Optimized synthesis of lipase-catalyzed hexyl acetate in n-hexane by response surface methodology

Hexyl acetate, a short-chain ester with fruity odor, is a significant green note flavor compound and widely used in the food industry. The ability for immobilized lipase from Mucor miehei (Lipozyme IM-77) to catalyze the transesterification of hexanol with triacetin was investigated in this study. Response surface methodology and five-level-five-factor central composite rotatable design were adopted to evaluate the effects of synthesis variables, such as reaction time (2-10 h), temperature (25-65 degrees C), enzyme amount (10-50%; 0.024-0.118 BAUN), substrate molar ratio of triacetin to hexanol (1:1 to 3:1), and added water content (0-20%) on percentage molar conversion of hexyl acetate. The results showed that reaction temperature and substrate molar ratio were the most important parameters and that added water content had less of an effect on percent molar conversion. On the basis of canonical analysis, optimum synthesis conditions were as follows: reaction time, 7.7 h; temperature, 52.6 degrees C; enzyme amount, 37.1% (0.089 BAUN); substrate molar ratio, 2.7:1; and added water, 12.5%. The predicted value was 88.9% molar conversion, and the actual experimental value was 86.6% molar conversion.     

Optimization of lipase-catalyzed synthesis of octyl hydroxyphenylpropionate by response surface methodology

The ability of immobilized lipase Candida antarctica (Novozyme 435) to catalyze the direct esterification of hydroxyphenylpropionic acid and octanol in a solvent-free system was investigated in this study. Response surface methodology (RSM) and five-level-four-factor central composite rotatable design (CCRD) were employed to evaluate the effects of synthesis parameters, such as reaction time, temperature, enzyme amount, and pH memory, on percentage molar conversion of phenolic acid esters. Reaction time, temperature, and enzyme amount were the most important variables. On the basis of canonical analysis and ridge max analysis, the optimum synthesis conditions with 95.9% molar conversion were reaction time of 58.2 h, temperature of 52.9 degrees C, enzyme amount of 37.8% (w/w), and pH memory of pH 7.     

Formulation of physical properties of methyl glucoside polyester by mixture response surface methodology

Methyl glucoside polyester (MGPE), consisting of a methyl glucoside molecule esterified with four fatty acids, is a potential fat substitute. A mixture response surface methodology was employed to model the physical properties (melting point, density, and viscosity) of MGPE prepared from composite blends of fatty acid methyl esters (FAME). The measured physical properties were similar to those of soybean oil, except for a higher viscosity. The physical properties correlated significantly with the degree of saturation of their composite fatty acids. Results showed that the physical properties of MGPE prepared from a FAME mixture derived from high oleic acid safflower oil and soybean oil (molar ratio 1:1) was similar to those of soybean oil. The physical properties of MGPE can be adjusted by modifying the saturated fatty acids of synthesized MGPE to simulate the physical properties of soybean oil.     

Enhancement in the synthesis of novel feruloyl lipids (feruloyl butyryl glycerides) by enzymatic biotransformation using response surface methodology

Response surface methodology was successfully employed to optimize lipase-catalyzed synthesis of feruloyl butyryl glycerides (FBGs). The effects of the reaction parameters, including the reaction time, reaction temperature, enzyme concentration, substrate molar ratio, and water activity, and the interaction parameters were examined. The analysis suggested that the conversion of the FBGs was significantly (p < 0.05) affected by independent factors of reaction time, reaction temperature, substrate molar ratio, and water activity as well as interactive terms of reaction temperature/reaction time, reaction temperature/enzyme concentration, substrate molar ratio/reaction temperature, water activity/reaction temperature, reaction time/enzyme concentration, and enzyme concentration/water activity. The highest conversion yield of FBGs was 81.2% at the following optimized reaction conditions: reaction temperature of 53.6 degrees C, reaction time of 5.5 days, enzyme concentration of 50.8 mg/mL, water activity of 0.14, and substrate molar ratio of 2.9. The conversion is higher as compared to that at the conditions before optimization.     

Optimal formation of hexyl laurate by Lipozyme IM-77 in solvent-free system

A medium-chain ester, hexyl laurate, with fruity flavor is primarily used in personal care formulations as an important emollient for cosmetic applications. To conform to the "natural" interests of consumers, the ability of immobilized lipase from Rhizomucor miehei (Lipozyme IM-77) to catalyze the direct esterification of hexanol and lauric acid by using a solvent-free system was investigated in this study. Response surface methodology (RSM) and four-factor-five-level central composite rotatable design (CCRD) were employed to evaluate the effects of synthesis parameters, such as reaction time (10-50 min), temperature (45-85 degrees C), lipase amount (10-30 mg/volume; 0.077-0.231 batch acidolysis units of Novo (BAUN), and pH memory (5-9), on percentage molar conversion of hexyl laurate by lipase-catalyzed direct esterification. Reaction time, temperature, and enzyme amount had significant effects on percent molar conversion. On the basis of ridge maximum analysis, the optimum synthesis conditions for hexyl laurate were a reaction time of 40.6 min, a temperature of 58.2 degrees C, an enzyme amount of 25.4 mg/volume (0.196 BAUN), and a pH memory of 5.9. The predicted percentage molar conversion of hexyl laurate was 69.7 +/- 1.4%.     

Optimization of lipase-catalyzed regioselective acylation of pyridoxine (vitamin B6)

Response surface methodology was successfully applied to optimize lipase-catalyzed regioselective esterification of pyridoxine (PN). Effects of various reaction conditions, including reaction temperature, time, enzyme loading, substrate molar ratio, and water activity, were investigated. A central composite design was employed to search for the optimal conversion of PN. A quadratic polynomial regression model was used for analysis of the experimental data at a 95% level (p < 0.05). The analysis confirmed that the water activity was the most significant factor affecting the conversion of PN. It was also suggested that the conversion was strongly affected by independent variables of temperature, time, substrate molar ratio, and water activity as well as interaction terms of temperature and enzyme loading/substrate molar ratio/water activity, time and enzyme loading/substrate molar ratio, substrate molar ratio, and water activity. The coefficient of determination of the model was found to be 0.963. Three sets of optimum reaction conditions were established, and the verified experimental trials were performed for validating the optimum points. A scale-up experiment was also done under the first set of optimal conditions.     

Candida rugosa lipase LIP1-catalyzed transesterification to produce human milk fat substitute

Structured lipids (SLs) containing palmitic and oleic acids were synthesized by transesterification of tripalmitin with either oleic acid or methyl oleate as acyl donor. This SL with palmitic acid at the sn-2 position and oleic acid at sn-1,3 positions is similar in structure to human milk fat triacylglycerol. LIP1, an isoform of Candida rugosa lipase (CRL), was used as biocatalyst. The effects of reaction temperature, substrate molar ratio, and time on incorporation of oleic acid were investigated. Reaction time and temperature were set at 6, 12, and 24 h, and 35, 45, and 55 degrees C, respectively. Substrate molar ratio was varied from 1:1 to 1:4. The highest incorporation of oleic acid (37.7%) was at 45 degrees C with methyl oleate as acyl donor. Oleic acid resulted in slightly lesser (26.3%) incorporation. Generally, higher percentage incorporation of oleic acid was observed with methyl oleate (transesterification) than with oleic acid (acidolysis). In both cases percentage incorporation increased with reaction time. Incorporation decreased with increase in temperature above 45 degrees C. Initially, oleic acid incorporation increased with increase in substrate molar ratio up to 1:3. LIP1 was also compared with Lipozyme RM IM as biocatalysts. The tested reaction parameters were selected on the basis of maximum incorporation of C18:1 obtained during optimization of LIP1 reaction conditions. Reaction temperature was maintained at 45, 55, and 65 degrees C. Lipozyme RM IM gave highest oleic acid incorporation (49.4%) at 65 degrees C with methyl oleate as acyl donor. Statistically significant (P < 0.05) differences were observed for both enzymes. SL prepared using Lipozyme RM IM may be more suitable for possible use in human milk fat substitutes.     

Enzymatically catalyzed synthesis of low-calorie structured lipid in a solvent-free system: optimization by response surface methodology

A kind of low-calorie structured lipid (LCSL) was obtained by interesterification of tributyrin (TB) and methyl stearate (St-ME), catalyzed by a commercially immobilized 1,3-specific lipase, Lipozyme RM IM from Rhizomucor miehei . The condition optimization of the process was conducted by using response surface methodology (RSM). The optimal conditions for highest conversion of St-ME and lowest content LLL-TAG (SSS and SSP; S, stearic acid; P, palmitic acid) were determined to be a reaction time 6.52 h, a substrate molar ratio (St-ME:TB) of 1.77:1, and an enzyme amount of 10.34% at a reaction temperature of 65 °C; under these conditions, the actually measured conversion of St-ME and content of LLL-TAG were 78.47 and 4.89% respectively, in good agreement with predicted values. The target product under optimal conditions after short-range molecular distillation showed solid fat content (SFC) values similar to those of cocoa butter substitutes (CBS), cocoa butter equivalent (CBE), and cocoa butters (CB), indicating its application for inclusion with other fats as cocoa butter substitutes.     

Solvent-free enzymatic preparation of feruloylated monoacylglycerols optimized by response surface methodology

The ability of immobilized lipase B from Candida antarctica (Novozym 435) to catalyze the direct esterification of glyceryl ferulate (FG) and oleic acid for feruloylated monoacylglycerols (FMAG) preparation in a solvent-free system was investigated. Enzyme screening and the effect of glycerol on the initial reaction rate of esterification were also investigated. Response surface methodology (RSM) was used to optimize the effects of the reaction temperature (55-65 degrees C), the enzyme load (8-14%; relative to the weight of total substrates), oleic acid/(FG + glycerol) (6:1-9:1; w/w), and the reaction time (1-2 h) on the conversion of FG and yield of FMAG. Validation of the RSM model was verified by the good agreement between the experimental and the predicted values of FG conversion and FMAG yield. The optimum preparation conditions were as follows: temperature, 60 degrees C; enzyme load, 8.2%; substrate ratio, 8.65:1 (oleic acid/(FG + glycerol), w/w); and reaction time, 1.8 h. Under these conditions, the conversion of FG and yield of FMAG are 96.7 +/- 1.0% and 87.6 +/- 1.2%, respectively.     

Lipase-catalyzed acidolysis of tripalmitin with hazelnut oil fatty acids and stearic acid to produce human milk fat substitutes

Structured lipids (SLs) containing palmitic, oleic, stearic, and linoleic acids, resembling human milk fat (HMF), were synthesized by enzymatic acidolysis reactions between tripalmitin, hazelnut oil fatty acids, and stearic acid. Commercially immobilized sn-1,3-specific lipase, Lipozyme RM IM, obtained from Rhizomucor miehei was used as the biocatalyst for the enzymatic acidolysis reactions. The effects of substrate molar ratio, reaction temperature, and reaction time on the incorporation of stearic and oleic acids were investigated. The acidolysis reactions were performed by incubating 1:1.5:0.5, 1:3:0.75, 1:6:1, 1:9:1.25, and 1:12:1.5 substrate molar ratios of tripalmitin/hazelnut oil fatty acids/stearic acid in 3 mL of n-hexane at 55, 60, and 65 degrees C using 10% (total weight of substrates) of Lipozyme RM IM for 3, 6, 12, and 24 h. The fatty acid composition of reaction products was analyzed by gas-liquid chromatography (GLC). The fatty acids at the sn-2 position were identified after pancreatic lipase hydrolysis and GLC analysis. The results showed that the highest C18:1 incorporation (47.1%) and highest C18:1/C16:0 ratio were obtained at 65 degrees C and 24 h of incubation with the highest substrate molar ratio of 1:12:1.5. The highest incorporation of stearic acid was achieved at a 1:3:0.75 substrate molar ratio at 60 degrees C and 24 h. For both oleic and stearic acids, the incorporation level increased with reaction time. The SLs produced in this study have potential use in infant formulas.     

Optimization of lipase-catalyzed synthesis of ginsenoside Rb1 esters using response surface methodology

In the lipase (Novozyme 435)-catalyzed synthesis of ginsenoside Rb1 esters, different acyl donors were found to affect not only the degree of conversion but also the regioselectivity. The reaction of acyl donors with short carbon chain was more effective, showing higher conversion than those with long carbon chain. Among the three solvent systems, the reaction in tert-amyl alcohol showed the highest conversion rate, while the reaction in the mixed solvent of t-BuOH and pyridine (1:1) had the lowest conversion rate. To allow the increase of GRb1 lipophilicity, we decided to further study the optimal condition of synthesis of GRb1 with vinyl decanoate with 10 carbon chain fatty acids in tert-amyl alcohol. Response surface methodology (RSM) was employed to optimize the synthesis condition. From the ridge analysis with maximum responses, the maximum GRb1 conversion was predicted to be 61.51% in a combination of factors (40.2 h, 52.95 degrees C, substrate mole ratio 275.57, and enzyme amount 39.81 mg/mL). Further, the adequacy of the predicted model was examined by additional independent experiments at the predicted maximum synthesis conditions. Results showed that the RSM was effective to optimize a combination of factors for lipase-catalyzed synthesis of ginsenoside Rb1 with vinyl decanoate.     

Studies of the lauroylation of wheat straw hemicelluloses under heating

Lauroylation of wheat straw hemicelluloses in the N, N-dimethylformamide/lithium chloride system under microwave irradiation was studied. The parameters optimized included lauroyl chloride concentration as the molar ratio of xylose unit in hemicelluloses/lauroyl chloride (1:1-1:4), 4-dimethylaminopyridine concentration (2-10%), reaction time (1-8 min), molar ratio of xylose unit in hemicelluloses/triethylamine (1:2), and reaction temperature (78 degrees C). The reaction efficiency was measured by the yield and degree of substitution (DS). Under an optimum reaction condition (molar ratio of xylose unit in hemicelluloses/lauroyl chloride 1:3, molar ratio of xylose unit in hemicelluloses/triethylamine 1:2, 5% 4-dimethylaminopyridine, 78 degrees C, 5 min), a DS of 1.63 was obtained. Changes in the structure of hemicelluloses were verified by FT-IR and 1H and 13C NMR spectroscopy. The results showed that the lauroylation occurred preferentially at the C-3 position of the xylose unit in hemicelluloses. The behavior of the lauroylated hemicelluloses was monitored by means of thermogravimetric (TG) and differential thermogravimetric (DTG) analysis. It was found that the product with low DS had a lower thermal stability than the native hemicelluloses, whereas the lauroylated polymers with high DS showed a higher thermal stability than the unmodified hemicelluloses.     

利用Biopro型设备转化废弃油脂制备生物柴油

为开发适应中国废弃油脂生物柴油转化的成套技术与装备,结合美国Biopro 380型设备,对中国的4种典型废弃油脂生物柴油转化工艺进行了系统研究,结果表明,甲醇回流温度65℃下,酯化反应时间2.5h、浓硫酸加入量0.5%、甲醇与游离脂肪酸摩尔比2.7∶1时,酯化混合物的酸值降至1～2?mg/g;转酯化反应在醇油摩尔比6∶1、催化剂NaOH的加入量1.0%（与废弃油脂的质量百分比）、反应时间60 min时,转酯化效果最佳;将该工艺条件应用于Biopro 380型设备中进行验证试验,获得的生物柴油产品质量指标基本符合中国生物柴油标准GB/T 20828-2007。     

Synthesis of structured triacylglycerols by lipase-catalyzed acidolysis in a packed bed bioreactor

Structured triacylglycerols (ST) from canola oil were produced by enzymatic acidolysis in a packed bed bioreactor. A commercially immobilized 1,3-specific lipase, Lipozyme IM, from Rhizomucormiehei, was the biocatalyst and caprylic acid the acyl donor. Parameters such as substrate flow rate, substrate molar ratio, reaction temperature, and substrate water content were examined. High-performance liquid chromatography was used to monitor the reaction and product yields. The study showed that all of the parameters had effects on the yields of the expected di-incorporated (dicaprylic) ST products. Flow rates below 1 mL/min led to reaction equilibrium, and lower flow rates did not raise the incorporation of caprylic acid and the product yield. Incorporation of caprylic acid and the targeted di-incorporated ST was increased by approximately 20% with temperature increase from 40 to 70 degrees C. Increasing the substrate molar ratio from 1:1 to 7:1 increased the incorporation of caprylic acid and the product yield slightly. Water content in the substrate also had a mild influence on the reaction. Water content at 0.08% added to the substrate gave the lowest incorporation and product yield. The use of solvent in the medium was also studied, and results demonstrated that it did not increase the reaction rate at 55 degrees C when 33% hexane (v/v) was added. The main fatty acids at the sn-2 position of the ST were C(18:1), 54. 7 mol %; C(18:2), 30.7 mol %; and C(18:3), 11.0 mol %.     

Studies on the preparation of biodiesel from Zanthoxylum bungeanum Maxim seed oil

To reduce the cost of biodiesel production, the feasibility of Zanthoxylum bungeanum Maxim seed oil (ZBMSO) was studied to produce biodiesel. A methyl ester biodiesel was produced from ZBMSO using methanol, sulfuric acid, and potassium hydroxide in a two-stage process. The main variables that affect the process were investigated. The high level of free fatty acids in ZBMSO was reduced to < 1% by an acid-catalyzed (2% H2SO4) esterification with methanol to oil molar ratios of 20-25:1 for 1 h. A maximum yield of 96% of methyl esters in ZBMSO biodiesel was achieved using a 6.5:1 molar ratio of methanol to oil, 0.9% KOH (percent oil), and reaction time of 0.5 h at 55 degrees C. Further investigation has also been devoted to the assessment of some important fuel properties of ZBMSO biodiesel produced under the optimized conditions according to specifications for biodiesel as fuel in diesel engines. The fuel properties of the ZBMSO biodiesel obtained are similar to those of no. 0 petroleum diesel fuel, and most of the parameters comply with the limits established by specifications for biodiesel.     

Production of ascorbyl palmitate by surfactant-coated lipase in organic media

The surface of a lipase from Burkholderia cepacia was coated with a nonionic surfactant, propylene glycol monostearate, and was used as a biocatalyst in the production of ascorbic acid in tert-butyl alcohol. The influence of various factors such as the type of surfactant, the pH of the buffer used for coating, the amount of surfactant in the coating, the organic solvent, and the temperature and molar ratio of the substrates used in the reaction on the conversion of ascorbyl palmitate were studied. After 24 h of reaction at 50 degrees C, a conversion of 47% was obtained using an ascorbic acid to palmitic acid molar ratio of 1:6. The native lipase showed only 6% conversion.     

菜籽油碱催化法制备生物柴油的工艺参数

以碱催化剂为媒介的转酯化反应制备生物柴油方法因其转化率高而倍受重视。该文以菜籽油为原料,在小型试验装置上,采用均相碱催化法,研究了菜籽油在碱性催化剂NaOH的作用下与甲醇经酯交换反应制备生物柴油的工艺条件。考察了醇油摩尔比（4︰1～8︰1）、催化剂用量（0.5%～2%）、反应温度（30～60℃）和反应时间（30～150 min）等工艺参数对酯交换反应的影响,对生物柴油的组成成分进行了气相色谱/质谱联用(GC-MS)分析。结果表明,在醇油摩尔比6︰1,催化剂用量为油质量的1%,反应温度为50～60℃,反应时间为60 min时,酯交换反应转化率最高可达到96.7%。该生物柴油主要由油酸甲酯、芥子酸甲酯、9,12-十八碳二烯酸甲酯、11-二十碳烯酸甲酯、亚麻酸甲酯等脂肪酸甲酯组成,其中油酸甲酯含量最高,相对质量分数高达50.30%。     

Activity and process stability of purified green pepper (Capsicum annuum) pectin methylesterase

Pectin methylesterase (PME) from green bell peppers (Capsicum annuum) was extracted and purified by affinity chromatography on a CNBr-Sepharose-PMEI column. A single protein peak with pectin methylesterase activity was observed. For the pepper PME, a biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters for activity and thermostability was performed. The optimum pH for PME activity at 22 degrees C was 7.5, and its optimum temperature at neutral pH was between 52.5 and 55.0 degrees C. The purified pepper PME required the presence of 0.13 M NaCl for optimum activity. Isothermal inactivation of purified pepper PME in 20 mM Tris buffer (pH 7.5) could be described by a fractional conversion model for lower temperatures (55-57 degrees C) and a biphasic model for higher temperatures (58-70 degrees C). The enzyme showed a stable behavior toward high-pressure/temperature treatments.     

Optimization of simultaneous saccharification and fermentation for the production of ethanol from lignocellulosic biomass

Simultaneous saccharification and fermentation (SSF) of alkaline hydrogen peroxide pretreated Antigonum leptopus (Linn) leaves to ethanol was optimized using cellulase from Trichoderma reesei QM-9414 (Celluclast from Novo) and Saccharomyces cerevisiae NRRL-Y-132 cells. Response surface methodology (RSM) and a three-level four-variable design were employed to evaluate the effects of SSF process variables such as cellulase concentration (20-100 FPU/g of substrate), substrate concentration (5-15% w/v), incubation time (24-72 h), and temperature (35-45 degrees C) on ethanol production efficiency. Cellulase and substrate concentrations were found to be the most significant variables. The optimum conditions arrived at are as follows: cellulase = 100 FPU/g of substrate, substrate = 15% (w/v), incubation time = 57.2 h, and temperature = 38.5 degrees C. At these conditions, the predicted ethanol yield was 3.02% (w/v) and the actual experimental value was 3.0% (w/v).     

碳基固体酸催化高酸值生物柴油原料降酸效果

以淀粉和对甲苯磺酸合成的碳基固体酸为催化剂,油酸模拟高酸值生物柴油原料进行酯化降酸的试验研究,考察醇油比、催化剂用量、反应温度、反应时间及重复利用性等因素对转化率的影响。通过单因素与正交试验确定最佳工艺条件：醇油质量比为1︰4,催化剂用量为油酸质量的6.5%,反应时间6 h,反应温度85℃,在此条件下转化率可达80.21%,重复使用6次,转化率仍保持在70%以上。碳基固体酸催化剂对高酸值原料酯化降酸有很好的催化活性,易于分离且具有良好的稳定性。