Stability of cutinase,wild type and mutants,in AOT reversed micellar system—effect of mixture components of alkyl esters production

BACKGROUND: Fusarium solani pisi cutinase was microencapsulated in reversed micelles of bis(2‐ethylhexyl) sodium sulfosuccinate (AOT) in isooctane for the production of alkyl esters, known as biodiesel; its stability in this system was also evaluated. Results using cutinase wild type and three mutants, L153Q, T179C and S54D, were compared. RESULTS: There is a loss of 45% of cutinase wild type activity when incubated in the micellar system for 3 h, indicating a denaturing effect of AOT towards the enzyme, and furthermore a loss of 90% of the activity is observed in the presence of methanol with only 10 min of incubation. In contrast, an improvement of cutinase performance was achieved when incubated with ethanol or butanol, indicating that these alcohols have a protective role regarding the enzyme. Mutant T179C displayed high stability in the presence of methanol with an activity loss of only 16%. CONCLUSIONS: Mutant T179C presented not only high resistance to the denaturing effect of AOT but also high stability in the presence of methanol. This is a promising biocatalyst for a continuous process. Copyright © 2010 Society of Chemical Industry     

Enzymatic Synthesis of Biodiesel from Transesterification Reactions of Vegetable Oils and Short Chain Alcohols

Biodiesel synthesis by alcoholysis of three vegetable oils (soybean, sunflower and rice bran) catalyzed by three commercial lipases (Novozym 435, Lipozyme TL-IM and Lipozyme RM-IM), and the optimization of the enzymes stability over repeated batches is described. The effects of the molar ratio of alcohol to oil and the reaction temperature with methanol, ethanol, propanol and butanol were also studied. All three enzymes displayed similar reaction kinetics with all three oils and no significant differences were observed. However, each lipase displayed the highest alcoholysis activity with a different alcohol. Novozym 435 presented higher activity in methanolysis, at a 5:1 methanol:oil molar ratio; Lipozyme TL-IM presented higher activity in ethanolysis, at a 7:1 ethanol:oil molar ratio; and Lipozyme RM-IM presented higher activity in butanolysis, at a 9:1 butanol:oil molar ratio. The optimal temperature was in the range of 30–35 °C for all lipases. The assessment of enzyme stability over repeated batches was carried out by washing the immobilized enzymes with different solvents (n-hexane, water, ethanol, or propanol) after each batch. When washing with n-hexane, approximately 90% of the enzyme activity remained after seven synthesis cycles.     

Acquisition of Water Solubility Diagrams in Ternary Systems (AOT/Organic Solvent/Alcohol) and Extraction of α-Lactalbumin Using Reverse Micellar Systems

In this study water solubility curves were constructed and calorimetric measurements obtained for reverse micellar systems consisting of an alcohol (isopropanol or butanol), surfactant (AOT) and organic solvent (isooctane or hexane). Also evaluated were the effects of alcohol and solvent type and surfactant concentration on the extraction of the α-lactalbumin (α–la). From the obtained solubility diagrams for ternary systems, it was concluded that isooctane presented the highest water solubility capacity in the center of the micelle systems with hexane, since isooctane has greater molecular volume and greater effect of the surfactant aggregation number. With respect to the alcohols, it was observed that isopropanol and butanol act in the system as a co-surfactant, since they prefer to adsorb at the water/solvent interface. It was also verified that butanol improved water solubility inside the reverse micellar due to its contribution to increase the critical packing parameter. The amount of α-la extracted increased proportionally with the AOT concentration for systems with isooctane and hexane. However, for systems with the latter solvent, the concentration of extracted protein first increases and then decreases. The extraction power of reverse micellar systems with isooctane was influenced by the type of alcohol with butanol showing better results. For systems containing hexane there was no effect of the alcohol added to the system on extraction power of α-lactalbumin.     

Characteristics of aqueous microenvironments in non‐ionic surfactant reverse micelles and their use for enzyme reactions in non‐aqueous media

The polarity of water solubilized in the interior of non‐ionic surfactant reverse micellar systems has been investigated using optical probes. Small amounts of water incorporated on the inside of various reverse micellar systems, polyoxyethylene sorbitan trioleate (Tween‐85)/isopropyl alcohol/n‐hexane mixture and polyoxyethylene tert‐octylphenyl ether (TX‐100)/cyclohexane, were found to have low polarity compared with bulk water. The polarity of water in both Tween‐85 and TX‐100 reverse micellar systems corresponded to that of methanol. The activity of the proteases α‐chymotrypsin and subtilisin in Tween‐85 and TX‐100 reverse micellar systems has also been investigated. It was found that the amounts of water in the reverse micellar system have a significant influence on the activity of enzymes. In the TX‐100 reverse micellar system, neither enzymes showed any activity. In contrast, the activity of enzymes in the Tween‐85 reverse micellar system was similar to that in the aqueous system and followed standard Michaelis–Menten kinetics. Copyright © 2004 Society of Chemical Industry     

Phase behavior and hydrated solid structure in lysophospholipid/long-chain alcohol/water system and effect of cholesterol addition

Phase behavior in lysophospholipid/long-chain alcohol/water system at 80°C was investigated using hexanol and oleyl alcohol as the long-chain alcohol. Similarly to hydrophilic surfactant, a micellar phase in a lysophospholipid/water system transitioned to a lamellar liquid-crystalline phase by the addition of long-chain alcohol. In the oleyl alcohol system the lamellar liquid-crystalline phase was observed in wider region compared to the hexanol system. The effect of cholesterol addition on the phase behavior was also studied. The region of liquid-crystalline phase and (reverse micellar + liquid-crystalline + water) phase shifted towards higher lysophospholipid concentrations. The structure of hydrated solid as well as the transition between lamellar liquid-crystalline phase and hydrated solid was analyzed by X-ray scattering measurement and differential scanning calorimetry measurement. It was revealed that the hydrated solid was α-type crystals with lamellar structure. The hydrated solid (gel)-liquid crystal transition temperature gradually decreased with increasing oleyl alcohol concentration and the decrement was enhanced by the addition of cholesterol.     

Pd@Al2O3‐Catalyzed Hydrogenation of Allylbenzene to Propylbenzene in Methanol and Aqueous Micellar Solutions

The palladium on alumina (Pd@Al₂O₃)‐catalyzed hydrogenation of allylbenzene to propylbenzene was studied in methanol and aqueous micellar solutions of sodium dodecyl sulfate (SDS), decyltrimethylammonium bromide (DTAB), and t‐octylphenoxypolyethoxyethanol (TX‐100). Over Pd@Al₂O₃, propylbenzene was obtained via direct hydrogenation of allylbenzene and isomerization to β‐methylstyrene which was hydrogenated afterwards. In aqueous micellar solutions, the reaction was faster than in pure water, but slower than in methanol due to lower hydrogen solubility. In the H₂O/SDS system, a higher activation energy was obtained than in methanol. For the investigated surfactants, the initial reaction rate in the micellar systems decreased in the order SDS > TX‐100 > DTAB.     

Production of bio-diesel fuels by transesterification of rice bran oil

Transesterification of rice bran oil was investigated to produce the bio-diesel oil. Experimental conditions included molar ratio of rice bran oil to alcohol (1:3, 1:5 and 1 :7), concentration of catalyst used (0.5, 1.0 and 1.5 wt%), types of catalysts (sodium methoxide, NaOH and KOH), reaction temperatures (30, 45 and 60°C) and types of alcohols (methanol, ethanol and butanol). The conversion of rice bran oil increased with the alcohol mixing ratio and with the reaction temperature. Sodium methoxide was the most effective among the catalysts. The conversion was increased with the concentration of catalyst, but slightly increased over 1.0 wt%. The best conversion was obtained using methanol with sodium methoxide. In that case, 98% conversion was achieved within 1 hr. The physical properties of rice bran oil for diesel fuel can be significantly improved by transesterification reaction.     

By-products of ethoxylation of simple alcohols in the presence of tripropylamine

Ethoxylation of simple alcohols such as methanol, ethanol and butanol in the presence of tripropylamine, in addition to the main reaction products, results in by-products from tripropylamine and ethylene oxide. This side-reaction consumes tripropylamine, forming dipropylamine ethoxylates and alcohol ethoxylates. These compounds remain in the product and influence the commercial properties of alcohol oxyethylates.     

乙酸与丁醇酯化反应和渗透蒸发耦合过程研究

以硫酸锆催化乙酸和丁醇的酯化反应为例对渗透蒸发和酯化反应耦合过程进行研究 ,考察了温度、反应物初始摩尔比、膜面积与反应液体积比和催化剂浓度对耦合过程的影响。     

Reverse micellar extraction of bromelain from Ananas comosus L. Merryl

BACKGROUND: A reverse micellar system (RMS) of ionic surfactants is used for the first time for the extraction and primary purification of fruit bromelain (EC 3.4.22.33) from the aqueous extract of pineapple (Ananas comosus L. Merryl). The effect of various process parameters on both forward and back extraction of bromelain is studied to improve the extraction efficiency of RMS. Most of the reverse micellar extraction (RME) studies reported so far are on model systems and its application to enzyme extraction from a natural source is rarely reported. RESULTS: Studies carried out with ionic surfactants sodium bis(2‐ethylhexyl)sulfosuccinate (AOT) and cetyltrimethylammonium bromide (CTAB) confirmed that electrostatic interaction was the main driving force for the extraction of fruit bromelain. Among the two surfactants studied, CTAB was found to be the most suitable for the extraction of fruit bromelain with respect to activity recovery (97.56%) and degree of purification (4.54 fold) when employed as a 150 mmol L^?1 CTAB/iso‐octane/5% (v/v) hexanol/15% (v/v) butanol system. Activity recovery with a counterionic system is higher (94.30%) in comparison with isopropyl alcohol added system (85.35%). CONCLUSION: RME could be used as an efficient primary purification step for the recovery of bromelain from pineapple juice. Reverse micellar phase components can easily be recovered and efficiently reused for fresh or subsequent extraction, which contributes favorably to the process economics and environmental issues. Copyright © 2007 Society of Chemical Industry     

非晶态Ni(OH)2电极材料的制备工艺

采用微乳液快速冷冻沉淀法制备非晶态Ni(OH)2. 通过单因素及正交实验研究反应体系的pH值、反应温度和时间等因素对制备的非晶态Ni(OH)2电化学性能的影响. 结果表明,主要影响因素为pH值,其次为反应温度和时间. 采用TX-100/正丁醇/环己烷/水体系,控制TX-100与正丁醇的体积比为1:15,W值(水与表面活性剂质量比)为15.1,pH为12,反应时间2 h,反应温度55℃的条件下进行反应,放入0~5℃的超低温恒温槽中快速冷冻沉淀,合成出Ni(OH)2非晶相粉体电极活性材料,该材料的放电比容量达333.22 mA×h/g,具有较高的电化学容量. 初步探讨了微乳液快速冷冻沉淀法制备非晶态Ni(OH)2粉体的作用机理.     

Exclusion and Tortuosity Effects for Alcohol/Water Separation by Zeolite-Filled PDMS Membranes

Abstract

A resistance model has been developed to describe the increased pervaporation flux and selectivity for the separation of ethanol/water mixtures with silicalite-filled silicone rubber (SR) membranes as compared to unfilled SR membranes. The model interprets the increased component flux for ethanol in terms of an increasing ethanol permeability of the membrane. Membrane permeability is given as a function of rubber and silicalite permeabilities and of the silicalite content of the membrane. It is shown that silicalite permeability varies with the type of alcohol and the alcohol concentration in the feed mixture. In the series methanol, ethanol, propanol, and butanol, the alcohol permeability of silicalite varies with the length of the alcohol molecule, the lowest permeability being found for butanol. In the presence of propanol and butanol, the silicalite particles are impermeable to water and obstruct water transport through the membrane.     

Lipase-Mediated Synthesis of Fatty Acid Esters Using a Blending Alcohol Consisting of Methanol and 1-Butanol

Lipase-mediated transesterification of soybean oil with a blending alcohol consisting of methanol and 1-butanol for synthesis of fatty acid esters was carried out. Lipase from Thermomyces lanuginosa (Lipozyme TL IM) was used as a biocatalyst. The lipase was purchased from Novozymes (Seoul, Republic of Korea). The effects of the molar proportions of methanol and 1-butanol in the blending alcohol, reaction temperature, enzyme loading and water content were investigated, for reaction optimization. The relative consuming rates of methanol and 1-butanol during the reaction were also explored. Among seven different ratios of alcohol blends employed in this study, that containing 80 mol% methanol gave the highest yield of fatty acid esters. Optimum reaction temperature, enzyme loading, and water content were 30 °C, 15% (based on the substrate weight), and 0.3% (based on the substrate weight), respectively. Water influenced significantly the reaction rate and yield. On the transesterification, the degree of reaction of methanol was higher than that of 1-butanol and the presence of 1-butanol contributed to increase of the reaction rate as well as yield. The maximum yield of ca. 98 wt% was achieved under the optimized condition.     

Synthesis of Chloroalkoxy eicosanoic and docosanoic acids from meadowfoam fatty acids by oxidation with aqueous sodium hypochlorite

Chloroalkoxy substituted C₂₀ and C₂₂ fatty acids can be synthesized from the unsaturated fatty acids in meadow-foam oil by reaction of the fatty acids with primary or secondary alcohols and an aqueous sodium hypochlorite solution (commercial bleach). The reactions are conducted at room temperature for 3 h. Chlorohydroxy fatty acid derivatives are formed as by-products owing to the presence of water in the reaction mixture. Chlorinated δ-lactones are also produced by direct reaction of sodium hypochlorite with the Δ5 unsaturated fatty acids present in meadowfoam or by ring closure of the 6-chloro-5-hydroxy fatty acids. The product yield of chloroalkoxy fatty acids is dependent on the nature and volume of the alcohol used in the reaction, as well as the concentration and pH of the sodium hypochlorite solution. Primary alcohols such as methanol and butanol produce maximal yields (50–60%) of chloroalkoxy fatty acids whereas the secondary alcohol 2-propanol gives a 30% yield. Chloroalkoxy fatty acid yields can be increased to 75–80% by elimination of water from the reaction mixture through a procedure that partitions sodium hypochlorite from water into hexane/ethyl acetate mixtures. All of the reaction products were fully characterized using nuclear magnetic resonance and gas chromatography-mass spectrometry.     

Elastomer solvent interactions III-effects of methanol mixtures on fluorocarbon elastomers

Fluorocarbon elastomers exhibit high volume swell in methanol, but not in ethanol or other alcohols. Proton nuclear magnetic resonance (NMR) data indicate that neat methanol exists as a hydrogen-bonded tetramer with a solubility parameter close to that of the fluorocarbon elastomer, thus causing the high swell. The NMR and volume swell data show further correlations: Increased temperature or dilution of methanol with nonpolar solvents or water breaks down the hydrogen-bonded structure and reduces the volume swell. Mixtures of methanol and ethanol form mixed tetramer species. In this case the volume swell can be predicted by statistical theory which indicates that an all-methanol tetramer and a tetramer containing one ethanol molecule are the only swelling species. Similarly, a tetramer singly substituted with butanol is too bulky to swell the elastomer. Thus the molecular structure of methanol and its mixtures as determined by NMR provides an explanation for the swelling of fluorocarbon elastomers in these environments.     

角质酶产生菌Thermobifida fusca WSH03-11诱变及高产突变株发酵条件优化

以Thermobifida fusca WSH03-11为出发菌株,经硫酸二乙酯诱变,获得一株遗传稳定性好的高产角质酶的突变菌株(酶活由2.3 U/mL提高为8.9 U/mL)。确定了以乙酸钠为碳源,并在2.5 L发酵罐中研究了不同pH值对突变株角质酶分批发酵的影响。根据不同pH值下发酵过程中细胞比生长速率及产物比生长速率的变化,确定了分阶段控制pH值的策略,即在发酵前20 h控制pH值7.3、20 h后控制pH值7.6,这种条件下角质酶活达到19.8 U/mL,比采用单一pH值7.0、7.3、7.6、7.9下的最大值分别提高了125%,64%,37%,47%。     

AOT逆胶束体系脂肪酶催化合成油酸乙酯

在二-(2-乙基己基)琥珀酸酯磺酸钠和异辛烷构建的逆胶束体系中,以Lipex脂肪酶为催化剂,合成了油酸乙酯,考察了各影响因素对其产率的影响,并进行了脂肪酶紫外荧光检测和体系粒度分析. 结果表明,Lipex脂肪酶具有良好的催化活性,反应条件优化选用异辛烷为溶剂,在反应温度25℃、缓冲液pH 6.5、水/表面活性剂(摩尔比)10、乙醇/油酸(摩尔比)20及Lipex脂肪酶浓度0.035 g/L、油酸浓度0.005 mol/L、摇床转速150 r/min、反应36 h的条件下,油酸乙酯产率达到71.25%. 逆胶束粒度和酶构象直接影响酶活性,最适逆胶束粒度约为80 nm.     

Operational stability of cutinase in organic solvent system: model esterification of alkyl esters

BACKGROUND: The present work aims to gain further insight on the use of Fusarium solani pisi cutinase from a Saccharomyces cerevisiae strain for the synthesis of short chain ethyl esters, a group of important fruit flavor compounds, in a non‐conventional environment. Synthesis is promoted by cutinase in organic media, in particular, in iso‐octane, an organic solvent recognized as a safe ingredient in food and beverage industrial processes. RESULTS: The effect of solvent and substrate components of the reaction mixture on the enzyme stability was measured separately. Focus was given to the effect of reaction medium on the operational stability of cutinase and on the esterification yield after 24h. The feasibility of the operation in fed batch mode was successfully evaluated. The effect of the addition of substrate in consecutive pulses in the activity and stability of the biocatalyst was again assessed. CONCLUSIONS: The bioconversion system used in this work allowed for the sustained production of short chain alkyl esters for more than 45 days, which is suggestive of the stability of cutinase in the organic environment evaluated. Copyright © 2010 Society of Chemical Industry     

磷钨杂多酸催化合成柠檬酸三丁酯

考察了以磷钨杂多酸为催化剂,催化柠檬酸与正丁酯合成柠檬酸三丁酯(TBC)的反应.结果表明,磷钨杂多酸对该反应表现出很高的催化活性.当醇酸比为4.0∶1,催化剂用量占醇酸总质量3％,反应时间3.5 h,反应温度150℃,TBC的收率达到97.96％.     

Reduction of Vegetable Oil-Derived Fatty Acid Methyl Esters toward Fatty Alcohols without the Supply of Gaseous H2

An alternative route to the conventional one for fatty alcohol synthesis was investigated. It was possible to synthesize lauryl alcohol from methyl laurate via reduction by transfer of hydrogen and hydride in liquid phase, in noncatalytic reactions and without the supply of H₂ gaseous. Pure NaBH₄ or alumina-supported NaBH₄ and methanol were used as co-reactants and 100% fatty alcohol selectivities were achieved. The aim of supporting the metal hydride was to increase its stability and achieve the full recovery of the solid at the end of reaction. When alumina-supported NaBH₄ was used, a final fatty alcohol yield of 93% was achieved. The use of methanol and NaBH₄ in amounts higher than stoichiometric is important to generate alkoxyborohydride anions which act as better reducing species than NaBH₄. The reaction conditions effect was investigated and the role of short carbon chain alcohol structure was elucidated. The effect of fatty acid methyl ester structure was also studied. Fatty acid methyl esters with shorter carbon chain length and without unsaturation (methyl laurate, methyl myristate) were easily reduced using NaBH₄/Al₂O₃ and methanol reaching high conversions and fatty alcohol selectivities. Unsaturated fatty acid methyl ester with longer carbon chain (methyl oleate) introduced steric hindrance which disfavoured interaction between ester and reducing solid surface and fatty acid methyl ester conversion was noticeably lower. A reaction mechanism based on alkoxyborohydride anions as the actual reducing species was proposed. This mechanism fully interprets results obtained during fatty acid methyl ester reduction using short carbon chain alcohols and metal hydride.