Efficient preparation of (R)-alpha-monobenzoyl glycerol by lipase catalyzed asymmetric esterification: optimization and operation in packed bed reactor

Optically active (R)-alpha-monobenzoyl glycerol (MBG) was synthesized by Candida antarctica lipase B (CHIRAZYME L-2) catalyzed asymmetric esterification of glycerol with benzoic anhydride in organic solvents. Various conditions, such as the type and composition of the organic solvent, water content of the system, reaction temperature, and concentrations of the substrates were systematically examined and optimized in screw-capped test tubes with respect to both the reaction rate and the enzyme selectivity. 1,4-Dioxane was found to be the best solvent and no additional water was needed for the system. The optimum temperature was around 30 degrees C, while the most suitable substrate concentrations were 100 mM each for glycerol and benzoic anhydride, respectively. However, when excessive anhydride (e.g., 200 mM) was used, the produced MBG could be further transformed into 1,3-dibenzoyl glycerol (DBG) by the same enzyme with a priority to (S)-MBG, resulting in a significant improvement of the product optical purity from ca. 50-70% e.e. Under optimal conditions (100 mM glycerol, 100-200 mM benzoic anhydride, dioxane, 25-30 degrees C), the enzymatic synthesis of (R)-MBG was successfully operated in a packed-bed reactor for about 1 week, with an average productivity of 0.79 g MBG/day/g biocatalyst in the case of continuous operation and 0.94 g MBG/day/g biocatalyst in the case of semicontinuous operation. After refinement and preferential crystallization of the crude product, (R)-MBG could be obtained in an almost optically pure form (>98% e.e.).     

Immobilization of steapsin lipase on macroporous immobead-350 for biodiesel production in solvent free system

Commercially available steapsin lipase was immobilized on macroporous polymer beads (IB-350) and further investigated for biodiesel production under solvent free conditions. The fatty acid methyl ester (biodiesel) synthesis was carried out by the methanolysis of fresh and used cooking sunflower oil. The enzymatic reaction for biodiesel synthesis was optimized with various reaction parameters and the obtained reaction conditions were 1: 6 molar ratio (oil: methanol), 50 mg biocatalyst and 20% water content at 45°C for 48 h under solvent free conditions. It was observed that 94% of biodiesel was produced under the optimized reaction conditions. The four step addition of methanol at the interval of 12 h was found to be more effective. Moreover the biocatalyst was effectively reused for four consecutive recycles and was appreciably stable for 90 days. The results obtained highlight potential of immobilized steapsin lipase for biodiesel production.     

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 synthesis of ethyl glucoside lactate in non-aqueous system

Ethyl glucoside lactate, a novel α-hydroxy acid derivative, was synthesized by transesterification in non-aqueous phase using immobilized lipase as biocatalyst. Parameters such as solvent type, substrate concentration, reaction temperature, and enzyme concentration were investigated to optimize the lipase-catalyzed transesterification. In solvent-free system with butyl lactate as both acyl donor and solvent, a 71% conversion was achieved. In order to investigate the effect of initial water content, the reactions were carried out in the mediums treated with molecular sieves. The results showed that conversion and initial rate decreased with the increase of water content. The conversion and initial rate reached to 95% and 67.4 mM/h, respectively, by carrying out the reaction under reduced pressure, which was employed to eliminate butanol and the initial water.     

Optimized butyl butyrate synthesis catalyzed by Thermomyces lanuginosus lipase

Butyl butyrate is an ester present in pineapple flavor, which is very important for the food and beverages industries. In this work, the optimization of the reaction of butyl butyrate synthesis catalyzed by the immobilized lipase Lipozyme TL‐IM was performed. n‐Hexane was selected as the most appropriate solvent. Other reaction parameters such as temperature, substrate molar ratio, biocatalyst content and added water, and their responses measured as yield, were evaluated using a fractional factorial design, followed by a central composite design (CCD) and response surface methodology. In the fractional design 2^4–1, the four variables were tested and temperature and biocatalyst content were statistically significant and then used for optimization on CCD. The optimal conditions for butyl butyrate synthesis were found to be 48°C; substrate molar ratio 3:1 (butanol:butyric acid); biocatalyst content of 40% of acid mass. Under these conditions, over 90% of yield was obtained in 2 h. Enzyme reuse was tested by washing the biocatalyst with n‐hexane or by direct reuse. The direct reuse produced a rapid decrease on enzyme activity, while washing with n‐hexane allowed reusing the enzyme for five reactions cycles keeping approximately 85% of its activity. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1416–1421, 2013     

Improvement of the enzymatic synthesis of ethyl valerate by esterification reaction in a solvent system

The present study reports the improved enzymatic synthesis of ethyl valerate (green apple flavor) by esterification reaction of ethanol and valeric acid in heptane medium. Lipase from Thermomyces lanuginosus (TLL) was immobilized by physical adsorption on polyhydroxybutyrate (PHB) particles and used as a potential biocatalyst. The effect of certain parameters that influence the ester synthesis was evaluated by factorial design. The experimental conditions that maximized the synthesis of ethyl valerate were 30.5°C, 18% m/v of biocatalyst (TLL–PHB), absence of molecular sieves, agitation of 234?rpm, and 1,000?mM of each reactant (ethanol and valeric acid). Under these conditions, conversion percentage ≈92% after 105?min of reaction was observed. Soluble TLL was also used as biocatalyst and the highest conversion was of 82% after 120?min of reaction. Esterification reaction performed in a solvent-free system exhibited conversion of 13% after 45?min of reaction catalyzed by immobilized lipase, while the soluble lipase did not exhibit catalytic activity. The synthesis of the ester was confirmed by Fourier transform infrared spectroscopy and gas chromatography–mass spectrometry analyses. After six consecutive cycles of ethyl valerate synthesis, the prepared biocatalyst retained ≈86% of its original activity.     

Immobilized cells biocatalyst for the production of S-acetylthio-2-methyl propionic acid

S-acetylthio-2-methylpropionic acid (S-AMPA) is an important chiral intermediary for numerous hypertension drugs such as captopril. S-AMPA can be produced by hydrolyzing the corresponding racemic methyl MAMP (S,R-methyl-β-acetylthioisobutyrate) by lipases or esterases that have the appropriate stereo specificity. Psudomonas fluorescens IFO 12055 possessing a highly specific lipase was used to process this reaction in the form of immobilized cells. Reaction kinetic and immobilization methods were also studied. Strong product inhibition was observed, that is, at 3% S-AMPA (namely 183 mM), activity was reduced by 50%. Spontaneous hydrolysis of the ester and thioester bonds was also observed, and was independent of the cells. Thus, reaction selectivity and yield must be optimized through adjusting the substrate concentration and total biocatalyst activity. Conventional calcium alginate (3% w/w) encapsulation was modified by adding 3% w/w polyethyleneimine (PEI) and cross-linked by a biologically derived agent, genipin (5.6 mM). This method was found to be satisfactory to produce stable and functioning biocatalyst and can maintain high reactivity for repeated 25 batches with e.e. values above 90%.     

复合脂肪酶协同催化制备生物柴油的研究

【目的】探讨复合酶协同催化体系在含水量较高的体系中催化油脂制备生物柴油的工艺条件。【方法】通过基因工程手段在毕赤酵母中分别高效分泌表达南极假丝酵母脂肪酶(CALB)和米根霉脂肪酶(ROL),构建CALB和ROL复合酶协同催化体系制备生物柴油,利用单因素实验优化工艺条件,以甲酯化得率作为复合酶协同催化体系效能的评价标准。【结果】优化工艺条件为:CALB?ROL最佳复合酶配比为7?3,每克大豆油中加入16 U的复合脂肪酶,甲醇与大豆油摩尔比为4?1,并按0 h时2?1醇油摩尔比,12 h和24 h时以1?1醇油摩尔比分批加入甲醇,含水量为30%-60%之间,40°C反应29-34 h,甲酯得率达到93%。【结论】该复合酶协同催化体系对环境友好,与常规酶法制备生物柴油工艺相比对酶的使用量和催化时间减少幅度都在50%以上,本复合酶协同催化体系能有效降低生物柴油制备成本,具有较好的工业化应用前景。     

Synthesis of monoacylglycerol containing pinolenic acid via stepwise esterification using a cold active lipase

High purity monoacylglycerol (MAG) containing pinolenic acid was synthesized via stepwise esterification of glycerol and fatty acids from pine nut oil using a cold active lipase from Penicillium camembertii as a biocatalyst. Effects of temperature, molar ratio, water content, enzyme loading, and vacuum on the synthesis of MAG by lipase‐catalyzed esterification of glycerol and fatty acid from pine nut oil were investigated. Diacylglycerol (DAG) as well as MAG increased significantly when temperature was increased from 20 to 40°C. At a molar ratio of 1:1, MAG content decreased because of the significant increase in DAG content. Water has a profound influence on both MAG and DAG content through the entire course of reaction. The reaction rate increased significantly as enzyme loading increased up to 600 units. Vacuum was an effective method to reduce DAG content. The optimum temperature, molar ratio, water content, enzyme loading, vacuum, and reaction time were 20°C, 1:5 (fatty acid to glycerol), 2%, 600 units, 5 torr, and 24 h, respectively. MAG content further increased via lipase‐catalyzed second step esterification at subzero temperature. P. camembertii lipase exhibited esterification activity up to ?30°C. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

High-performance continuous operation for enanthioselective esterification of menthol by use of acid anhydride and free lipase in organic solvent

By using propionic anydride as a highly reactive and non-water-producing acyl donor, and free lipase OF 360 suspended in cyclohexane as a biocatalyst under the optimized conditions, efficient and stable enantioselective esterification of (+-)-menthol is possible and has been carried out very stably for 2 months in a continuous stirred-tank reactor at 30°C, retaining a 47%–35% conversion of menthol and high optical purity of the (–)-menthyl ester produced (95%–98% e.e.). However, when the corresponding propionic acid was used as an acyl donor, the conversion ratio of menthol decreased rapidly after the start of continuous operation, indicating a higher productivity with the anhydride than with the free acid. In the case of the acid anhydride, the water content of the reaction medium, which has a great effect on the reactivity and stability of lipase, was easily controlled in long-term operation by the occasional micro-adjustment of the flow rate or concentration of the feed solution of the acid anhydride, and could essentially be kept constant (2–4 mM), avoiding the inactivation of the enzyme.     

Immobilization of Candida cylindracea lipase on poly lactic acid,polyvinyl alcohol and chitosan based ternary blend film: Characterization,activity, stability and its application for N-acylation reactions

The ecofriendly ternary blend polymer film was prepared from the chitosan (CH), polylactic acid (PLA) and polyvinyl alcohol (PVA). Immobilization of Candida cylindracea lipase (CCL) was carried out on ternary blend polymer via entrapment methodology. The ternary blend polymer and immobilized biocatalyst were characterized by using N₂ adsorption–desorption isotherm, SEM, FTIR, DSC, and (%) water content analysis through Karl Fischer technique. Biocatalyst was then subjected for the determination of practical immobilization yield, protein loading and specific activity. Immobilized biocatalyst was further applied for the determination of biocatalytic activity for N-acylation reactions. Various reaction parameters were studied such as effect of immobilization support (ratio of PLA:PVA:CH), molar ratio (dibutylamine:vinyl acetate), solvent, biocatalyst loading, time, temperature, and orbital speed rotation. The developed protocol was then applied for the N-acylation reactions to synthesize several industrially important acetamides with excellent yields. Interestingly, immobilized lipase showed fivefold higher catalytic activity and better thermal stability than the crude extract lipase CCL. Furthermore various kinetic and thermodynamic parameters were studied and the biocatalyst was efficiently recycled for four successive reuses. It is noteworthy to mention that immobilized biocatalyst was stable for period of 300 days.     

Continuous enantioselective esterification of trans-2-phenyl-1-cyclohexanol using a new Candida rugosa lipase in a packed bed bioreactor

Enantioselective resolution of trans-2-phenyl-1-cyclohexanol (TPCH) by a Candida rugosa lipase, obtained by fermentation in the laboratory, and immobilised on EP100 polypropylene powder has been carried out using isooctane as solvent and propionic acid as esterifying agent. The study have included the utilisation of this biocatalyst in a batch process and the optimisation of the esterification conditions by means of a Box-Hunter-based experimental design. The main variables controlling the process, concentration of acid and alcohol, have been numerically optimised using initial esterification rate as objective function. The optimal concentrations for the batch process were 50 mM for the alcohol and 71 mM for the acid. This esterification reaction kinetics corresponded to a reversible Michaelis-Menten kinetic law for the optimal conditions, which has permitted to select a plug-flow packed bed bioreactor as the most appropriate configuration to minimise the residence time and to avoid shear stress effect on the biocatalyst. The behaviour of the continuous packed bed bioreactor at two different residence times (302 and 582 min) was in accordance with predictions from batch experiments, with slightly deviations (less than 10%). Continuous experiments maintained high values of enantioselectivity (enantiomeric factor was practically 1) and conversion near equilibrium value (35%) when long-time operation was carried out. Besides, long-time stability of biocatalyst has permitted to scale-up the production of enantioenriched (1R,2S)-TPCH propionate to yield gram quantities.     

Promiscuous enzyme-catalyzed cascade reaction: Synthesis of xanthone derivatives

Based on the screening of biocatalysts and reaction conditions including organic solvent, water content, lipase loading, reaction temperature and time, lipase TLIM exhibited the prominent promiscuity for the Knoevenagel-Michael cascade reactions of 1, 3-diketones with aromatic aldehydes to synthesize xanthone derivatives. This procedure provides satisfactory advantages such as environmental begin, simple work-up, generality, obtaining in excellent yields (80–97%), and potential for recycling of biocatalyst.     

Plant lipases: biocatalyst aqueous environment in relation to optimal catalytic activity in lipase-catalyzed synthesis reactions.

Adsorption and desorption isotherms of two commercial enzyme preparations of papain and bromelain were determined with a Dynamic Vapor System. The Guggenheim-Anderson-deBoer (GAB) modeling of the obtained sorption isotherms allowed the definition of different levels of hydration of those samples. Afterward, these enzyme preparations were used as biocatalysts in water and solvent-free esterification and alcoholysis reactions. The evolution of the obtained fatty acid ester level as a function of the initial hydration level of the biocatalyst, i.e., thermodynamic water activity (a(w)) and water content, was studied. The results show an important correlation between the initial hydration level of the biocatalyst and its catalytic activity during the lipase-catalyzed synthesis reactions. Thus, the Carica papaya lipase (crude papain preparation) catalytic activity is highly dependent on the biocatalyst hydration state. The optimized synthesis reaction yield is obtained when the a(w) value of the enzyme preparation is stabilized at 0.22, which corresponds to 2% water content. This optimal level of hydration occurs on the linear part of the biocatalyst's sorption isotherm, where the water molecules can form a mono- or multiple layer with the protein network. The synthesis reaction yield decreases when the a(w) of the preparation is higher than 0.22, because the excess water molecules modify the system equilibrium leading to the reverse and competitive reaction, i.e., hydrolysis. These results show also that an optimal storage condition for the highly hydrophilic crude papain preparation is a relative humidity strictly lower than 70% to avoid an irreversible structural transition leading to a useless biocatalyst. Concerning the bromelain preparation, no effect of the hydration level on the catalytic activity during esterification reactions was observed. This biocatalyst has too weak a catalytic activity which makes it difficult to observe any differences. Furthermore, the bromelain preparation is far more hydrophobic as it adsorbs only 18 g of water per 100 g of dry material at a(w) around 0.90. No deliquescence of this enzymatic preparation is observed at this a(w) value.     

A new method to determine the aw range in which immobilized lipases display optimum activity in organic media

Summary We describe a qualitative method to predict the pre-equilibration a_w, system value in which, covalent immobilized lipase B from Candida antarctica to sepharose and silica, displayed best synthetic activity. The methodology is based in the analysis of the water adsorption isotherms of the biocatalyst in air and in the organic solvent. The biocatalyst is active at pre-equilibration a_w values higher than the divergence point between both isotherms. In addition, native and immobilized lipase display highest activity if the biocatalyst is pre-equilibrated at a_w=P point. For preparative purposes, the validity of the method was proved in the esterification of racemic 2-(4-isobutyl phenyl) propionic acid with 1-propanol in isooctane at long reaction time.     

Effect of water on lipase NS81006-catalyzed alcoholysis for biodiesel production

Biodiesel production catalyzed by free lipase has been drawing attention for its lower cost and faster reaction rate compared to immobilized lipase. It has been found that free lipase NS81006 could efficiently catalyze alkyl esters production and a certain amount of water is demonstrated to be necessary for the catalytic process. The effect of water content on liquid lipase NS81006-mediated methanolysis and ethanolysis for biodiesel production was first explored respectively in this paper. It was found that with water content ranging from 3% to 10% (based on oil weight), there was no significant difference in the final alkyl ester yield either in NS81006-mediated methanolysis or ethanolysis process, while the quality of biodiesel varied obviously. The acid value as well as the contents of monoglyceride and diglyceride were much lower in the lower water-containing system. With the water content decreasing from 10% to 3%, the acid value reduced from 8.24 to 4.89 mg KOH/g oil, and the content of MAG and DAG dropped to 0.31 and 0.22, from 0.62 and 0.74, respectively. Lipase could maintain rather good stability with proper alcohol adding strategy and the gradual reduction in biodiesel yield in the repeated uses resulted from the accumulation of by-product glycerol. The continuous running of lipase-mediated methanolysis of waste cooking oil was successfully realized at 30L reactor and a final methyl ester yield of over 90% could be obtained.     

Kinetic biosynthesis of l-ascorbyl acetate by immobilized Thermomyces lanuginosus lipase (Lipozyme TLIM)

Thermomyces lanuginosus lipase (Lipozyme TLIM)-catalyzed esterification of l-ascorbic acid was studied. It was suggested that Lipozyme TLIM was a suitable biocatalyst for enzymatic esterification of l-ascorbic acid. Three solvents were investigated for the reaction, and acetone was found to be a suitable reaction medium. Furthermore, it was found that water activity could notably affect the conversion. Moreover, pH memory of Lipozyme TLIM lipase for catalyzing l-ascorbic acid esterification in acetone was observed and the effect of pH on the reaction was estimated. In addition, the influences of other parameters such as substrate mole ratio, enzyme loading, and reaction temperature and reusability of lipase on esterification of l-ascorbic acid were also analyzed systematically and quantitatively. Kinetic characterization of Lipozyme TLIM showed that K _m,a and V _max were 80.085 mM and 0.747 mM min⁻¹, respectively. As a result, Lipozyme TLIM-catalyzed esterification of l-ascorbic acid gave a maximum conversion of 99%.     

Whole cell biocatalyst for biodiesel fuel production utilizing Rhizopus oryzae cells immobilized within biomass support particles

As part of a research program aimed at producing biodiesel fuel from plant oils enzymatically cells of Rhizopus oryzae (R. oryzae) IFO4697 (with a 1,3-positional specificity lipase) immobilized within biomass support particles (BSPs) were investigated for the methanolysis of soybean oil. The R. oryzae cells easily became immobilized within the BSPs during batch operation. To enhance the methanolysis activity of the immobilized cells under the culture conditions used, various substrate-related compounds were added to the culture medium. Among the compounds tested, olive oil or oleic acid was significantly effective. In contrast, no glucose was necessary. Immobilized cells were treated with several organic solvents, but none gave higher activity than untreated cells. When methanolysis was carried out with stepwise additions of methanol using BSP-immobilized cells, in the presence of 15% water the methyl esters (MEs) content in the reaction mixture reached 90% - the same level as that using the extracellular lipase. The process presented here, using a whole cell biocatalyst, is considered to be promising for biodiesel fuel production in industrial applications.     

Acidolysis between triolein and short-chain fatty acid by lipase in organic solvents

Ten kinds of lipases were examined as biocatalysts for the incorporation of short-chain fatty acids (acetic, propionic, and butyric acids) into triolein in order to produce one kind of reduced-calorie structured lipids. Trans-esterification (acidolysis) was successfully done in n-hexane by several microbial lipases. Among them, lipase from Aspergillus oryzae was used to investigate the effects of incubation time, substrate molar ratio, and water content on acidolysis. Finally, more than 80% of triolein was incorporated by butyric acid (molar ratio of triolein to butyric acid, 1:10) in the dried n-hexane at 52 degrees C for 72 h. More than 90% of the products was monosubstituent, which was esterified with this short chain fatty acid at the 1-position of the glycerol moiety of triolein. These results suggest that A. oryzae lipase would be a powerful biocatalyst for the synthesis of low caloric oil, such as triacylglycerol containing a mixture of long- and short-chain aliphatic acids.     

Adsorptive control of water in esterification with immobilized enzymes: I. Batch reactor behavior

Reducing the influence of an undesired product in an enzymatic reaction could have a significant impact on the productivity of such systems. Here, we focus on the removal of water formed during an enzymatic esterification in a batch reactor. A commercial immobilized lipase preparation, known as Lipozyme, is used as the biocatalyst and propionic acid and isoamyl alcohol dissolved in hexane are the substrates. In this system, the water formed will partition between the catalyst and the medium. As the more polar reactants are converted into the less polar ester product, the water is partitioned more towards the biocatalyst and the accumulation of water eventually causes lower reaction rates. Addition of a strong-acid cation exchange resin in sodium form is found to control the water accumulation on the biocatalyst without stripping the essential water needed for the enzyme to function and substantial improvements in conversion are achieved. A mathematical model is developed to describe the batch reaction behavior with and without added absorbent, which successfully predicts the behavior of water and its effects.