: Adipate esters are used as low‐temperature and low‐viscosity plasticizers for polyvinyl chloride and its copolymers. In this work, optimization of lipase‐catalyzed production of dilauryl adipate was carried out using response surface methodology (RSM) based on a four‐factor‐five‐level central composite rotatable design (CCRD). Immobilized lipase from Candida antarctica (Novozym 435) was used as catalyst in this reaction. Various reaction parameters affecting the synthesis of adipate ester, including alcohol/acid molar ratio, amount of enzyme, temperature and reaction time, were investigated. RESULTS: Statistical analysis showed that the amount of enzyme was less significant than the other three factors. The optimal conditions for the enzymatic reaction were obtained at 5.7:1 substrate molar ratio using 0.18 g of enzyme at 53.1 °C for 282.2 min. Under these conditions the esterification percentage was 96.0%. CONCLUSIONS: The results demonstrated that response surface methodology can be applied effectively to optimize the lipase‐catalyzed synthesis of adipate ester. The optimum conditions can obtained be used to scale up the process. Copyright © 2008 Society of Chemical Industry     

Acidolysis Reaction of Terebinth Fruit Oil with Palmitic and Caprylic Acids to Produce Low Caloric Spreadable Structured Lipid

The lipase (Lipozyme IM from Rhizomucor miehei) catalyzed acidolysis reaction of terebinth (Pistacia terebinthus L.) fruit oil with caprylic and palmitic acid in hexane was investigated in a batch system. The effect of reaction conditions and relationship among them were analyzed and optimized by response surface methodology with a four-factor five-level central composite rotatable experimental design. The four major factors chosen were enzyme load (10–20 wt%), reaction time (12–20 h), reaction temperature (45–60 °C) and substrate mol ratio (TO:PA:CA, 1:2.3–4.1:1.15–2.05). Optimum reaction conditions for reaction time, temperature, enzyme load and substrate mole ratio were 12 h, 45 °C, 10 wt% and 1:4.1:2.05, respectively. The maximum yield of desired triacylglycerols (TAG) obtained at these optimum conditions was 50.87 %. Produced structured lipid had a caloric value which was 1.5 % lower than that of terebinth fruit oil. Its solid fat content was found comparable with commercially available margarines. The relative activity of lipase was well maintained in up to 10 repeated cycles.     

Synthesis and Optimization of 2-ethylhexyl Ester as Base Oil for Drilling Fluid Formulation

A stable ester was synthesized to overcome the ester hydrolysis problem during the drilling of oil or gas wells using a conventional ester-based drilling fluid. The thermal and hydrolytic stability of the produced ester was high owing to the transesterification method employed in this study. The reaction was performed using 2-ethylhexanol and methyl laureate esters in the presence of sodium methoxide as a catalyst. In order to obtain the optimum synthesis conditions, a response surface methodology (RSM) was appraised based on the central composite design (CCD). The optimum conditions were determined as follows: 0.6 wt.% catalyst, 70°C reaction temperature, 1:1.5 molar ratio, and 11.5 min of reaction time. The results of 77 wt.% 2-ethylhexyl ester (2-EH) illustrated a high agreement between the experimental and RSM models. The reaction product contained 77 wt.% 2-EH and 23% 2-ethylhexanol. The kinematic viscosity was 5 mm²/s at 40°C and 1.5 mm²/sec at 100°C; the specific gravity was 0.854, flash point was 170°C, and pour point was ?7°C. The produced product showed similar properties to the available commercial product. However, it was observed that the mud formulation using the synthesized base oil had superior rheological properties at 121°C.     

Characterization of Terebinth Fruit Oil and Optimization of Acidolysis Reaction with Caprylic and Stearic Acids

Characterization of the fatty acid and triacylglycerol composition of terebinth fruit oil and the synthesis of structured lipids (SL) were performed in this study. Interesterification reaction of terebinth fruits oil (Pistacia terebinthus L.) with caprylic acid (CA) and stearic acid (SA) to produce a SL was performed in n-hexane using immobilized sn-1,3 specific lipase from Mucor miehei. The effect of reaction conditions and relationship among them were analyzed by response surface methodology (RSM) with a four-factors five-level central composite rotatable experimental design. The four major factors chosen were enzyme load (10–30 wt% based on substrates), reaction time (7–18 h), reaction temperature (40–60 °C) and substrate mole ratio (terebinth oil:SA:CA 1:1:1–1:1:3). The best fitting quadratic model was determined by regression and backward elimination. Based on the fitted model, the optimal reaction conditions for the incorporation of CA and SA were found to be temperature 50 °C; time 18 h; enzyme load 30 wt%; substrate ratio 1:1:3. Under these optimum conditions, the incorporation of SA and CA could be obtained as 19 and 14%, respectively.     

Optimization of Lipase-Catalyzed Synthesis of Cetyl Octanoate in Supercritical Carbon Dioxide

Cetyl octanoate, a wax ester of 24 carbons, is widely used in the cosmetic industry as a base oil. The current work focuses on lipase-catalyzed synthesis of cetyl octanoate in supercritical carbon dioxide (SC-CO₂) by esterification of cetyl alcohol and octanoic acid. Three immobilized lipases were screened, and 15 reaction conditions were tested in order to find the combination for maximal yield. The results showed that Novozym^® 435 was the best catalyst for the synthesis, and a reaction time of 20 min was adequate for a maximal yield. Response surface methodology (RSM) with a 3-factor-3-level Box-Behnken design was employed to evaluate the effects of synthesis parameters, including reaction temperature (35–75 °C), pressure (8.27–12.41 MPa), and enzyme amount (5–15% wt of cetyl alcohol). A model for the synthesis was developed and the optimum conditions could be predicted to be reaction pressure of 10.22 MPa, reaction temperature of 63.70 °C, and enzyme amount of 11.20%. An experiment was performed under this optimum condition and a yield of 99.5% was obtained. This experimental yield correlated well with the predicted value of yield (97.6%). We concluded that, in a SC-CO₂ system, nearly 100% molar conversion of cetyl octanoate could be obtained by immobilized Novozym^® 435 in a short reaction time (20 min) under the predicted optimal conditions.     

Lipase-Catalyzed Irreversible Transesterification of <Emphasis Type="Italic">Jatropha Curcas</Emphasis> L. Seed Oil to Fatty Acid Esters: An Optimization Study

In this work, fatty acid ethyl esters were produced from the lipase-catalyzed irreversible transesterification reaction between Jatropha oil and diethyl carbonate (DEC). Response surface methodology (RSM) based on central composite design (CCD) was used to optimize the five important reaction variables for the irreversible transesterification of Jatropha oil in a solvent-free system. The optimum conditions for the transesterification were a reaction time of 13.3 h, a temperature of 44.5 °C, a lipase amount of 13.7% (w/w), a DEC to Jatropha oil molar ratio of 3.75:1 and no need for adding water. The optimal predicted yield of fatty acid esters was 97.7% and the actual value was 96.2%. The results showed that the RSM based on CCD was adaptable for a fatty acid esters yield study for the current transesterification system.     

A continuous ultrasound‐assisted packed‐bed bioreactor for the lipase‐catalyzed synthesis of caffeic acid phenethyl ester

BACKGROUND: The focus of this paper is the ultrasound‐assisted synthesis of caffeic acid phenethyl ester (CAPE) from caffeic acid and phenyl ethanol in a continuous packed‐bed bioreactor. Immobilized Novozym^® 435 (from Candida antarctica) is used as the catalyst. A three‐level–three‐factor Box–Behnken design and a response surface methodology (RSM) are employed to evaluate the effects of temperature, flow rate, and ultrasonic power on the percentage molar conversion of CAPE. RESULTS: Based on ridge max analysis, it is concluded that the optimum condition for synthesis is reaction temperature 72.66 °C, flow rate 0.046 mL min^?1, and ultrasonic power 1.64 W cm^?2. The expected molar conversion value is 97.84%. An experiment performed under these optimal conditions resulted in a molar conversion of 92.11 ± 0.75%. The enzyme in the bioreactor was found to be stable for at least 6 days. CONCLUSIONS: The lipase‐catalyzed synthesis of CAPE by an ultrasound‐assisted packed‐bed bioreactor uses mild reaction conditions. Enzymatic synthesis of CAPE is suitable for use in the nutraceutical and food production industries. Copyright © 2011 Society of Chemical Industry     

Modification of Stearidonic Acid Soybean Oil by Immobilized Rhizomucor miehei Lipase to Incorporate Caprylic Acid

Immobilized sn-1,3 specific Rhizomucor miehei lipase (RML) was used to catalyze the incorporation of caprylic acid (C8:0) into high stearidonic acid (SDA, C18:4ω3) soybean oil (SDASO) to form structured lipids (SL). The effects of type of biocatalyst (Celite-, octyl-Sepharose-, and Duolite-immobilized RML) and reaction temperature (30, 40, 50, and 60 °C) on acidolysis and acyl migration were studied. Celite-immobilized RML (C-RML) at 50 °C maximized C8:0 incorporation and minimized acyl migration compared to other treatments. Optimal levels of substrate molar ratio (C8:0 to SDASO), incubation time, and enzyme load for SL synthesis by C-RML at 50 °C was determined by response surface methodology to be 6:1, 24 h, and 20 % weight of substrates, respectively. This optimum treatment was scaled-up in hexane or solvent-free reaction media using SDASO or an SDA-enriched acylglycerol mixture as substrate. This yielded various SL with C8:0 contents ranging from 17.0 to 32.5 mol% and SDA contents ranging from 20.6 to 42.3 mol%. When digested, these SL may deliver C8:0 for quick energy and SDA for heart health making them potentially valuable for medical and nutraceutical applications.     

Free‐radical copolymerization of [(4‐isopropyl phenyl) oxycarbonyl] methyl methacrylate with acrylonitrile and methyl methacrylate

The present investigation has been achieved in accordance with the Diels–Alder reaction (1,4 cycloaddition) to produce a new halogenated bicyclic adduct. ortho‐Bromoallylbenzoate is a new dienophile that was prepared in a pure form, and its structure was confirmed. The Diels–Alder syntheses of hexachlorocyclopentadiene and the new dienophile were studied to determine the optimum condensation reaction conditions under a temperature range of 90–160°C, reaction times of 1–8 h, and molar diene/dienophile ratios from 1:1 to 5:1 as a consequence. The optimum conditions reached were a temperature of 140°C, an initial diene/dienophile molar ratio of 3:1, and a duration time of 6 h. The maximum stoichometric yield under these optimum conditions (82.5%) was obtained. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 2331–2338, 2003     

Response Surface Optimized Ultrasonic-Assisted Extraction of Quercetin and Isolation of Phenolic Compounds From Hypericum perforatum L. by Column Chromatography

The effects of ultrasonic-assisted extraction factors for the main phenolic compound (quercetin) from Hypericum perforatum L. were optimized using the Box–Behnken design (BBD) combined with response surface methodology. The BBD was employed to evaluate the effects of extraction temperature (30–70°C), extraction time (20–80 min), methanol concentration (20–80%, v/v), and HCl concentration (0.8–2.0 M) on the content of one of the major phenolic compounds of quercetin. The extracts were analyzed by high performance liquid chromatography (HPLC). The major phenolic compounds of H. perforatum were isolated and the antioxidant capacity and total phenol content were determined in crude extract and fractions. The optimum conditions were determined as extraction temperature 67°C, extraction time 67 min, methanol concentration 77% (v/v), and HCl concentration 1.2 M. The predicted content of quercetin was 10.81 mg/g dried plant under the optimal conditions and the subsequent verification experiment with 11.09 mg/g dried plant confirmed the validity of the predicted model. The isolated compounds were identified as quercetin, cyanidin, protocatechuic acid, and kaempferol.     

Optimisation study of large‐scale enzymatic synthesis of oleyl oleate,a liquid wax ester,by response surface methodology

An optimisation study of the large‐scale enzymatic synthesis of a liquid wax ester from oleic acid and oleyl alcohol using Novozym 435 was carried out. Investigations were performed in batch mode with a stirred tank reactor (STR) with one multi‐bladed impeller. Response surface methodology (RSM) based on a five‐level, three‐variable central composite rotatable design (CCRD) was used to evaluate the interactive effects of various parameters. The parameters are amount of enzyme (A) (90–120 g), impeller speed (B) (100–400 rpm) and temperature (C) (40–60 °C). The optimum conditions derived via RSM at a fixed reaction time of 1 h were successfully optimised as A = 104 g, B = 388.0 rpm and C = 49.7 °C. The actual experimental yield was 96.7% under the optimum conditions, which compared well with the maximum predicted value of 97.6%. Copyright © 2005 Society of Chemical Industry     

BIODIESEL PRODUCTION FROM PALM OIL VIA HETEROGENEOUS TRANSESTERIFICATION: OPTIMIZATION STUDY

Fatty acid methyl ester (FAME) prepared by transesterification process using a heterogeneous catalyst has received a lot of interest lately as it could overcome the limitations of the current commercial homogeneous catalytic process. Apart from that, palm oil, being the cheapest edible oil in the world, will always remain the most economical source of FAME. Therefore, in this study, the use of sulfated zirconia alumina as a heterogeneous catalyst to catalyze the transesterification of palm oil with methanol to FAME was carried out using design of experiment (DOE), specifically response surface methodology (RSM) based on four-variable central composite design (CCD) with α = 2. The transesterification process variables are reaction temperature (60–180°C), reaction period (1–5 h), methanol-to-oil ratio (4–12 mol mol^?1), and amount of catalyst (2–10 wt.%). In this study involving many multiple process variables, the design of experiment approach was found to be superior to the conventional one-variable-at-one-time approach. Interactions between variables were found to have significant effect on the yield of FAME. At the conditions of 3 h of reaction period, 127°C reaction temperature, methanol-to-palm oil ratio of 8, and 6 wt.% of catalyst, an optimum FAME yield of 83.3% can be obtained, indicating that sulfated zirconia alumina has potential as a heterogeneous catalyst for the production of FAME from palm oil.     

Optimization of Lipase-Catalyzed Fractionation of Two Conjugated Linoleic Acid (CLA) Isomers

The separation of two isomers of conjugated linoleic acid is highly significant since each exhibits different biochemical properties. The aim of this study was to investigate and optimize several factors affecting the esterification of l-menthol with the c9,t11-CLA isomer in an organic solvent-free system using lipase from Candida rugosa (Lipase AY-30). D-optimal design with 5 factors and 3 levels were employed to evaluate the effects of synthesis parameters; reaction time (8–24 h), temperature (30–50 °C), enzyme content (2–20 U/ml), substrate molar ratio of conjugated linoleic acid oil to l-menthol (2:1–1:2) and pH (6–8) on esterification of c9,t11-CLA with l-menthol. Based on the analysis of the residual amount of c9,t11-CLA in the free fatty acid fraction after just one-step esterification, the optimum synthesis conditions were as follows: reaction time 23.12 h, temperature 32.65 °C, enzyme amount 135.40 U, molar ratio of CLA oil to l-menthol at 1:1.7 and pH at 7.7; the lowest purity of c9,t11-CLA in free fatty acid fraction based on the total content of c9,t11 and t10,c12-CLA isomers was 8.6 %.