Different enzyme requirements for the synthesis of biodiesel: Novozym 435 and Lipozyme TL IM

Enzymatic syntheses of biodiesel via alcoholysis of different vegetable oils (sunflower, borage, olive and soybean) have been studied. Loss of lipase activity induced by the nucleophile is greater with methanol than with ethanol, and is greater for Lipozyme TL IM than for Novozym 435. The optimum volume of ethanol depends on the loading of solid biocatalyst and is higher for preparations of Novozym 435 than for Lipozyme TL IM. Maximum rates were obtained with Lipozyme TL IM, for a molar ratio of alcohol to FA residues of 0.33. By contrast, Novozym 435 requires at least a 2:1 ratio. Alcoholysis of the vegetable oils is faster with Lipozyme TL IM than with Novozym 435. Use of a high loading of Novozym 435 (50% w/w) and a large molar excess of ethanol are required to obtain an initial rate similar to that obtained with Lipozyme TL IM at a lower enzyme loading (10% w/w) and an equimolar ratio of ethanol and FA residues. Novozym 435 produces quantitative conversions in only 7h at 25 degrees C, but complete conversions are not obtained with Lipozyme TL IM. Three stage stepwise addition of ethanol yields 84% conversion to ethyl esters for Lipozyme TL IM. Hence use of Novozym 435 is preferred. After nine cycles in a batch reactor Novozym 435 retained 85% of its initial activity.     

Enzymatic Synthesis of Cinnamic Acid Derivatives

Using Novozym 435 as catalyst, the syntheses of ethyl ferulate (EF) from ferulic acid (4-hydroxy 3-methoxy cinnamic acid) and ethanol, and octyl methoxycinnamate (OMC) from p-methoxycinnamic acid and 2-ethyl hexanol were successfully carried out in this study. A conversion of 87% was obtained within 2 days at 75 °C for the synthesis of EF. For the synthesis of OMC at 80 °C, 90% conversion can be obtained within 1 day. The use of solvent and high reaction temperature resulted in better conversion for the synthesis of cinnamic acid derivatives. Some cinnamic acid esters could also be obtained with higher conversion and shorter reaction times in comparison to other methods reported in the literature. The enzyme can be reused several times before significant activity loss was observed. Revisions requested 10 January 2006; Revisions received 17 January 2006     

Kinetics of enzymatic synthesis of L-ascorbyl acetate by Lipozyme TLIM and Novozym 435

L-ascorbyl acetate was synthesized through lipase-catalyzed esterification using Lipozyme TLIM and Novozym 435. Four solvents, including methanol, ethanol, acetonitrile, and acetone were investigated for the reaction, and acetone and acetonitrile were found to be suitable reaction media. The influences of several parameters such as water activity (a _w), substrate molar ratio, enzyme loading, and reaction temperature on esterification of L-ascorbic acid were systematically and quantitatively analyzed. Through optimizing the reaction, lipase-catalyzed esterification of L-ascorbic acid gave a maximum conversion of 99%. The results from using Lipozyme TLIM and Novozym 435 as biocatalysts both showed that a _w was an important factor for the conversion of L-ascorbic acid. The effect of pH value on lipase-catalyzed L-ascorbic acid esterification in acetone was also investigated. Furthermore, results from a kinetic characterization of Lipozyme TLIM were compared with those for Novozym 435, and suggested that the maximum reaction rate for Lipozyme TLIM was greater than that for Novozym 435, while the enzyme affinity for substrate was greater for Novozym 436.     

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.     

Immobilization of lipase B from Candida antarctica on porous styrene-divinylbenzene beads improves butyl acetate synthesis

A new biocatalyst of lipase B from Candida antarctica (MCI-CALB) immobilized on styrene-divinylbenzene beads (MCI GEL CHP20P) was compared with the commercial Novozym 435 (immobilized lipase) in terms of their performances as biocatalysts for the esterification of acetic acid and n-butanol. The effects of experimental conditions on reaction rates differed for each biocatalyst, showing different optimal values for water content, temperature, and substrate molar ratio. MCI-CALB could be used at higher acid concentrations, up to 0.5 M, while Novozym 435 became inactivated at these acid concentrations. Although Novozym 435 exhibited 30% higher initial activity than MCI-CALB for the butyl acetate synthesis, the reaction course was much more linear using the new preparation, meaning that the MCI-CALB allows for higher productivities per cycle. Both preparations produced around 90% of yield conversions after only 2 h of reaction, using 10% (mass fraction) of enzyme. However, the main advantage of the new biocatalyst was the superior performance during reuse. While Novozym 435 was fully inactivated after only two batches, MCI-CALB could be reused for six consecutive cycles without any washings and keeping around 70% of its initial activity. It is proposed that this effect is due to the higher hydrophobicity of the new support, which does not retain water or acid in the enzyme environment. MCI-CALB has shown to be a very promising biocatalyst for the esterification of small-molecule acids and alcohols.     

Enzymatic coproduction of biodiesel and glycerol carbonate from soybean oil and dimethyl carbonate

The enzymatic coproduction of biodiesel and glycerol carbonate by the transesterification of soybean oil was studied using lipase as catalyst in organic solvent. To produce biodiesel and glycerol carbonate simultaneously, experiments were designed sequentially. Enzyme screening, the molar ratio of dimethyl carbonate (DMC) to soybean oil, reaction temperature and solvent effects were investigated. The results of enzyme screening, at 100 g/L Novozym 435 (immobilized Candida antarctica lipase B), biodiesel and glycerol carbonate showed conversions of 58.7% and 50.7%, respectively. The optimal conditions were 60 °C, 100 g/L Novozym 435, 6.0:1 molar ratio with tert-butanol as solvent: 84.9% biodiesel and 92.0% glycerol carbonate production was achieved.     

Continuous lipase-catalyzed esterification of soybean fatty acids under ultrasound irradiation

This work investigates the continuous production of alkyl esters from soybean fatty acid (FA) charges using immobilized Novozym 435 as catalyst. The experiments were performed in a packed-bed bioreactor evaluating the effects of FA charge to alcohol (methanol and ethanol) molar ratio, from 1:1 to 1:6, substrate flow rate in the range of 0.5–2.5 mL/min and output irradiation power up to 154 W, at fixed temperature of 65 °C, on the reaction conversion. Results showed that almost complete conversions to fatty acids ethyl esters were achieved at mild ultrasonic power (61.6 W), FA to ethanol molar ratio of 1:6, operating temperature (65 °C) and remained nearly constant for long-term reactions without negligible enzyme activity losses.     

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.     

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.     

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%.     

Improved enzymatic production of phenolated acylglycerols through alkyl phenolate intermediates

A novel approach is reported for the synthesis of dihydrocaffoylated glycerols that consists of two steps: enzymatic synthesis of octyl dihydrocaffeate (as a synthetic intermediate) from octanol and dihydrocaffeic acid, and enzymatic interesterification of triacylglycerols with octyl dihydrocaffeate. Due to the good compatibility of the intermediate with triacylglycerols, an improved volumetric productivity [147 mol h⁻¹(kg Novozym 435)⁻¹] and high enzyme specific activity [up to 9.6 μmol⁻¹ min⁻¹(g Novozym 435)⁻¹] have been obtained.     

Ultrasonic irradiation with vibration for biodiesel production from soybean oil by Novozym 435

The production of biodiesel with soybean oil and methanol through transesterification by Novozym 435 (Candida antarctica lipase B immobilized on polyacrylic resin) were conducted under two different conditions—ultrasonic irradiation and vibration to compare their overall effects. Compared with vibration, ultrasonic irradiation significantly enhanced the activity of Novozym 435. The reaction rate was further increased under the condition of ultrasonic irradiation with vibration (UIV). Effects of reaction conditions, such as ultrasonic power, water content, organic solvents, ratio of solvent/oil, ratio of methanol/oil, enzyme dosage and temperature on the activity of Novozym 435 were investigated under UIV. Under the optimum conditions (50% of ultrasonic power, 50 rpm vibration, water content of 0.5%, tert-amyl alcohol/oil volume ratio of 1:1, methanol/oil molar ratio of 6:1, 6% Novozym 435 and 40 °C), 96% yield of fatty acid methyl ester (FAME) could be achieved in 4 h. Furthermore, repeated use of Novozym 435 after five cycles showed no obvious loss in enzyme activity, which suggested this enzyme was stable under the UIV condition. These results indicated that UIV was a fast and efficient method for biodiesel production.     

Ultrasound-assisted enzymatic transesterification of methyl benzoate and glycerol to 1-glyceryl benzoate in organic solvent

The aim of this work is to report the enzymatic transesterification production of 1-glyceryl benzoate under ultrasound irradiation, using a commercial immobilized lipase, Novozym 435. Firstly, a preliminary evaluation was carried out at 2, 4 and 6h, at constant temperature of 50 °C, methyl benzoate to glycerol molar ratio of 1:1 and 5.5 wt% of enzyme concentration. After analyzing the results obtained, the experimental design technique was used to evaluate the effects of temperature, substrates molar ratio, enzyme concentration, solvent volume and ultrasonic power on the 1-glyceryl benzoate production. The highest conversion, around 16%, was obtained at 65 °C, 1:1 of methyl benzoate to glycerol molar ratio, 15 wt% of enzyme concentration, 7 mL of solvent and 40% ultrasonic power in 4h of reaction. A preliminary kinetic experiment carried out varying the enzyme concentration (15 and 20 wt%) keeping fixed the temperature at 35 °C, 1:1 of substrates molar ratio, 3 mL of solvent and 40% of maximum ultrasonic power led to lower (around 15% after 12 h of reaction) conversions compared to that achieved in the experimental design.     

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.     

Reactor Design for the Novozym 435 ® -catalysed Enantioselective Esterification of 4-methyloctanoic Acid

The bench scale Novozym 435 ® catalysed esterification of 4-methyloctanoic acid with ethanol was studied at 35°C. Esterification in a batch reactor (molar ratio of 1:8 (acid:EtOH)) resulted in the isolation of the enantiomerically enriched product (ee p =81%) and substrate (ee s =93%). In order to integrate reaction and separation, liquid-vapour equilibria calculations were performed showing that an excess of ethanol results in a very low ester fraction in the vapour phase. Since this is undesirable for an integrated process of reaction and product removal, a repeated batch reaction was performed using a molar ratio of 10:1 (acid:EtOH). After six cycles (45% conversion) the ee of 4-methyloctanoic acid ethyl ester turned out to be 80%. For different E values the ee p was calculated for batch and repeated batch reactions. It was shown that in all cases the ee p was higher for the repeated batch reaction. However, the product is not enantiopure since the E value of the reaction is rather low at the low ethanol concentration used. An alternative approach would be the continuous separation of the product during the reaction. A mathematical model was developed to describe esterification in a packed bed reactor integrated with product separation. This model shows that integration of reaction and product removal in advance is not suitable either to obtain an enantiomerically pure product. Since the optimal reaction conditions (high ethanol concentration) and the optimal separation system (low ethanol concentration) do not match in this reaction, the preference is given to the batch reaction at high ethanol concentrations because in that case the highest enantioselectivity of the enzyme is obtained.     

Reactor Design for the Novozym 435 ® -catalysed Enantioselective Esterification of 4-methyloctanoic Acid

The bench scale Novozym 435 ® catalysed esterification of 4-methyloctanoic acid with ethanol was studied at 35°C. Esterification in a batch reactor (molar ratio of 1:8 (acid:EtOH)) resulted in the isolation of the enantiomerically enriched product (ee p =81%) and substrate (ee s =93%). In order to integrate reaction and separation, liquid-vapour equilibria calculations were performed showing that an excess of ethanol results in a very low ester fraction in the vapour phase. Since this is undesirable for an integrated process of reaction and product removal, a repeated batch reaction was performed using a molar ratio of 10:1 (acid:EtOH). After six cycles (45% conversion) the ee of 4-methyloctanoic acid ethyl ester turned out to be 80%. For different E values the ee p was calculated for batch and repeated batch reactions. It was shown that in all cases the ee p was higher for the repeated batch reaction. However, the product is not enantiopure since the E value of the reaction is rather low at the low ethanol concentration used. An alternative approach would be the continuous separation of the product during the reaction. A mathematical model was developed to describe esterification in a packed bed reactor integrated with product separation. This model shows that integration of reaction and product removal in advance is not suitable either to obtain an enantiomerically pure product. Since the optimal reaction conditions (high ethanol concentration) and the optimal separation system (low ethanol concentration) do not match in this reaction, the preference is given to the batch reaction at high ethanol concentrations because in that case the highest enantioselectivity of the enzyme is obtained.     

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.     

超声波辅助下脂肪酶催化高酸值废油脂制备生物柴油

探讨了超声波辅助条件下脂肪酶催化高酸值废油脂转化为生物柴油的反应。来源于Aspergillus oryzae和Candida antarctica的固定化脂肪酶,在超声波辅助下,对高酸值废油脂转化为生物柴油具有高的催化活性。以来自于C.antarctica的固定化脂肪酶Novozym435为催化剂,以酸价为157mg KOH/g的高酸值废油脂为原料在超声波辅助下与丙醇反应,在脂肪酶用量为油质量的8%、初始醇油摩尔比为3∶1、反应温度控制在40~45℃、超声波频率和功率分别采用28kHz和100W的条件下,反应50min转化率达到94.86%。在此条件下,不同碳原子数(C1~C5)的直链和支链醇均有较高的转化率,在短链醇的选择上具有宽广的适应性。超声波还减少了反应产物和反应体系中其他黏性杂质在固定化脂肪酶表面的吸附,回收的Novozym435相较单纯机械搅拌条件下回收的外观干净、分散良好无结块现象、易于洗涤和再次利用,具有良好的操作稳定性。     

Optimization of ultrasound-accelerated synthesis of enzymatic octyl hydroxyphenylpropionate by response surface methodology

The ultrasound-accelerated enzymatic synthesis of octyl hydroxyphenylpropionate (OHPP) from p-hydroxyphenylpropionic acid (HPPA) and octanol was investigated in this study. A commercially available immobilized lipase from Candida antarctica, Novozym 435, was used as the biocatalyst. A three-level-three-factor Box-Behnken design experiment and response surface methodology were used to evaluate the effects of temperature, reaction time, and enzyme activity on percent yield of OHPP. The results indicated that temperature and enzyme activity significantly affected percent yield, whereas reaction time did not. A model for the synthesis of OHPP was established. Based on a ridge max analysis, the optimum conditions for OHPP synthesis were predicted to use a reaction temperature of 58.8°C, a reaction time of 14.6 h, and an enzyme activity of 410.5 PLU with a yield of 98.5%. A reaction was performed under these optimal conditions, and a yield of 97.5% ± 0.1% was obtained.