Lipase‐catalyzed ethanolysis of soybean oil in a solvent‐free system using central composite design and response surface methodology

BACKGROUND: In this work we describe the synthesis of ethyl esters, commonly known as biodiesel, using refined soybean oil and ethanol in a solvent‐free system catalyzed by lipase from Thermomyces lanuginosus. Central composite design and response surface methodology (RSM) were employed to optimize the biodiesel synthesis parameters, which were: reaction time, temperature, substrate molar ratio, enzyme content, and added water, measured as percentage of yield conversion. RESULTS: The optimal conditions obtained were: temperature, 31.5 °C; reaction time, 7 h; substrate molar ratio, 7.5:1 ethanol:soybean oil; enzyme content, 15% (g enzyme g⁻¹ oil); added water, 4% (g water g⁻¹ oil). The experimental yield conversion obtained under these conditions was 96%, which is very close to the maximum predicted value of 94.4%. The reaction time‐course at the optimal values indicated that 5 h was necessary to obtain high yield conversions. CONCLUSION: A high yield conversion was obtained under the optimized conditions, with relative low enzyme content and short time. Comparison of predicted and experimental values showed good correspondence, implying that the empirical model derived from RSM can be used to adequately describe the relationship between the reaction parameters and the response (yield conversion) in lipase‐catalyzed biodiesel synthesis. Copyright © 2008 Society of Chemical Industry     

Enzymatic synthesis of monoacylglycerol citrate optimized by response surface methodology

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

Enzymatic Hydrolysis of Corn Stover after Pretreatment with Dilute Sulfuric Acid

Although many previous studies have been carried on the enzymatic hydrolysis of corn stover after pretreatment with dilute sulfuric acid, this paper emphasizes the use of different conditions to attain the highest yields of two sugars, xylose and glucose, from both stages. The pretreatment was performed at a range of sulfuric acid concentrations of 2, 4 and 6 % at 80, 100 and 120 °C. Up to 77 % xylose yield was obtained while the glucose yield was only 8.4 %. The corresponding solid phase was hydrolyzed by cellulase and the influences of five factors and their interactions on enzyme hydrolysis were evaluated by response surface methodology based on one‐factor‐at‐a‐time experiments. The optimal levels for each variable to obtain the highest reducing sugar yield were as follows: enzyme concentration of 22 FPU/g substrate, substrate concentration of 77 g/L, temperature of 49 °C, pH 4.8 and reaction time of 38 h. A reducing sugar yield of 42.11 g/100 g substrate was achieved, which was consistent with the predicted value of 42.13 g/100 g substrate. Compared with the one‐factor‐at‐a‐time experiments, there was a 9.4 % increase in reducing sugar yield when the enzyme concentration was decreased to 3 FPU/g substrate, the substrate concentration increased to 17 g/L and the reaction time dropped to 22 h. The total sugar released from the two stages was 62.81 g/100 g substrate.     

碱性蛋白酶水解林蛙卵粕蛋白的工艺条件优化

研究了碱性蛋白酶水解林蛙卵粕蛋白制备混合氨基酸的工艺,分析了反应温度、酶加量、底物浓度和反应时间对蛋白水解度的影响.通过单因素实验和正交实验确定碱性蛋白酶水解林蛙卵粕蛋白的最佳工艺条件为:反应温度60℃、酶加量2000 U·g-1、底物浓度4％、反应时间120min,此时的蛋白水解度为10.18％.     

Removal of formaldehyde from overactivated‐carbon‐fiber‐loaded biological enzyme

To remove formaldehyde effectively, we used activated carbon fiber as a carrier to prepare the composite materials (biological enzyme/activated carbon fiber) by loading biological enzyme on its surface. A one‐factor‐at‐a‐time design and orthogonal design were used in the experimental design methods to optimize the biological enzyme based on soya protein. The experimental results indicate that the pH value was the most significant condition for optimizing the biological enzyme. The optimum orthogonal design conditions were proposed to be a reaction temperature of 80°C, a pH of 4, a reaction time of 6 h, and an amino nitrogen content of up to 5.4%. In addition, the morphologies of the composite materials were characterized with scanning electron microscopy, and the removal efficiency of formaldehyde was investigated with the acetyl acetone method. The results show that the formaldehyde removal efficiency was the best and the removal rate was 80% when the loading time was 8 h. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 2619–2623, 2013     

Lipase‐catalyzed production of medium‐chain triacylglycerols from palm kernel oil distillate: Optimization using response surface methodology

Optimization of lipase‐catalyzed esterification for the production of medium‐chain triacylglycerols (MCT) from palm kernel oil distillate and glycerol was carried out in order to determine the factors that have significant effects on the reaction system and MCT yield. Novozyme 435 from Candida antarctica lipase was found to have the highest activity at 52.87 ± 0.03 U/g. This lipase also produced the highest MCT yield, which is 56.67%. The effect of different variables on MCT synthesis was studied with a two‐level five‐factor fractional factorial design. The various variables include (1) reaction temperature, (2) enzyme load, (3) molecular sieves concentration, (4) reaction time and (5) molar substrate ratio. Reaction temperature, reaction time and molar substrate ratio strongly affect MCT synthesis (p <0.05). However, enzyme load and molecular sieve concentration did not have a significant (p >0.05) influence on MCT yield. Therefore, the significant variables such as reaction temperature, reaction time and molar substrate ratio were further optimized through central composite rotatable design (CCRD). Comparisons between predicted and experimental values from the CCRD optimization procedures revealed good correlation, implying that the quadric response model satisfactorily expressed the percentage yield of MCT in the lipase‐catalyzed esterification. The optimum MCT yield is 73.3% by using 2 wt‐% enzyme dosage, a molecular sieves concentration of 1 wt‐%, a reaction temperature of 90 °C, a reaction time of 10 h and a molar substrate ratio of 4 : 1 (medium‐chain fatty acid/glycerol). Experiments to confirm the predicted results using the optimal parameters were conducted and an MCT yield of 70.21 ± 0.18% (n = 3) was obtained.     

Numerical Modeling of Differential Kinetics in the Asymmetric Hydrogenation of Acetophenone by Noyori's Catalyst

A combination of numerical integration of the rate equations and experimental measurement of the time‐dependence of rates rather than concentrations is described for the analysis of the catalytic cycle by which Noyori's catalyst, trans‐RuCl₂[(S)‐binap][(S,S)‐dpen], hydrogenates acetophenone. The method is simpler in practice than usual kinetic methods and yields furthermore rate constants for activation, dihydrogen cleavage, and hydride transfer to substrate under realistic catalytic conditions. The specific results for the asymmetric hydrogenation of acetophenone show that the turnover‐limiting step changes from dihydrogen cleavage to hydride transfer if the H₂ pressure is increased, but also during the course of the reaction when the hydrogenation is performed under typical conditions.     

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

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

A Redox‐Based Superoxide Generation System Using Quinone/Quinone Reductase

Superoxide (O₂^.?) generation in biological systems is achieved through some of the most complex enzymatic systems. Of these, only xanthine/xanthine oxidase has been used for in vitro biochemical studies. However, it suffers from limitations such as a lack of suitable heterologous expression system for xanthine oxidase and the irreversible consumption and low solubility of xanthine under physiological conditions. Herein, we report a redox‐based, enzyme‐catalyzed system, in which autoxidation of hydroquinone to quinone via semiquinone results in superoxide generation. Quinone is reduced back to hydroquinone by using the NfsB (oxygen‐insensitive nitroreductase) enzyme of Escherichia coli strain K‐12 and nicotinamide adenine dinucleotide phosphate hydride (NADPH; which is regenerated by using the glucose/glucose dehydrogenase system). This new system relies on quinones that can be recycled and have superior water solubility, as well as enzymes that are heterologously expressed. By using a variety of quinones and reaction conditions, along with a comparison of real‐time fluorescence, menadione has been identified as the optimal substrate for superoxide generation. The new redox‐based system presents a viable alternative for studying the biochemistry of superoxide under different physiological and pathological conditions.     

维生素C乳酸酯酶催化合成的响应曲面分析

以脂肪酶Novozym 435催化维生素C和乳酸乙酯转酯合成维生素C乳酸酯。采用基于四因素五水平的中心组合设计实验的响应曲面法,分析了温度(30~70℃)、酶量(10~30 mg)、维生素C质量浓度(9.36~28.07g/L)和反应时间(24~72 h)以及它们的交互作用对维生素C乳酸酯产率的影响。获得的最佳实验条件为:温度50℃,维生素C的质量浓度18.71 g/L,脂肪酶质量20 mg和反应时间48 h。在最佳条件下,维生素C乳酸酯的预计产率为71.20%,与实际产率71.12%符合较好。     

Stability and activity of chymotrypsin immobilized on magnetic nanogels covered with carboxyl groups

α‐chymotrypsin as model enzyme was anchored to the carboxyl‐functionalized magnetic nanogels prepared by in situ photochemical polymerization. Furthermore, to explore the optimum immobilization, the effects of immobilization time, pH of the reaction mixture, and proportion of enzyme to the magnetic nanogels were studied. The immobilized enzyme was stable in the presence of enzyme denaturation surfactants, and maintained their activity against protein‐digesting enzyme. The immobilized enzyme exhibited hydrolysis activity against the substrate of casein, and cleaved the substrate into small fragments. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Engineering model for intumescent coating behavior in a pilot‐scale gas‐fired furnace

In the event of a fire, intumescent fire protective coatings expand and form a thermally insulating char that protects the underlying substrate from heat and subsequent structural failure. The intumescence includes several rate phenomena, which have been investigated and quantified in the literature for several decades. However, various challenges still exist. The most important one concerns mathematical model validation under realistic exposure conditions and/or time scales. Another is the simplification of advanced models to overcome the often‐seen lack of a complete set of input and adjustable model parameters for a given coating, thereby providing models for industrial applications. In this work, these two challenges are addressed. Three experimental series, with an intumescent coating inside a 0.65 m³ gas‐fired furnace, heating up according to so‐called cellulosic fire conditions, were conducted and a very good repeatability was evident. The experiments were run for almost 3 h, reaching a final gas temperature of about 1100°C. Measurements include transient temperature developments inside the expanding char, at the steel substrate, and in the mineral wool insulation placed behind the substrate. A mathematical model, describing the intumescent coating behavior and temperatures in the furnace using a single overall reaction was developed and validated against experimental data. By including a decomposition front movement through the char, a good qualitative agreement was obtained. After further validation against experiments with other coating formulations, it has potential to become a practical engineering tool. © 2016 American Institute of Chemical Engineers AIChE J, 62: 3947–3962, 2016     

Monitoring the reaction progress of a high‐performance phenylethynyl‐terminated poly(etherimide). Part I: Cure kinetics modeling

The cure kinetics of a phenylethynyl‐terminated poly(etherimide) are examined via differential scanning calorimetry (DSC) measurements. Isothermal holds at temperatures ranging from 325°C to 360°C provided the necessary information to develop reaction kinetics models using both first‐order reaction kinetics and combination reaction kinetics. The first‐order reaction kinetics model works well for quick estimates of degree of cure versus time over the experimental temperature range. However, significantly more accurate predictions of degree of cure versus time were provided by the combination reaction kinetics model. The lack of accuracy in the first order model is due to the fact that the reaction cannot be described by a simple order. The experimental procedures followed to obtain the cure kinetics data and the construction of the models from this data are described; the predictions of these models are compared with the experimental results.     

Study on the enzymatic hydrolysis of racemic methyl ibuprofen ester

The hydrolysis of racemic methyl ibuprofen ester in the presence of lipase from Candida rugosa was investigated in shake flasks. Experiments were performed to study the effect of temperature, pH and shaking speed on the reaction rate. Different hydrophobic co‐solvents were screened for the highest reaction rate and the presence of enzyme inhibition by substrate and products was examined. A kinetic expression was then proposed to describe the reaction. Kinetic parameters were determined for the optimum operating conditions and the proposed model was verified with the experimental results. Next, this reaction was scaled up to a fed batch stirred tank reactor. Batch reactor and fed batch reactor configurations were compared for better conversions. The effects of aqueous phase hold‐up, substrate concentration and feed flow rate on the conversion of the reaction were also studied. Higher conversions were obtained in a fed batch reactor when compared with the batch reactor. In the fed batch reactor, increased conversions were observed with lower feed flowrates and high aqueous phase hold‐up. © 2001 Society of Chemical Industry     

Selective labeling of proteins by using protein farnesyltransferase

The challenging task of identifying and studying protein function has been greatly aided by labeling proteins with reporter groups. Here, we present a strategy that utilizes an enzyme that labels a four-residue sequence appended onto the C terminus of a protein, with an alkyne-containing substrate. By using a bio-orthogonal cycloaddition reaction, a fluorophore that carried an azide moiety was then covalently coupled to the alkyne appended on the protein. FRET was used to calculate a F?rster (R) distance of 40 A between the eGFP chromophore and the newly appended Texas Red fluorophore. This experimental value is in good agreement with the predicted R value determined by using molecular modeling. The small recognition tag, the high specificity of the enzyme, and the orthogonal nature of the derivatization reaction will make this approach highly useful in protein chemistry.     

Production of heat-sensitive monoacylglycerols by enzymatic glycerolysis in tert-pentanol: Process optimization by response surface methodology

The aim of this study was to optimize production of MAG by lipase-catalyzed glycerolysis in a tert-pentanol system. Twenty-nine batch reactions consisting of glycerol, sunflower oil, tert-pentanol, and commercially available lipase (Novozym^®435) were carried out, with four process parameters being varied: Enzyme load, reaction time, substrate ratio of glycerol to oil, and solvent amount. Response surface methodology was applied to optimize the reaction system based on the experimental data achieved. MAG, DAG, and TAG contents, measured after a selected reaction time, were used as model responses. Well-fitting quadratic models were obtained for MAG, DAG, and TAG contents as a function of the process parameters with determination coefficients (R²) of 0.89, 0.88, and 0.92, respectively. Of the main effects examined, only enzyme load and reaction time significantly influenced MAG, DAG, and TAG contents. Both enzyme amount and reaction time showed a surprisingly nonlinear relationship between factors (process parameters) and responses, indicating a local maximum. The substrate ratio of glycerol to oil did not significantly affect the MAG and TAG contents; however, it had a significant influence on DAG content. Contour plots were used to evaluate the optimal conditions for the complex interactions between the reaction parameters and responses. The optimal conditions established for MAG yield were: enzyme load, 18% (w/w of oil); glycerol/oil ratio, 7∶1 (mol/mol); solvent amount, 500% (vol/wt of oil); and reaction time, 115 min. Under these conditions, a MAG content of 76% (w/w of lipid phase) was predicted. Verification experiments under optimized reaction conditions were conducted, and the results agreed well with the range of predictions.     

Fate of horseradish peroxidase during oxidation of monobrominated phenols

BACKGROUND: Peroxidase‐catalyzed polymerization of phenols is accompanied by substantial enzyme precipitation with reaction products. The enzyme fate during the polymerization of monobromophenols by horseradish peroxidase (HRP) was studied. Enzyme fate was simultaneously monitored by protein, total nitrogen mass balance and gel electrophoresis (SDS‐PAGE) analysis of both soluble and precipitate fractions. RESULTS: SDS‐PAGE analysis revealed that molecular weight bands of protein in the precipitate shifted upwards toward higher molecular weights, compared with protein control. When co‐polymerization was practiced higher HRP precipitation occurred compared with polymerization of a single substrate, regardless of substrate combination applied. Addition of polyethylene glycol (PEG) to the reaction mixture decreased the extent of HRP precipitation. At 2 mmol L^?1 H₂O₂, corresponding to the stoichiometric equivalent concentration, 50% precipitation occurred after 1 h (～70% after 24 h) compared with 97–98% (～100% after 24 h) without PEG. Nevertheless, further increase of H₂O₂ increased HRP precipitation regardless of PEG (85% at 4 mmol L^?1 and 95% at 5 mmol L^?1). The lowest degree of enzyme inactivation was observed for metabromophenol, which displayed the lowest transformation yield, compared to the other congeners. CONCLUSIONS: Results from SDS‐PAGE indicate that an interaction stronger than hydrophobic, resisting the denaturative conditions, may take place between HRP and the reaction products, suggesting the occurrence of a covalent link between them. Oxidation was enhanced by inclusion of PEG, which partially suppressed product‐dependent inactivation. The extent of enzyme inactivation depends on the substrate used, while highest inactivation occurred when co‐polymerization was practiced. Copyright © 2009 Society of Chemical Industry     

废纸的利用探讨———酶法水解反应生产还原糖

通过纤维素酶水解复印废纸生产还原糖的实验,测定了还原糖得率与酶解时间的关系么及温度、ｐＨ值、酶浓度、底物浓度对酶解还原糖得率的影响。研究表明：废纸酶解回收利用具有很大的开发潜能。     

反胶束中中性蛋白酶AS1.398反应机理

枯草杆菌中性蛋白酶AS1.398可用于反胶束萃取全脂豆粉分离大豆蛋白和油脂.揭示酶的催化作用机理可用于优化萃取条件.以大豆分离蛋白为底物,研究了AS1.398在AOT/异辛烷反胶束体系中的反应机理. 由于底物分子在反胶束中呈泊松分布,通过计算底物和酶分子数比例,推算出酶催化为单底物反应的概率占绝对优势.借鉴水相酶反应模型,考虑反胶束中分子交换作用,提出了反胶束中酶的单底物分子反应的作用模型,模型推导出水相的米氏常数比反胶束相的大1个数量级.实验测定酶在水相和反胶束相的米氏常数分别为3.2×10^-3g&#8226;ml^-1和4.2×10^-4g&#8226;ml^-1.两者比值与模型推导出的关系较吻合,表明此模型可用来表示反胶束中AS1.398的催化反应.     

Enzymatic acyl modification of phosphatidylcholine using immobilized lipase and phospholipase A2

Ethanol‐soluble (ES) lecithin mainly contains phosphatidylcholine (PC). The incorporation of caprylic acid into PC using immobilized phospholipase A₂ (PLA₂) and lipase was investigated. The Rhizomucor meihei lipase and the porcine pancreatic PLA₂ were immobilized on the hydrophobic resin Diaion HP‐20 and the modification was carried out in hexane as solvent. HPTLC with densitometer technique was successfully used for monitoring the production of structured phospholipids (PL) (ML‐type PC, MM‐type PC, and lysophosphatidylcholine; L: long‐chain fatty acid, M: medium‐chain fatty acid). The various parameters such as the effects of reaction temperature, enzyme loading, and the effect of molar proportion of substrate were studied in order to determine the optimum reaction conditions for the acidolysis reaction. The optimal operating conditions for the PLA₂‐catalyzed reaction were obtained as 50°C temperature, 50% (wt/wt of substrate) enzyme loading, and a 1:12 molar proportion of PC/caprylic acid. For the lipase‐catalyzed reaction, the optimized temperature was the same as for PLA₂, but the enzyme loading and molar proportion were slightly lower, i.e., 40 % w/w of substrate and 1:9 PC/caprylic acid, respectively. The effects of these parameters on the production of structured PL were compared. Under these optimal conditions, the ML‐type PC content was higher in the PLA₂‐catalyzed reaction, i.e., 45.29 mol%, and in the lipase‐catalyzed reaction it was 38.74 mol%.