Enzymatic synthesis of 2-ethylhexyl esters of fatty acids by immobilized lipase from Candida sp. 99–125

The 2-ethylhexyl esters of fatty acids were synthesized by immobilized lipase from Candida sp. 99–125. The reuse stability of immobilized lipase was at least four batches. The conditions of enzymatic synthesis of 2-ethylhexyl palmitate were optimized. In the system of petroleum ether, 10% (w/w) immobilized lipase was used in the esterfication of 2-ethyl hexanol (7.8 mmol) and palmitic acid (7.8 mmol) at 40 °C with silica gel as the water absorbent. The esterification degree was 91% under these conditions. The purity of 2-ethylhexyl palmitate was 98% after purification consisting washing by water and evaporation to remove the organic solvent.     

Enhancing operational stability and exhibition of enzyme activity by removing water in the immobilized lipase‐catalyzed production of erythorbyl laurate

Erythorbyl laurate was continuously synthesized by esterification in a packed‐bed enzyme reactor with immobilized lipase from Candida antarctica. Response surface methodology based on a five‐level three‐factor central composite design was adopted to optimize conditions for the enzymatic esterification. The reaction variables, such as reaction temperature (10–70°C), substrate molar ratio ([lauric acid]/[erythorbic acid], 5–15), and residence time (8–40 min) were evaluated and their optimum conditions were found to be 56.2°C, 14.3, and 24.2 min, respectively. Under the optimum conditions, the molar conversion yield was 83.4%, which was not significantly different (P < 0.05) from the value predicted (84.4%). Especially, continuous water removal by adsorption on an ion‐exchange resin in a packed‐bed enzyme reactor improved operational stability, resulting in prolongation of half‐life (2.02 times longer compared to the control without water‐removal system). Furthermore, in the case of batch‐type reactor, it exhibited significant increase in initial velocity of molar conversion from 1.58% to 2.04%/min. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:882–889, 2013     

Construction of a non-support bioreactor: optical resolution of 2-(4-chlorophenoxy)propanoic acid in an organic solvent system

Summary A non-support bioreactor, a novel column reactor packed with a free non-supported enzyme was constructed by applying the insolubility of the enzyme in organic solvents. Stereoselective esterification of 2-(4-chlorophenoxy)propanoic acid by lipase OF 360 from Candida cylindracea with n-tetradecanol was selected as a model reaction. Non-supported lipase revealed threefold higher activity than Celite-adsorbed lipase by maintaining high stereoselectivity in a batch reaction. In continuous operation, a non-support bioreactor produced the ester with fourfold higher productivity to that of a column reactor packed with Celite-adsorbed lipase (an adsorbed bioreactor). However, the optical purity of the remaining (S)-acid was low even when the conversion ration was kept at approximately 50%. Lipase recovered from the non-support bioreactor after continuous operation retained the original stereoselectivity in a batch reaction. Therefore, semi-continuous operation was conducted by recycling the substrate solution at a high flow rate. The non-support reactor showed high stereoselectivity and ten times the productivity compared with the adsorbed bioreactor. The reason for this high performance is discussed. Offprint requests to: A. Tanaka     

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

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

Enzymatic conversion of sunflower oil to biodiesel in a solvent-free system: Process optimization and the immobilized system stability

The feasibility of using the commercial immobilized lipase from Candida antarctica (Novozyme 435) to synthesize biodiesel from sunflower oil in a solvent-free system has been proved. Using methanol as an acyl acceptor and the response surface methodology as an optimization technique, the optimal conditions for the transesterification has been found to be: 45 ^oC, 3% of enzyme based on oil weight, 3:1 methanol to oil molar ratio and with no added water in the system. Under these conditions, >99% of oil conversion to fatty acid methyl ester (FAME) has been achieved after 50 h of reaction, but the activity of the immobilized lipase decreased markedly over the course of repeated runs. In order to improve the enzyme stability, several alternative acyl acceptors have been tested for biodiesel production under solvent-free conditions. The use of methyl acetate seems to be of great interest, resulting in high FAME yield (95.65%) and increasing the half-life of the immobilized lipase by about 20.1 times as compared to methanol. The reaction has also been verified in the industrially feasible reaction system including both a batch stirred tank reactor and a packed bed reactor. Although satisfactory performance in the batch stirred tank reactor has been achieved, the kinetics in a packed bed reactor system seems to have a slightly better profile (93.6 ± 3.75% FAME yield after 8–10 h), corresponding to the volumetric productivity of 48.5 g/(dm³ h). The packed bed reactor has operated for up to 72 h with almost no loss in productivity, implying that the proposed process and the immobilized system could provide a promising solution for the biodiesel synthesis at the industrial scale.     

Continuous hydrolysis of oil by immobilized lipase in a countercurrent reactor

Lipase from Pseudomonas fluorescens biotype I was immobilized by adsorption of anion exchange resin using glutaraldehyde to enhance the adsorption. The activity yield of the immobilized lipase was very low (below 1%) when lipase activity was measured using emulsion substrate. The activity yield was 10-70% when lipase activity was measured using non-emulsion substrate. Countercurrent reactors for hydrolysis of oil using non-emulsion substrate were studied. A fluidized bed reactor was found to be superior to a fixed bed one since in a fixed bed reactor the separation rate of the two layers was slow and the flow rate of the reactor had to be slower than the separation rate. A fluidized bed reactor system equipped with settling compartments and stirring compartments was devised. Continuous lipolysis at 60 degrees C and continuous separation of oily product and water soluble product were performed. After continuous operation for more than 3 months, 70% of the initial activity of the immobilized lipase was observed at the end of the reaction.     

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.     

Enzymatic Synthesis of Ethyl-glucoside Monooleate with Lipase in Solvent-free Medium

Ethylglucoside monooleate was synthesized by esterification between ethylglucoside and oleic acid with immobilized lipase from Candida antarctica in a solvent-free system. It was shown that a stirred tank reactor was suitable for the enzymatic reaction process involving substrates with low miscibility, in which the biocatalyst was recycled five times without significant activity loss. Removal of the co-product, water, from the reaction medium by carrying out the reaction under reduced pressure benefited the esterification reaction and increased the monooleate yield up to 97% within 8 hours.     

Assessment of process variables on 2-ethylhexyl palmitate production using Novozym 435 as catalyst in a solvent-free system

This work reports the optimization of 2-ethylhexyl palmitate production by esterification reaction in a solvent-free system using a commercial lipase as catalyst. For this, a sequential strategy was performed applying three experimental designs. An empirical model was built so as to assess the effects of process variables on the reaction conversion. Afterward, the operating conditions that optimized 2-ethylhexyl palmitate production were determined to be acid to alcohol molar ratio of 1:5.5, 70 °C, 150 rpm and 10.5 wt% of enzyme, leading to a reaction conversion as high as 93%. From this point, a kinetic study was carried out evaluating the influence of acid to alcohol molar ratio, enzyme concentration and temperature on product yield. Results obtained in this step allow to conclude that an excess of alcohol (acid to alcohol molar ratio of 1:6), relatively low enzyme concentration (10 wt%) and temperature of 70 °C led to nearly complete reaction conversion.     

微水——有机溶剂两相体系中消旋萘普生的酶法拆分

研究了微水－有机溶剂两相体系中固定化脂肪酶催化的萘甲酯的立体选择性水解反应,固定化酶活性受载体极性、水含量、有机溶剂的ｌｏｇＰ值,产物抑制的影响,据此构建了一种可以连续拆分产生（Ｓ）－（＋）－萘普生的微水－有机溶剂两相体系。反应在一个具有回路的连续流搅拌反应器中进行,反应器中添加有采用吸附法固定化的脂肪酶,截体为一种弱极性的合成载体,水相连同固定化酶颗粒一起永久保持在反应器中,有机流动相带入底物,     

Biodiesel production using Aspergillus niger as a whole‐cell biocatalyst in a packed‐bed reactor

Enzymatic lipase transesterification of palm oil to biodiesel in a packed‐bed reactor (PBR) using a novel strain of the fungus Aspergillus niger, immobilized within polyurethane biomass support particles (BSPs), was investigated. A three‐step addition of methanol was used to reduce lipase inhibition by immiscible methanol. The influence of water content and PBR flow rate was investigated. FAME yield was enhanced with an increase of PBR flow rate in the range of 0.15–30 L h^?1, where inefficient mixing of the reaction mixture at lower flow rates resulted in low conversion rates i.e. 69% after 72‐h reaction. Adding the third mole equivalent of methanol resulted in lipase inhibition due to methanol migration into the accumulated glycerol layer. Glutaraldehyde (GA) solution (0.5 vol.%) was used to stabilize lipase activity, which led to a high FAME yield (>90%) in the PBR after 72‐h of reaction time at a flow rate of 15 L h^?1, and a water content of 15%. Moreover, a high conversion rate (>85%) was maintained after four palm oil batch conversion cycles in the PBR. In contrast, lipase activity of non‐GA‐treated cells decreased with each PBR batch cycle, where only 70% FAME was produced after the forth PBR cycle. Transesterification of palm oil in a PBR system using BSPs‐immobilized A. niger as a whole‐cell biocatalyst is a viable process for enzymatic biodiesel production.     

Immobilized biocatalytic process development and potential application in membrane separation: a review

Biocatalytic membrane reactors have been widely used in different industries including food, fine chemicals, biological, biomedical, pharmaceuticals, environmental treatment and so on. This article gives an overview of the different immobilized enzymatic processes and their advantages over the conventional chemical catalysts. The application of a membrane bioreactor (MBR) reduces the energy consumption, and system size, in line with process intensification. The performances of MBR are considerably influenced by substrate concentration, immobilized matrix material, types of immobilization and the type of reactor. Advantages of a membrane associated bioreactor over a free-enzyme biochemical reaction, and a packed bed reactor are, large surface area of immobilization matrix, reuse of enzymes, better product recovery along with heterogeneous reactions, and continuous operation of the reactor. The present research work highlights immobilization techniques, reactor setup, enzyme stability under immobilized conditions, the hydrodynamics of MBR, and its application, particularly, in the field of sugar, starch, drinks, milk, pharmaceutical industries and energy generation.     

Enantioselective esterification of butanol-2 in an enzyme membrane reactor

The enzymatic esterification of octanoic acid with racemic butanol-2 was investigated. Esterifications of the acid were performed in a forced flow enzyme membrane reactor. The used membrane was prepared by a phase inversion process in polyamide-6 solution followed by the chemical immobilization of a lipase-catalyst. Influences of water content and pH were estimated. Their optimum values are equal to 0.5% w/w and pH 8. The reaction rate (at 303 K) of 5.1 × 10^?5 mol/h·cm² of the membrane area, and at least 85% enantiomeric excess in the produced ester mixture were obtained. The activity of immobilized lipase in the membrane process is about two times higher than that of the native lipase in the esterification performed in a tank reactor.     

Enzymatic Synthesis of Ethyl-glucoside Monooleate with Lipase in Solvent-free Medium

Ethylglucoside monooleate was synthesized by esterification between ethylglucoside and oleic acid with immobilized lipase from Candida antarctica in a solvent-free system. It was shown that a stirred tank reactor was suitable for the enzymatic reaction process involving substrates with low miscibility, in which the biocatalyst was recycled five times without significant activity loss. Removal of the co-product, water, from the reaction medium by carrying out the reaction under reduced pressure benefited the esterification reaction and increased the monooleate yield up to 97% within 8 hours.     

Optimization of lactic acid production by immobilized <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1

Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l⁻¹, the optimal production medium had the following initial concentrations of nutrients (g l⁻¹): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 °C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l⁻¹ h⁻¹. Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l⁻¹ h⁻¹. The packed bed could be used in repeated batch runs to produce lactic acid.     

A novel process for enzymatic synthesis of N-lauroyl-β-amino propionitrile using packed bed reactor coupled with on-line separation

N-Lauroyl-β-amino propionitrile is an intermediate for synthesis of sodium N-lauroyl-β-alanine, an antimicrobial surfactant. We provide a novel process for enzymatic synthesis of N-lauroyl-β-amino propionitrile, using a cascade connection of an enzyme packed bed reactor (EPBR) with a crystallization separator for on-line separation. The substrate solution was fed to the reactor inlet. High-purity crystal product was obtained from the separator outlet with a yield of 91.7% under the optimum conditions. The immobilized lipase can be utilized repeatedly. The solvent and unreacted substrates were recovered and reused on-line.     

Stereoselective enzymatic esterification of 3-benzoylthio-2-methylpropanoic acid

Summary A key intermediate, S-(–)-3-benzoylthio-2-methylpropanoic acid (1) was made in high optical purity by the lipase-catalyzed stereoselective esterification of racemic 1 with methanol in an organic solvent system. Among various lipases evaluated, Amano P-30 lipase from Pseudomonas sp. efficiently catalyzed the esterification of 1 to yield R-(+) methyl ester and unreacted S-(–) 1. A reaction yield of 40 mol% and an optical purity of 97.2% were obtained for compound 1 at a substrate concentration of 0.1 m (22 mg/ml). Lipase P-30 was immobilized on Accurel polypropylene (PP) and the immobilized enzyme was reused (23 cycles) in the esterification reaction without loss of enzyme acitivity, productivity or optical purity. Among various solvents evaluated, toluene was found to be the most suitable organic solvent and methanol was the best alcohol for the esterification of racemic 1 by immobilized lipase. Substrate concentrations as high as 1.0 m were used in the esterification reaction. When the temperature was increased from 28° C to 60° C, the reaction time required for the esterification of 0.1 m substrate decreased from 16 h to 2 h. On increasing the methanol to substrate molar ratio from 1:1 to 4:1, the rate of esterification decreased. A lipase fermentation using Pseudomonas sp. ATCC 21 808 was developed. In the batch-fermentation process, 56 units/ml of extracellular lipase activity was obtained. A fed-batch process using soybean oil gave a significant increase in the lipase activity (126 units/ml). Crude lipase recovered from the filtrate by ethanol precipitation and immobilized on Accurel PP was also effective: S-(–) compound 1 was obtained in 35 mol% yield and 95% optical purity. Offsprint requests to: R. N. Patel     

Efficient production of diltiazem chiral intermediate using immobilized lipase from <Emphasis Type="Italic">Serratia marcescens</Emphasis>

The lipase from Serratia marcescence ECU1010 (Sml) was capable of enantioselectively catalyzing the synthesis of many chiral drug precursors. This paper investigated the immobilization of Sml on appropriate supporting materials and its performance in bioreactor. Chitosan, Celite 545, and DEAE-cellulose were found to be the ideal supports among 8 carriers tested with respect to enzyme load and activity recovery of lipase. When Sml was immobilized, significant improvements of stability against pH, thermal, and operational deactivation were observed with all the 3 better supports, and the best stability was observed when the lipase was immobilized on glutaraldehyde activated chitosan. As for the effect of organic solvent in the biphasic reaction system, the hydrolytic activity of the immobilized lipase on trans-3-(4′-methoxyphenyl)glycidic acid methyl ester ((±)-MPGM) observed in isopropyl ether was 6 and 3 times higher than those in toluene and methyl tert-butyl ether. The lipasecatalyzed production of (−)-MPGM by enzymatic resolution of (±)-MPGM with chitosan-Sml in isopropyl etherwater biphasic system was carried out in a 2 L stirred-tank reactor. The batch operation was more efficient operation mode for the enantioselective hydrolysis of (±)-MPGM, affording enantiopure (−)-MPGM in 44.3% overall yield, in contrast to 29.3% in a continuous reactor.     

Biodiesel synthesis in a solvent-free system by recombinant Rhizopus oryzae: comparative study between a stirred tank and a packed-bed batch reactor

A simultaneous synthesis of biodiesel, as fatty acid methyl esters, and monoacylglycerols catalysed by the recombinant Rhizopus oryzae lipase immobilized by adsorption on Relizyme OD/403M is presented. The use of this 1(3)-positional specific lipase prevents the formation of glycerol as a by-product, thus avoiding its drawbacks. The synthesis was carried out in a solvent-free system and it has been studied in two different reactor systems: stirred tank and packed-bed reactor. Stirred tank reactor presented a high-initial reaction rate and achieved a 33.6% yield, which corresponds to a value of 50.4% of the maximum yield that can be achieved with a 1(3)-positional specific lipase. In packed-bed reactor there was a smaller initial reaction rate, but it was achieved a 49.1% yield, which corresponds to a 73.6% of the maximum yield. When a second batch is performed, the yield decreased only 4% when packed-bed reactor is employed whereas a drastic decrease is observed in a stirred tank operation. Therefore, packed-bed reactor showed a best performance and minor damage to the biocatalyst.     

Hydrolysis of rice bran oil using an immobilized lipase from Candida rugosa in isooctane

The kinetics of enzymatic hydrolysis of rice bran oil in isooctane by immobilized Candida rugosa lipase in a batch reactor showed competitive inhibition by isooctane with a dissociation constant, K1, of 0.92 M. Continuous hydrolysis of rice bran oil was performed in recycling, packed bed reactor with 4352 U of immobilized lipase; the optimum recycle ratio was 9 and the operational half-life was 360 h without isooctane but 288 h with 25% (v/v) isooctane in rice bran oil.