High yield lipase-catalyzed synthesis of Engkabang fat esters for the cosmetic industry

Engkabang fat esters were produced via alcoholysis reaction between Engkabang fat and oleyl alcohol, catalyzed by Lipozyme RM IM. The reaction was carried out in a 500 ml Stirred tank reactor using heptane and hexane as solvents. Response surface methodology (RSM) based on a four-factor-five-level Central composite design (CCD) was applied to evaluate the effects of synthesis parameters, namely temperature, substrate molar ratio (oleyl alcohol: Engkabang fat), enzyme amount and impeller speed. The optimum yields of 96.2% and 91.4% were obtained for heptane and hexane at the optimum temperature of 53.9 °C, impeller speeds of 309.5 and 309.0 rpm, enzyme amounts of 4.82 and 5.65 g and substrate molar ratios of 2.94 and 3.39:1, respectively. The actual yields obtained compared well with the predicted values of 100.0% and 91.5%, respectively. Meanwhile, the properties of the esters show that they are suitable to be used as ingredient for cosmetic applications.     

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.     

Effect of alcohol chain length on the optimum conditions for lipase-catalyzed synthesis of adipate esters

Immobilized Candida antarctica lipase B, Novozym® 435, was used in the esterification of adipic acid and alcohols with different chain lengths (C₁–C₁₈). Optimum conditions for the synthesis of adipate esters were obtained using response surface methodology (RSM) with respect to important reaction parameters including time, temperature, substrate molar ratio and amount of enzyme. Alcohol chain length specificity of the enzyme in the synthesis of adipate esters was also determined. Minimum reaction time (215 min) for achieving maximum ester yield was obtained for butyl alcohol. Methanol required an increased time (358 min) and enzyme amount (10.2%, w/w) for attaining maximum yield. The maximum required temperature and time of 65°C and 523 min, respectively, were obtained for the synthesis of dioctadecyl adipate. The results demonstrate that alcohol chain length is a determining parameter in optimization of the lipase-catalyzed synthesis of adipate esters. Reactions under optimized conditions yielded a high percentage of esterification (>97%). The optimum conditions can be used to scale up the process.     

Lipase-catalyzed acylation of naringin with palmitic acid in highly concentrated homogeneous solutions

Acylation reactions of naringin with palmitic acid were performed by a lipase after formation of highly concentrated homogeneous solutions. Their initial naringin concentration was 840–950 mM, which is 20–60 times greater than that in organic solvent media. The overall productivity of highly concentrated solutions was more than 15 times greater than those of organic phase media. The addition of DMSO (20–40%, w/w) to substrate mixtures lowered the melting temperature of a naringin–palmitic acid mixture (1:1 molar ratio) to about 40 °C. Reactions at 80 °C apparently followed Michaelis–Menten kinetics despite extremely high substrate concentrations. As the temperature increased from 60 °C to 80 °C, the apparent viscosity of the highly concentrated solution decreased remarkably from 4.31 Pa s to 0.063 Pa s. An activation energy of 7.65 kcal/mol obtained in a range of 60–75 °C suggests a diffusion-control. On the other hand, an activation energy of 17.09 kcal/mol in a range of 75–90 °C indicates a reaction-control. The highest product conversion yield of 33% (mol/mol) was obtained in a 10 h reaction at 80 °C. Addition of activated molecular sieves to the highly concentrated solution increased the product conversion yield by 7% (mol/mol), suggesting that the original equilibrium was disrupted by removing water and then a new equilibrium was reached.     

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.     

Optimized enzymatic synthesis of geranyl butyrate with lipase AY from Candida rugosa

Response surface methodology (RSM) and five-level, five-variable central composite rotatable design (CCRD) were used to evaluate the effects of synthetic variables, such as reaction time (1-9 h), temperature (25-65 degrees C), enzyme amount (10-50%), substrate molar ratio of geraniol to tributyrin (1:0.33-1:1), and added water amount (0-20%) on molar percent yield of geranyl butyrate, using lipase AY from Candida rugosa. Reaction time and temperature were the most important variables and substrate molar ratio had no effect on percent molar conversion. Based on contour plots, optimum conditions were: reaction time 9 h, temperature 35 degrees C, enzyme amount 50%, substrate molar ratio 1:0.33, and added water 10%. The predicted value was 100% and actual experimental value was 96.8% molar conversion. (c) 1996 John Wiley & Sons, Inc.     

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 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 and kinetic study on the synthesis of palm oil ester using Lipozyme TL IM

Enzymatic synthesis of palm oil esters (POE) was carried out via alcoholysis of palm oil (PO) and oleyl alcohol (OA) catalyzed by Lipozyme TL IM. The optimum reaction conditions were: temperature: 60 °C; enzyme load: 24.7 wt%; substrate ratio: 1:3 (PO/OA), impeller speed: 275 rpm and reaction time: 3 h. At the optimum condition, the conversion of POE was 79.54%. Reusability study showed that Lipozyme TL IM could be used for 5 cycles with conversion above 50%. The alcoholysis reaction kinetic follows the Ping-Pong Bi-Bi mechanism characterized by the V_max, K_m(PO), and K_m(OA) values of 32.7 mmol/min, 0.3147 mmol/ml and 0.9483 mmol/ml, respectively. The relationship between initial reaction rate and temperature was also established based on the Arrhenius law.     

Transesterification of palm oil to methyl ester on activated carbon supported calcium oxide catalyst

In this study, methyl ester (ME) was produced by transesterification of palm oil (CPO) (cooking grade) using activated carbon supported calcium oxide as a solid base catalyst (CaO/AC). Response surface methodology (RSM) based on central composite design (CCD) was used to optimize the effect of reaction time, molar ratio of methanol to oil, reaction temperature and catalyst amount on the transesterification process. The optimum condition for CPO transesterification to methyl ester was obtained at 5.5 wt.% catalyst amount, 190 °C temperature, 15:1 methanol to oil molar ratio and 1 h 21 min reaction time. At the optimum condition, the ME content was 80.98%, which is well within the predicted value of the model. Catalyst regeneration studies indicate that the catalyst performance is sustained after two cycles.     

Enzymatic synthesis of biodiesel from palm oil assisted by microwave irradiation

Optimal conditions for enzymatic synthesis of biodiesel from palm oil and ethanol were determined with lipase from Pseudomonas fluorescens immobilized on epoxy polysiloxane–polyvinyl alcohol hybrid composite under a microwave heating system. The main goal was to reduce the reaction time preliminarily established by a process of conventional heating. A full factorial design assessed the influence of ethanol-to-palm oil (8:1–16:1) molar ratio and temperature (43–57 °C) on the transesterification yield. Microwave irradiations varying from 8 to 15 W were set up according to reaction temperature. Under optimal conditions (8:1 ethanol-to-oil molar ratio at 43 °C), 97.56 % of the fatty acids present in the palm oil were converted into ethyl esters in a 12-h reaction, corresponding to a productivity of 64.2 mg ethyl esters g^?1 h^?1. This represents a sixfold increase from the process carried out under conventional heating, thus proving to be a potential tool for enhancing biochemical modification of oils and fats. In general, advantages of the new process include: (1) microwaves speed up the enzyme-catalyzed reactions; (2) there are no destructive effects on the enzyme properties, such as stability and substrate specificity, and (3) the microwave assistance allows the entire reaction volume to be heated uniformly. These bring benefits of a low energy demand and a faster conversion of palm oil into biodiesel.     

Optimized lipase-catalyzed synthesis of adipate ester in a solvent-free system

Immobilized Candida antarctica lipase-catalyzed esterification of adipic acid and oleyl alcohol was investigated in a solvent-free system (SFS). Optimum conditions for adipate ester synthesis in a stirred-tank reactor were determined by the response surface methodology (RSM) approach with respect to important reaction parameters including time, temperature, agitation speed, and amount of enzyme. A high conversion yield was achieved using low enzyme amounts of 2.5% w/w at 60°C, reaction time of 438 min, and agitation speed of 500 rpm. The good correlation between predicted value (96.0%) and actual value (95.5%) implies that the model derived from RSM allows better understanding of the effect of important reaction parameters on the lipase-catalyzed synthesis of adipate ester in an organic solvent-free system. Higher volumetric productivity compared to a solvent-based system was also offered by SFS. The results demonstrate that the solvent-free system is efficient for enzymatic synthesis of adipate ester.     

Immobilization of thermostable β-glucosidase from Sulfolobus shibatae by cross-linking with transglutaminase

Thermostable β-glucosidase from Sulfolobus shibatae was immobilized on silica gel modified or not modified with 3-aminopropyl-triethoxysilane using transglutaminase as a cross-linking factor. Obtained preparations had specific activity of 3883 U/g of the support, when measured at 70 °C using o-nitrophenyl β-d-galactopyranoside (GalβoNp) as substrate. The highest immobilization yield of the enzyme was achieved at pH 5.0 in reaction media. The most active preparations of immobilized β-glucosidase were obtained at a transglutaminase concentration of 40 mg/ml at 50 °C. The immobilization was almost completely terminated after 100 min of the reaction and prolonged time of this process did not cause considerable changes of the activity of the preparations. The immobilization did not influence considerably on optimum pH and temperature of GalβoNp hydrolysis catalyzed by the investigated enzyme (98 °C, pH 5.5). The broad substrate specifity and properties of the thermostable β-glucosidase from S. shibatae immobilized on silica-gel indicate its suitability for hydrolysis of lactose during whey processing.     

Continuous biosynthesis of biodiesel from waste cooking palm oil in a packed bed reactor: optimization using response surface methodology (RSM) and mass transfer studies

This study aimed to develop an optimal continuous procedure of lipase-catalyzes transesterification of waste cooking palm oil in a packed bed reactor to investigate the possibility of large scale production further. Response surface methodology (RSM) based on central composite rotatable design (CCRD) was used to optimize the two important reaction variables packed bed height (cm) and substrate flow rate(ml/min) for the transesterification of waste cooking palm oil in a continuous packed bed reactor. The optimum condition for the transesterification of waste cooking palm oil was as follows: 10.53 cm packed bed height and 0.57 ml/min substrate flow rate. The optimum predicted fatty acid methyl ester (FAME) yield was 80.3% and the actual value was 79%. The above results shows that the RSM study based on CCRD is adaptable for FAME yield studied for the current transesterification system. The effect of mass transfer in the packed bed reactor has also been studied. Models for FAME yield have been developed for cases of reaction control and mass transfer control. The results showed very good agreement compatibility between mass transfer model and the experimental results obtained from immobilized lipase packed bed reactor operation, showing that in this case the FAME yield was mass transfer controlled.     

Biocatalytic properties of native and immobilized subtilisin 72 in aqueous-organic and low water media

Subtilisin 72 was immobilized on cryogel of poly(vinyl alcohol), the macroporous carrier prepared by the freeze-thaw-treatment of concentrated aqueous solution of the polymer. The obtained biocatalyst was active and stable in aqueous, aqueous-organic, as well as in low water media. The stability of immobilized biocatalyst was substantially higher than that of native enzyme in all mixtures especially in aqueous buffer containing 5–8 M Urea and in acetonitrile/60–90%DMF mixtures. The ability of native and immobilized subtilisin to catalyze peptide bond formation between Z-Ala-Ala-Leu-OMe and Phe-pNA was studied in non-aqueous media. Considerable enzyme stabilization in acetonitrile/90%DMF mixture, induced by the immobilization, resulted in higher product yield (57%) than in case of native subtilisin suspension (32%). Detailed study of synthesis reaction revealed that notable increase in product yield could be reached using increase in both substrate concentrations up to 200 mM.     

Characterization of sol–gel encapsulated lipase using tetraethoxysilane as precursor

Lipase from Candida rugosa was encapsulated within a chemically inert sol–gel support prepared by polycondensation of the precursor tetraethoxysilane (TEOS) in the presence of polyethylene glycol (PEG) as additive. The properties of silica and their derivatives with regard to mean pore diameter, specific surface area, mean pore size, weight loss upon heating (thermogravimetric analysis, TGA) and ²⁹Si and ¹³C NMR are reported. The pH optimum shifted from 7.8 to 6.7 and optimum temperature jumped from 36 to 60 °C upon enzyme encapsulation. Encapsulated lipase in presence of PEG (EN-PEG) exhibited higher stability in the range of 37–45 °C, but from 50 to 65 °C the EN-PEG was inactivated after seven cycles. Hydrolytic activity during long-term storage at room temperature decreased to 50% after 94 days. High diffusional resistance was observed for large oil concentration reducing hydrolytic effectiveness by 60% in the case of the encapsulated lipase. NMR, pore size and specific surface area data suggested an active participation of the lipase enzyme during gelling of the silica matrix. This lead to reduction of available Si–OH groups, larger pores and smaller surface area. Larger pores increase substrate diffusion that correlates well with higher hydrolytic activity of the TEOS–PEG sol–gel matrix encapsulated enzyme in comparison with other sol–gel supports.     

Synthesis of alkylgalactosides using whole cells of Bacillus pseudofirmus species as catalysts

Whole cells of alkaliphilic Bacillus pseudofirmus AR-199, induced for β-galactosidase activity, were used for the synthesis of 1-hexyl-β- -galactoside and 1-octyl-β- -galactoside, respectively, by transglycosylation reaction between lactose and the corresponding alcohol acceptor. The product yield was strongly influenced by the initial water content in the reaction mixture. Water content of 10% (v/v) was optimal providing 3.6–36 mM hexyl galactoside from 10 to 150 mM lactose, and no secondary product hydrolysis. Product yield could be enhanced by supplementing the reaction mixture with more cells or partly replacing the product with fresh substrate, but was decreased with time to the initial equilibrium level. Cell permeabilisation or disruption resulted in increased reaction rate and higher product yield but was followed by product hydrolysis. Octyl galactoside synthesis using whole cells was optimal at water content of 2% (v/v) with a yield of 26%. The cells were immobilised in cryogels of polyvinyl alcohol for use in continuous process, where hexyl galactoside was produced with a constant yield of 50% from 50 mM lactose for at least a week.     

Kinetic characterization of chiral biocatalysis of cycloarenes by the camphor 5-monooxygenase enzyme system

Redox enzyme mediated biocatalysis has the potential to regio- and stereo-specifically oxidize hydrocarbons producing valuable products with minimal by-product formation. In vitro reactions of the camphor (cytochrome P-450) 5-monooxygenase enzyme system with naphthalene-like substrates yield stereospecifically hydroxylated products from nonactivated hydrocarbons. Specifically, the enzyme system catalyzes the essentially stereospecific conversion of the cycloarene, tetralin (1,2,3,4-tetrahydronaphthalene) to (R)-1-tetralol ((R)-(−)-1,2,3,4-tetrahydro-1-naphthol). It is shown that this reaction obeys Michaelis–Menten kinetics and that interactions between the enzyme subunits are not affected by the identity of the substrate. This subunit independence extends to the efficiency of NADH usage by the enzyme system—subunit ratios do not effect efficiency, but substrate identity does. Tetralin is converted at an efficiency of 13±3%, whereas (R)-1-tetralol is converted at 7.8±0.7%. A model of this system based on Michaelis–Menten parameters for one subunit (Pdx: K_M=10.2±2 μM) and both substrates (tetralin: K_M=66±26 μM, ν_max=0.11±0.04 s⁻¹, and (R)-1-tetralol: K_M=2800±1300 μM, ν_max=0.83±0.22 s⁻¹) is presented and used to predict the consumption and production of all substrates, products and cofactors.     

Vinyl imidazole carrying metal-chelated beads for reversible use in yeast invertase adsorption

Poly(ethylene glycol dimethacrylate-n-vinyl imidazole) [poly(EGDMA–VIM)] hydrogel (average diameter 150–200 μm) was prepared copolymerizing ethylene glycol dimethacrylate (EGDMA) with n-vinyl imidazole (VIM). Poly(EGDMA–VIM) beads had a specific surface area of 59.8 m²/g. Poly(EGDMA–VIM) beads were characterized by swelling studies and scanning electron microscope (SEM). Cu²⁺ ions were chelated on the poly(EGDMA–VIM) beads (452 μmol Cu²⁺/g), then the metal-chelated beads were used in the adsorption of yeast invertase in a batch system. The maximum invertase adsorption capacity of the poly(EGDMA–VIM)–Cu²⁺ beads was observed as 35.2 mg/g at pH 4.5. The adsorption isotherm of the poly(EGDMA–VIM)–Cu²⁺ beads can be well fitted to the Langmuir model. Adsorption kinetics data were tested using pseudo-first- and -second-order models. Kinetic studies showed that the adsorption followed a pseudo-second-order reaction. The value of the Michaelis constant K_m of invertase was significantly larger upon adsorption, indicating decreased affinity by the enzyme for its substrate, whereas V_max was smaller for the adsorbed invertase. The optimum temperature for the adsorbed preparation of poly(EGDMA–VIM)–Cu²⁺-invertase at 50 °C, 10 °C higher than that of the free enzyme at 40 °C. Storage stability was found to increase with adsorption. Adsorbed invertase retains an activity of 82% after 10 batch successive reactions, demonstrating the usefulness of the enzyme-loaded beads in biocatalytic applications.     

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%.