Lipase catalyzed acetylation of 3,5,4′-trihydroxystilbene: optimization and kinetics study

The use of immobilized lipase from Candida antarctica (Novozym(?) 435) to catalyze acetylation of trans-3,5,4'-trihydroxystilbene was investigated in this study. Response surface methodology and 5-level-4-factor central composite rotatable design were adopted to evaluate the effects of synthesis variables, including reaction time (24-72 h), temperature (25-65 °C), substrate molar ratio (1:15-1:75), and enzyme amount (600-3,000 PLU) on the percentage molar conversion of trans-4'-O-acetyl-3,5-dihydroxystilbene. The results showed that reaction temperature and enzyme amount were the most important parameters on percentage molar conversion. Based on ridge max analysis, the optimum conditions for synthesis were: reaction time 60 h, reaction temperature 64 °C, substrate molar ratio 1:56 and enzyme amount 2,293 PLU. The molar conversion of actual experimental values was 95% under optimal conditions. The synthesis product was analyzed using HPLC, mass and NMR. The results revealed that the major product was trans-4'-O-acetyl-3,5-dihydroxystilbene. The reaction kinetics was found to follow the Ping-Pong mechanism; substrate inhibition was not found at high vinyl acetate concentration.     

Optimization of lipase-catalyzed biodiesel by response surface methodology

Biodiesel prepared by catalyzed mild transesterification has become of much current interest for bioenergy. The ability of a commercial immobilized lipase (Novo Industries--Bagsvaerd, Denmark) from Rhizomucor miehei (Lipozyme IM-77) to catalyze the transesterification of soybean oil and methanol was investigated in this study. Response surface methodology and 5-level-5-factor central composite rotatable design were employed to evaluate the effects on reaction time, temperature, enzyme amount, molar ratio of methanol to soybean oil, and added water content on percentage weight conversion to soybean oil methyl ester by transesterification. Based on ridge max analysis, the optimum synthesis conditions giving 92.2% weight conversion were: reaction time 6.3 h, temperature 36.5 degrees C, enzyme amount 0.9 BAUN (Batch Acidolysis Units NOVO), substrate molar ratio 3.4:1, and added water 5.8%.     

Optimization of immobilized Candida rugosa lipase LIP2-catalyzed resolution to produce l-menthyl acetate

Esterification of l-menthol by lipase is a highly selective method for the resolution of dl-menthol. The present work focuses on the reaction parameters that affect lipase-catalyzed synthesis of l-menthyl acetate in n-hexane using triacetin as acyl donor. Genetically engineered LIP2, an isoform of Candida rugosa lipase, was used as a biocatalyst in the present study. The main objectives of the work were to develop an approach that would enable a better understanding of relationships between the variables (reaction time, temperature, enzyme amount, substrate molar ratio) and the response (molar conversion) for l-menthyl acetate synthesis, and to obtain the optimum conditions for synthesis. By using central composite rotatable design (CCRD) and response surface methodology (RSM) analysis, we found that substrate molar ratio and enzyme amount were the most important variables for the reaction. Based on ridge max analysis, the optimum synthesis conditions were found to be: reaction time 2.2 days, temperature 34.3°C, enzyme amount 0.09 g and substrate molar ratio (dl-menthol:triacetin) 1:1.9, and molar conversion of dl-menthol to l-menthyl acetate was calculated to be 50%. An experiment under optimum conditions was carried out and molar conversion of 48.3% was obtained.     

Optimization of enzymatic synthesis of L-ascorbyl palmitate by solvent engineering and statistical experimental designs

Statistical experimental designs combined with solvent engineering for optimization of enzymatic synthesis of L-ascorbyl palmitate were developed. First, the composition of the solvent for co-dissolving polar and apolar substrates was determined. The co-solvent mixture of tert-pentanol: DMSO at a ratio of 9:1 (v/v) and the optimal biocatalyst were obtained. Then, the Plackett-Burman design was implemented to screen the variables that significantly influence the conversion. The method of steepest ascent was used to approach the proximity of optimum. After determining the Plackett-Burman and steepest ascent designs, the optimum values were determined by central composite design under response surface methodology. The statistical analysis showed that the optimum reaction conditions (temperature 50°C, enzyme concentration 5.8 g/L, and substrate molar ratio 11:1, stirring rate 160 rpm, amount of molecular sieve 50 g/L, time 18 h) led to the maximum conversion (66.44%) and production concentration (20.63 g/L). A very satisfactory conversion (64.74%) and production concentration (20.13 g/L) could be achieved in short time (6 h).     

Optimized synthesis of lipase-catalyzed l-ascorbyl laurate by Novozym® 435

l-Ascorbyl laurate is a fatty acid derivative of l-ascorbic acid which can be widely used as a natural antioxidant in both lipid containing food and cosmetic applications. To avoid any possible harmful effects from chemically synthesized product, the enzymatic synthesis appears to be the best way to satisfy the consumer demand for natural antioxidants. The ability of immobilized lipase from Candida antarctica (Novozym^® 435) to catalyze the direct esterification between l-ascorbic acid and lauric acid was investigated. Response surface methodology (RSM) and 5-level-4-factor central composite rotatable design (CCRD) were employed to evaluate the effects of synthesis parameters, such as reaction time (2–10 h), temperature (25–65 °C), enzyme amount (10–50% w/w of l-ascorbic acid), and substrate molar ratio of l-ascorbic acid to lauric acid (1:1–1:5) on percentage molar conversion to l-ascorbyl laurate. Based on the analysis result of ridge max, the optimal enzymatic synthesis conditions were predicted as follows: reaction time 6.7 h, temperature 30.6 °C, enzyme amount 34.5%, substrate molar ratio 1:4.3; and the optimal actual yield was 93.2%.     

Optimized enzymatic synthesis of ascorbyl esters from lard using Novozym 435 in co-solvent mixtures

A mild and efficient method for the conversion of fatty acid methyl esters from lard into ascorbyl esters via lipase-catalyzed transesterification in co-solvent mixture is described. A solvent engineering strategy was firstly applied to improve fatty acid ascorbyl esters production. The co-solvent mixture of 30% t-pentanol:70% isooctane (v/v) was optimal. Response surface methodology (RSM) and central composite design (CCD) were employed to estimate the effects of reaction parameters, such as reaction time (12–36 h), temperature (45–65 °C), enzyme amount (10–20%, w/w, of fat acid methyl esters), and substrate molar ratio of fatty acid methyl esters to ascorbic acid (8:1–12:1) for the synthesis of fatty acid ascorbyl esters in co-solvent mixture. Based on the RSM analysis, the optimal reaction conditions were determined as follows: reaction time 34.32 h, temperature 54.6 °C, enzyme amount 12.5%, substrate molar ratio 10.22:1 and the maximum conversion of fatty acid ascorbyl esters was 69.18%. The method proved to be applicable for the synthesis of ascorbyl esters using Novozym 435 in solvent.     

Optimization of lipase-catalyzed synthesis of caffeic acid phenethyl ester in ionic liquids by response surface methodology

Lipase-catalyzed caffeic acid phenethyl ester (CAPE) synthesis in ionic liquid, 1-ethyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ([Emim][Tf₂N]), was investigated in this study. The effects of several reaction conditions, including reaction time, reaction temperature, substrate molar ratio of phenethyl alcohol to caffeic acid (CA), and weight ratio of enzyme to CA, on CAPE yield were examined. In a single parameter study, the highest CAPE yield in [Emim][Tf₂N] was obtained at 70 °C with a substrate molar ratio of 30:1 and weight ratio of enzyme to CA of 15:1. Based on these results, response surface methodology (RSM) with a 3-level-4-factor central composite rotatable design (CCRD) was adopted to evaluate enzymatic synthesis of CAPE in [Emim][Tf₂N]. The four major factors were reaction time (36–60 h), reaction temperature (65–75 °C), substrate molar ratio of phenethyl alcohol to CA (20:1–40:1), and weight ratio of enzyme to CA (10:1–20:1). A quadratic equation model was used to analyze the experimental data at a 95 % confidence level (p < 0.05). A maximum conversion yield of 99.8 % was obtained under the optimized reaction conditions [60 h, 73.7 °C, substrate molar ratio of phenethyl alcohol to CA (27.1:1), and weight ratio of enzyme to CA (17.8:1)] established by our statistical method, whereas the experimental conversion yield was 96.6 ± 2 %.     

Optimization of lipase-catalyzed synthesis of acetylated EGCG by response surface methodology

(−)-Epigallocatechin-3-O-gallate (EGCG) acetylated derivatives, which can be widely used as a natural antioxidant in both lipid containing food and cosmetic applications, were prepared by lipase catalyzed acylation of EGCG with vinyl acetate. Response surface methodology (RSM) and 5-level-4-factor central composite rotatable design (CCRD) were employed to evaluate the effects of synthesis parameters, such as reaction time (6–10 h), temperature (30–50 °C), enzyme amount (1.5–2.5% (w/w) of substrate), and substrate molar ratio of EGCG to vinyl acetate (0.5–1.5) on conversion of EGCG. By using multiple regression analysis, the experimental data were fitted to a second order polynomial model. The most suitable combination of variables was 40 °C, 2.12%, 10 h and 1.13 for the reaction temperature, the enzyme amount, the reaction time, and EGCG/vinyl acetate mole ratio, respectively. At these optimal conditions, the conversion yield reached 87.37%. The presence of mono-, di- and tri-acetylated derivatives in acetylated EGCG was confirmed by LC–MS-MS and identified as 5″-O-acetyl-EGCG, 3″, 5″-2-O-acetyl-EGCG and 5′, 3″, 5″-3-O-acetyl-EGCG by NMR.     

Study on response surface methodology (RSM) of lipase-catalyzed synthesis of palm-based wax ester

The synthesis of wax ester using refined, bleached and deodorized (RBD) palm oil and oleyl alcohol catalyzed by lipozyme IM was carried out. Response surface methodology (RSM) based on a five-level, four-variable central composite rotatable design (CCRD) was used to evaluate the interactive effects of synthesis, of reaction time (2.5–10 h), temperature (30–70 °C), amount of enzyme (0.1–0.2 g) and substrate molar ratio (palm oil to oleyl alcohol, 1:1–1:5) on the percentage yield of wax esters. The optimum conditions derived via RSM were: reaction time 7.38 h, temperature 53.9 °C, amount of enzyme 0.149 g, and substrate molar ratio 1:3.41. The actual experimental yield was 84.6% under optimum condition, which compared well to the maximum predicted value of 85.4%.     

Kinetics of lipase-catalysed methyl gallate production in the presence of deep eutectic solvent

Production of methyl gallate (MG), which is an important phenolic acid ester for pharmaceutical industry, was carried out by Novozym 435-catalysed transesterification of propyl gallate (PG) with methanol in a deep eutectic solvent. Reaction parameters governing substrate molar ratio, enzyme concentration, temperature and agitation rate were investigated batch-wise in choline chloride:glycerol-water binary mixture. The results were evaluated in terms of conversion of PG, yield of MG and hydrolysis of PG to gallic acid. 10% (w/w) of water was found to be favourable in the reaction medium for low hydrolysis percent. The highest conversion (17.4%) and yield (60.4%) but the lowest hydrolysis (2%) after 120?h of transesterification were found at PG/methanol molar ratio of 1:6, enzyme concentration of 40?g/L, 50?°C and 200?rpm. A kinetic model based on the Ping-Pong Bi–Bi mechanism for transesterification of PG was proposed with the assumption that there were no internal and external mass transfer resistances.     

Enzymatic production of linalool esters in organic and solvent-free system

This work investigated the influence of temperature, enzyme concentration, substrates molar ratio, in the absence and presence of organic solvent, at two molar ratios of the substrates on the enzymatic production of linalil esters using the immobilized lipase Novozym 435 as catalyst, different acids and linalool and Ho-Sho essential oil as substrates. The best reaction conversion was obtained at the highest temperature (70 °C), for both solvent free (3.81%) and with solvent addition (2.25%), for a solvent to substrates molar ratio of 2:1, enzyme concentration of 5 wt% and acid to alcohol molar ratio of 1:1. The reaction kinetics revealed that Ho-Sho essential oil afforded the greatest conversions when compared with pure linalool. Higher linalil esters production were achieved after 10 h reaction (5.58%) in 2:1 solvent to substrates molar ratio, with enzyme concentration of 5 wt%, at 70 °C and anhydride to alcohol molar ratio of 1:1 using Ho-Sho essential oil as substrate.     

Enzymatic esterification of beta-methylglucoside with acrylic/methacrylic acid in organic solvents

The enzymatic esterifications of beta-methylglucoside with acrylic acid/methacrylic acid were carried out using Novozym 435. t-Butanol indicating the highest conversion value was determined as an optimal solvent. The molar ratio (beta-methylglucoside:acids) of 1:15 was most favorable to the esterification. The enzyme concentration of 5% (w/v), and the temperature (50 degrees C for beta-methylglucoside:acrylic acid, 45 degrees C for beta-methylglucoside:methacrylic acid) resulted in the highest final conversion. Beta-methylglucoside of 60gl(-1) was found to be most effective in terms of short reaction time as well as product concentrations. Under these conditions, the maximum conversions for the esterification of beta-methylglucoside with acrylic acid and beta-methylglucoside with methacrylic acid were 59.3% after 12h and 71.3% after 72h, respectively. The structural analysis of the products was performed by FT-IR spectroscopy and (1)H NMR.     

Assessment of process variables on 2-ethylhexyl palmitate production using Novozym 435 as catalyst in a solvent-free system

This work reports the optimization of 2-ethylhexyl palmitate production by esterification reaction in a solvent-free system using a commercial lipase as catalyst. For this, a sequential strategy was performed applying three experimental designs. An empirical model was built so as to assess the effects of process variables on the reaction conversion. Afterward, the operating conditions that optimized 2-ethylhexyl palmitate production were determined to be acid to alcohol molar ratio of 1:5.5, 70 °C, 150 rpm and 10.5 wt% of enzyme, leading to a reaction conversion as high as 93%. From this point, a kinetic study was carried out evaluating the influence of acid to alcohol molar ratio, enzyme concentration and temperature on product yield. Results obtained in this step allow to conclude that an excess of alcohol (acid to alcohol molar ratio of 1:6), relatively low enzyme concentration (10 wt%) and temperature of 70 °C led to nearly complete reaction conversion.     

Modeling and optimization of lipase-catalyzed production of succinic acid ester using central composite design analysis

Esterification of succinic acid with oleyl alcohol catalyzed by immobilized Candida antarctica lipase B (Novozym 435) was investigated in this study. Response surface methodology (RSM) based on a five-level, four-variable central composite design (CCD) was used to model and analyze the reaction. A total of 21 experiments representing different combinations of the four parameters including temperature (35–65°C), time (30–450 min), enzyme amount (20-400 mg), and alcohol:acid molar ratio (1:1-8:1) were generated. A partial cubic equation could accurately model the response surface with a R² of 0.9853. The effect and interactions of the variables on the ester synthesis were also studied. Temperature was found to be the most significant parameter that influenced the succinate ester synthesis. At the optimal conditions of 41.1°C, 272.8 min, 20 mg enzyme amount and 7.8:1 alcohol:acid molar ratio, the esterification percentage was 85.0%. The model can present a rapid means for estimating the conversion yield of succinate ester within the selected ranges.     

β-galactosidase-catalyzed synthesis of 3-O-β-D-galactopyranosyl-sn-glycerol: Optimization by response surface methodology

In this study, the synthesis of 3-O-β-D-galactopyranosyl-sn-glycerol (GG) was performed by the reverse hydrolysis of D-galactose and glycerol using β-galactosidase from Kluyveromyces lactis. Four process variables, reaction temperature (30.0–45.0?°C), reaction time (24–48?h), enzyme concentration (150.00–350.00?U/mL), and substrate molar ratio (glycerol:D-galactose, 7.5:12.5?mmol/mmol) were investigated and optimized via response surface methodology (RSM) for optimal GG synthesis. Both quadratic equations and the optimal reaction conditions were established. Results showed that the four variables, i.e., reaction temperature, reaction time, enzyme concentration, and substrate molar ratio had significant (p?β-galactosidase concentration and 8.65:1.00 of substrate molar concentration ratio (glycerol: D-galactose) at 39.8?°C and 48?h of reaction. Under these conditions, the GG concentration was 140.03?g/L and GG yield was 55.71%, which both were close to the predicted values (143.26?g/L and 56.73%). This finding proves the RSM to be a useful tool in optimizing process conditions for GG synthesis.     

An efficient system for the asymmetric acylation of (R,S)-3-n-butylphthalide catalyzed by novozyme 435

Novozyme 435 could be a highly efficient catalyst in the asymmetric acylation of (R,S)-3-n-butylphthalide in tetrahydrofuran-hexane solvents. The effect of various reaction parameters such as agitation velocity, water content, mixed media, temperature, concentration of Novozyme 435, molar ratio of acetic anhydride to (R,S)-3-n-butylphthalide, reaction time, enantiomeric excess of substrate (ee(S)), enantiomeric excess of product (ee(P)), and enantioselective ratio (E) were studied. Tetrahydrofuran markedly improved (R,S)-3-n-butylphthalide conversion, enantiomeric excess of remaining 3-n-butylphthalide, and enantiomeric ratio. The optimum media were 50% (v/v) tetrahydrofuran and 50% (v/v) hexane. Other ideal reaction conditions were an agitation velocity of 150 rpm, 0.4% (v/v) water content, temperature of 30 °C, 8 mg/mL dosage of Novozyme 435, 8:1 (0.4 mmol: 0.05 mmol) molar ratio of acetic anhydride to (R,S)-3-n-butylphthalide, and a reaction time of 48 hr. Under the optimum conditions, 96.4% ee(S) and 49.3% conversion of (R,S)-3-n-butylphthalide were achieved. In addition, enantiomeric excess of the product was above 98.0%.     

Optimization and kinetic study of immobilized lipase-catalyzed synthesis of ethyl lactate

Abstract

Enzymatic synthesis of ethyl lactate catalyzed by immobilized lipase has been investigated. The reaction variables (including the molar ratio of ethanol to acid, total substrate amount, temperature, reaction time and rotation speed) were selected in accordance with the Plackett–Burman design and were further optimized via response surface methodology. The molar ratio of ethanol to acid, total substrate amount and reaction time were screened out as significant variables for the optimization study. A 20-run, full-factorial, central composite design was used to construct the statistical model and the optimal conditions obtained were as follows: molar ratio of ethanol to acid of 8.3:1, total substrate amount of 0.4 g, reaction time of 26.87 h with temperature of 55°C and rotation speed of 150 rpm. Under the optimal conditions, the yield of ethyl lactate was up to 24.32%; close to the 25.13% obtained using the commercial lipase, Novozym 435. Due to the low cost and simple immobilization process, the lipase prepared in the present work could have great potential in enzymatic applications. Additionally, a kinetic model with inhibition by both ethanol and lactic acid following a ping-pong bi-bi mechanism was proposed.     

Optimisation and Characterisation of Lipase-Catalysed Synthesis of a Kojic Monooleate Ester in a Solvent-Free System by Response Surface Methodology

Kojic acid is widely used to inhibit the browning effect of tyrosinase in cosmetic and food industries. In this work, synthesis of kojic monooleate ester (KMO) was carried out using lipase-catalysed esterification of kojic acid and oleic acid in a solvent-free system. Response Surface Methodology (RSM) based on central composite rotatable design (CCRD) was used to optimise the main important reaction variables, such as enzyme amount, reaction temperature, substrate molar ratio, and reaction time along with immobilised lipase from Candida Antarctica (Novozym 435) as a biocatalyst. The RSM data indicated that the reaction temperature was less significant in comparison to other factors for the production of a KMO ester. By using this statistical analysis, a quadratic model was developed in order to correlate the preparation variable to the response (reaction yield). The optimum conditions for the enzymatic synthesis of KMO were as follows: an enzyme amount of 2.0 wt%, reaction temperature of 83.69°C, substrate molar ratio of 1:2.37 (mmole kojic acid:oleic acid) and a reaction time of 300.0 min. Under these conditions, the actual yield percentage obtained was 42.09%, which is comparably well with the maximum predicted value of 44.46%. Under the optimal conditions, Novozym 435 could be reused for 5 cycles for KMO production percentage yield of at least 40%. The results demonstrated that statistical analysis using RSM can be used efficiently to optimise the production of a KMO ester. Moreover, the optimum conditions obtained can be applied to scale-up the process and minimise the cost.     

Enzymatic synthesis of benzyl benzoate using different acyl donors: Comparison of solvent-free reaction techniques

In this study, benzyl benzoate was successfully synthesized via enzymatic acylation using three immobilized enzymes as biocatalysts. Different acyl donors (benzoic acid and benzoic anhydride), operation regimes (batch, fed-batch), mixing modes (conventional mechanical stirring and ultrasound), process parameters (temperature, substrate molar ratio of acyl donor to acyl acceptor), presence or absence of solvents, enzyme amount and type were evaluated. Benzoic acid is a solid that is difficult to solubilize and, thus, was not efficient as acyl donor for the synthesis of benzyl benzoate. On the other hand, benzoic anhydride was very effective for the acylation of benzyl benzoate, and the presence of an excess of benzyl alcohol was essential to ensure the solute-solvent intermolecular attractions and good substrate solubilization, allowing the ester synthesis to be performed in the absence of organic solvents. The ultrasound was effective in increasing increase the initial reaction rate and the final conversion (88 %). However, the Lipozyme TL-IM and RM-IM supports were damaged, and the reuse was unfeasible. The batch and fed-batch approaches in conventional stirring ensured high conversions of 92 and 90 %, respectively, for batch (anhydride: alcohol 1:6) and fed-batch (1:3) using the Lipozyme TL-IM as biocatalyst. The controlled addition of the anhydride (fed-batch) allowed the reduction of alcohol molar ratio but decreased the reaction rates, and the maximum conversions were reached only after 24 h, while the batch approach had 92 % of conversion after 6 h. The yield of benzyl benzoate was high at 6 wt.% of enzyme, low temperature (50 °C), and simple reactor operation (batch). Results show the feasibility of the synthesis of benzyl benzoate via acylation using a green process that may be an alternative route to the chemical synthesis.     

Enzymatic synthesis of betulinic acid ester as an anticancer agent: Optimization study

Abstract

Immobilized Candida antarctica lipase, Novozym 435, was used to catalyze the esterification reaction between betulinic acid and phthalic anhydride to synthesize 3-O-phthalyl betulinic acid in n-hexane/chloroform. Response surface methodology based on a five-level, four-variable central composite rotatable design was employed to evaluate the effects of synthesis parameters such as reaction time, reaction temperature, enzyme amount and substrate molar ratio on the yield of ester. Based on the response surface model, the optimal enzymatic synthesis conditions were predicted to be: reaction time 20.3 h, reaction temperature 53.9°C, enzyme amount 145.6 mg, betulinic acid to phthalic anhydride molar ratio 1:1.11. The predicted yield was 65.8% and the actual yield was 64.7%.