Hydrolysis of butteroil by immobilized lipase using a hollow-fiber reactor: part II. Uniresponse kinetic studies

A lipase from Aspergillus niger immobilized by adsorption on microporous, polypropylene hollow fibers was used to effect the hydrolysis of the glycerides of melted butterfat at 40 degrees C and pH 7.0. Mcllvane buffer was pumped through the lumen and melted butterfat was pumped courrently through the shell side of a shell-and-tube reactor. Nonlinear regression methods were employed to determine the kinetic parameters of three nested rate expressions derived from a Ping Pong Bi Bi enzymatic mechanism coupled with three nested rate expressions for the thermal deactivation of the enzyme. For the reaction conditions used in this research, a four-parameter rate expression (which includes a two-parameter deactivation rate expression and a two-parameter hydrolysis rate expression) is sufficient to model the overall release of free fatty acids from the triglycerides of butterfat as a function of space time and time elapsed after immobilization. At a space time of 3.7 h immediately after immobilization of lipase, 50% of the fatty acid residues esterified in the sn-1,3 positions of the triglycerides can be released in the hollow-fiber reactor.     

Hydrolysis of butteroil by immobilized lipase using a hollow-fiber reactor: part III. Multiresponse kinetic studies

A lipase from Aspergillus niger immobilized by adsorption on microporous, polypropylene hollow fibers was used to effect the continuous hydrolysis of the glycerides of butter oil at 40 degrees C and pH 7.0. The effluent concentrations of 10 different free fatty acid products were measured by highperformancee liquid chromatography (HPLC). Multiresponse nonlinear regression methods were used to fit the data to a multisubstrate rate expression derived from a Ping Pong Bi Bi mechanism in which the rate-controlling step is deacylation of the lipase. Thermal deactivation of the enzyme was also included in the mathematical model of reactor performance. A postulated normal distribution of v(max) with respect to the chain length of the fatty acid (with an additive correction for the degree of unsaturation) was tested for statistical significance. The model is useful for predicting the free fatty acid profile of the lipolyzed butteroil product over a wide range of flow rates.     

Hydrolysis of butteroil by immobilized lipase using a hollow-fiber reactor: IV. Effects of temperature

A lipase from Aspergillus niger, immobilized by adsorption on microporous polypropylene hollow fibers, was used to effect the hydrolysis of the glycerides of melted butterfat at pH. 7.0 at 40, 50, 55, and 60 degrees C. Mcllvane buffer was pumped upward through the lumen, and melted butterfat was pumped upward through the shell side of a hollow fiber reactor. Nonlinear regression methods were employed to determine the kinetic parameters of models based on combinations of three nested rate expressions for the hydrolysis reaction with three nested rate expressions for thermal deactivation of the enzyme. A rate expression containing four lumped parameters is sufficient to model the release of free fatty acids as a function of reactor space time and time elapsed after immobilization. Nonlinear regression methods were also employed in global fits of the data to rate expressions containing an explicit dependence on temperature. For the reaction conditions used in this research, a 14-parameter rate expression is necessary to accurately model the overall release of free fatty acids as a continuous function of the absolute temperature, initial substrate concentrations, reactor space time, and time elapsed after immobilization of the lipase.     

Production of lipolyzed butteroil by a calf pregastric esterase immobilized in a hollow fiber reactor: III. Effect of glycerol

Lipolysis of butter oil in a hollow fiber reactor containing an immobilized calf pregastric esterase was studied at 40 degrees C, a pH of 6.0, and glycerol concentrations of 0, 150, and 500 g/L in the buffer solution. The concentrations of 10 fatty acid species in the lipolyzed product were determined using high-performance liquid chromatography. The rate of loss of enzyme activity and the relative selectivities of this esterase depended on the glycerol concentration. By contrast, the overall rate of release of fatty acids was not affected by the glycerol concentration. Loss of enzyme activity was modeled using first-order kinetics. The models for deactivation and reaction kinetics were fit simultaneously to the data. The model was successful in describing the rates of release of all 10 fatty acid species for a range of space times from 0 to 25 h. The parameters of the model were tested for dependence on glycerol concentration.     

Use of a lipase immobilized in a membrane reactor to hydrolyze the glycerides of butteroil

A lipase from A spergillus niger, immobilized by adsorption on a microporous, polypropylene flat-sheet membrane, was used to effect the continous hydrolysis of the glycerides of melted butterfat at 35°C. For the reaction conditions used in this research, a pseudo-zero order rate expression can be used to model the kinetics of the overall hydrolysis of butterfat. Multiresponse nonlinear regression methods were employed to determine the kinetic parameters of a multisubstrate rate expression derived fro ma mechanism based on the general Michaëlis–Menten approach. For the multiresponse data taken at pH 7.0, the dependence of the maximum rate of release of each fatty acid residue of butterfat on its carbon chain length is accurately described by a skewed, bell-shaped (or Γ-type) distribution. Data taken at five different pH values were fit assuming a Dixon–Webb diprotic model for the pH dependence of the reaction rate. The thermal deactivation of the immobilized lipase obeyed first-order kinetics with a half-life of 19.9 days at 35°C. The multisubstrate model is useful for the prediction of the free fatty acid profile of lipolyzed butterfat, whereas the lumped-substrate model provides an estimate of the overall degree of hydrolysis as a function of the reactor space time.     

Biotechnology for the production of nutraceuticals enriched in conjugated linoleic acid: I. Uniresponse kinetics of the hydrolysis of corn oil by a Pseudomonas sp. lipase immobilized in a hollow fiber reactor

The kinetics of the hydrolysis of corn oil in the presence of a lipase from Pseudomonas sp. immobilized within the walls of a hollow fiber reactor can be modeled in terms of a three‐parameter rate expression. This rate expression consists of the product of a two‐parameter rate expression for the hydrolysis reaction itself (which is of the general Michaelis–Menten form) and a first‐order rate expression for deactivation of the enzyme. Optimum operating conditions correspond to 30°C and buffer pH values of 7.0 during both immobilization of the enzyme and the hydrolysis reaction. Under these conditions, the total fatty acid concentration in the effluent oil stream for a fluid residence time of 4 h is approximately 1.6 M. This concentration corresponds to hydrolysis of approximately 50% of the glyceride bonds present in the feedstock corn oil. The fatty acid of primary interest in the effluent stream is linoleic acid. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 568–579, 1999.     

Hydrolysis of menhaden oil by a Candida cylindracea lipase immobilized in a hollow-fiber reactor

A lipase from Candida cylindracea immobilized by adsorption on microporous polypropylene fibers was used to selectively hydrolyze the saturated and monounsaturated fatty acid residues of menhaden oil at 40 degrees C and pH 7.0. At a space time of 3.5 h, the shell and tube reactor containing these hollow fibers gives a fractional release of each of the saturated and monounsaturated fatty acid residues (i.e., C14, C16, C16:1, C18:1) of ca. 88% of the corresponding possible asymptotic value. The corresponding coproduct glycerides retained over 90% of the initial residues of both eicosapentaenoic (EPA; C20:5) and docosahexaenoic (DHA; C22:6) acids. The half-life of the immobilized lipase was 170 h when the reactor was operated at the indicated (optimum) conditions. Rate expressions associated with a generic ping-pong bi-bi mechanism were used to fit the experimental data for the lipase catalyzed reaction. Both uni- and multiresponse nonlinear regression methods were employed to determine the kinetic parameters associated with these rate expressions. The best statistical fit of the uniresponse data was obtained for a rate expression, which is formally equivalent to a general Michaelis-Menten mechanism. After reparameterization, this rate expression reduced to a pseudo-first-order model. For the multiresponse analysis, a model that employed a normal distribution of the ratio of Vmax/Km with respect to the chain length of the fatty acid residues provided the best statistical fit of the experimental data.     

Hydrolysis of rice bran oil using immobilized lipase in a stirred batch reactor

Candida cylindracea lipase was immobilized by adsorption on acid washed glass beads. It was observed that protein loading of the support depends on the size of the particle, with smaller particle containing higher amount of protein per unit weight. Initial reaction rate linearly varied up to enzyme concentration of 17.25 U/mL. Amount of free fatty acids produced was linearly proportional up to the enzyme loading of 1650 μg/g of bead. Achievement of chemical equilibrium took longer time in the case of less protein loading. Degree of hydrolysis was found to decrease in second and third consecutive batch operations on repeated use of immobilized lipase.     

Lipase kinetics: hydrolysis of triacetin by lipase from Candida cylindracea in a hollow-fiber membrane reactor

The aptitude of a hollow-fiber membrane reactor to determine lipase kinetics was investigated using the hydrolysis of triacetin catalyzed by lipase from Canadida cylindracea as a model system. The binding of the lipase to the membrane appears not to be very specific (surface adsorption), and probably its conformation is hardly altered by immobilization, resulting in an activity comparable to that of the enzyme in its native form. The reaction kinetics defined on the membrane surface area were found to obey Michaelis-Menten kinetics. The specific activity of the lipase in the membrane reactor was found to be significantly higher than in an emulsion reactor. The activity and stability of the enzyme immobilized on a hydrophilic membrane surface seem not to be influenced significantly by the choice of the membrane material. The hollow-fiber membrane reactor is a suitable tool to assess lipase kinetics in a fast and convenient way.     

Continuous interesterification of butteroil and conjugated linoleic acid in a tubular reactor packed with an immobilized lipase

Modified milkfats were produced via interesterification (acidolysis) reactions of butteroil and conjugated linoleic acid (CLA) in a packed bed reactor containing an immobilized lipase preparation from Candida antarctica. The rate expression for the interesterification reaction is of the generalized Michaelis–Menten form. Significant enrichment of butteroil in CLA residues was accomplished at reactor space times (fluid residence times) of 2–4 h at 40–60°C, but the optimum operating temperature was ca. 50°C. Approximately 80–90% of the free CLA fed to the reactor can be converted to its esterified form.     

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.     

Continuous production of monoacylglycerols from palm olein in packed-bed reactor with immobilized lipase PS

A packed-bed reactor (PBR) system using immobilized lipase PS as biocatalyst was developed for continuous monoacylglycerols (MAG) production. The condition for continuous MAG production using immobilized lipase PS (IM-PS) of 1.5 g (550 U) in PBR (0.68 cm i.d., 25 cm long) was optimized. The effect of molar ratio of glycerol to palm olein, water content in glycerol and residence time on MAG production was investigated. The optimal glycerol to palm olein molar ratio and water content in glycerol were 12:1 and 10% (w/w), respectively. The yield of MAG increased with increasing residence time. At a residence time of 7.5 h gave the highest yield of MAG of 60%. The long-term operation gave the highest yield of MAG 61.5% at 24 h of the operation time with the productivity of 1.61 g MAG/day. A half-life of the long-term process was 35 days of the operation time with the productivity of 0.81 g MAG/day. Furthermore, the large scale of MAG production was performed continuously with IM-PS of 15 g (5500 U) in PBR (1.5 cm i.d., 50 cm long). The highest yield of MAG in large-scale operation of 70.1% and the 11-fold increasing in productivity of 18.3 g MAG/day were obtained at 24 h of the operation time.     

Lipase-mediated acidolysis of butteroil with free conjugated linoleic acid in a packed bed reactor

Acidolysis of butteroil with free conjugated linoleic acid (CLA) was studied in a packed bed reactor containing an immobilized Candida antarctica (fraction B) lipase. Kinetic data were used to develop quantitative reversible rate expressions of the general Michaelis-Menten form that also incorporate a term for first-order deactivation of the enzyme. The extent of incorporation of CLA in the triacylglycerols of butteroil was characterized for reactions carried out at several temperatures (namely 45 degrees, 50 degrees, and 55 degrees C) with different weight ratios of butteroil to CLA (namely 10:1 and 2:1). At the optimum operating temperature of 50 degrees C, similar levels of incorporation of CLA (60% to 85%) were achieved at low space times (<3 h) for both 10:1 and 2:1 (w/w) ratios of butteroil to CLA.     

Hydrolysis of oils by using immobilized lipase enzyme: A review

This review focuses on the use of immobilized lipase technology for the hydrolysis of oils. The importance of lipase catalyzed fat splitting process, the various immobilization procedures, kinetics, deactivation kinetics, New immobilized lipases for chiral resolution, reactor configurations, and process considerations are all reviewed and discussed.     

Use of stable emulsion to improve stability, activity, and enantioselectivity of lipase immobilized in a membrane reactor

The enantiocatalytic performance of immobilized lipase in an emulsion membrane reactor using stable emulsion prepared by membrane emulsification technology was studied. The production of optical pure (S)-naproxen from racemic naproxen methyl ester was used as a model reaction system. The O/W emulsion, containing the substrate in the organic phase, was fed to the enzyme membrane reactor from shell-to-lumen. The enzyme was immobilized in the sponge layer (shell side) of capillary polyamide membrane with 50 kDa cut-off. The aqueous phase was able to permeate through the membrane while the microemulsion was retained by the thin selective layer. Therefore, the substrate was kept in the enzyme-loaded membrane while the water-soluble product was continuously removed from the reaction site. The results show that lipase maintained stable activity during the entire operation time (more than 250 h), showing an enantiomeric excess (96 +/- 2%) comparable to the free enzyme (98 +/- 1%) and much higher compared to similar lipase-loaded membrane reactors used in two-separate phase systems (90%). The results demonstrate that immobilized enzymes can achieve high stability as well as high catalytic activity and enantioselectivity.     

间歇及连续式固定化酶反应生产生物柴油

探讨了利用本实验室自制的Candida sp99．125脂肪酶转酯化合成生物柴油的过程。在利用间歇式反应得到最佳反应条件的情况下利用固定床反应器生产生物柴油,经过初步优化的试验结果表明,在采用分级流加甲醇下,生物柴油的转化率可以达到93％左右,并且固定化酶的使用寿命超过480h。     

Continuous production of isovaleraldehyde through extractive bioconversion in a hollow-fiber membrane bioreactor

A membrane bioreactor was developed to perform an extractive bioconversion aimed at the production of isovaleraldehyde by isoamyl alcohol oxidation with whole cells of Gluconobacter oxydans. A liquid/liquid extractive system using isooctane as extractant and assisted by a hollow-fiber hydrophobic membrane was chosen to recover the product. The aqueous bioconversion phase and the organic phase were maintained apart with the aid of the membrane. The extraction of alcohol and aldehyde was evaluated by performing equilibrium and mass transfer kinetic studies. The bioprocess was then performed in a continuous mode with addition of the substrate to the aqueous phase. Fresh solvent was added to the organic phase and exhausted solvent was removed at the same flow rate. The extractive system enabled a fast and selective in situ removal of the aldehyde from the water to the organic phase. High conversions (72–90%) and overall productivity (2.0–3.0 g l⁻¹ h⁻¹) were obtained in continuous experiments performed with different rates of alcohol addition (1.5–3.5 g l⁻¹ h⁻¹). Cell deactivation was observed after 10–12 h of operation.     

Hydrolysis of fish oil by hyperactivated rhizomucor miehei lipase immobilized by multipoint anion exchange

Rhizomucor miehei lipase (RML) is greatly hyperactivated (around 20‐ to 25‐fold toward small substrates) in the presence of sucrose laurate. Hyperactivation appears to be an intramolecular process because it is very similar for soluble enzymes and covalently immobilized derivatives. The hyperactivated enzyme was immobilized (in the presence of sucrose laurate) on cyanogen bromide‐activated Sepharose (very mild covalent immobilization through the amino terminal residue), on glyoxyl Sepharose (intense multipoint covalent immobilization through the region with the highest amount of Lys residues), and on different anion exchangers (by multipoint anionic exchange through the region with the highest density of negative charges). Covalent immobilization does not promote the fixation of the hyperactivated enzyme, but immobilization on Sepharose Q retains the hyperactivated enzyme even in the absence of a detergent. The hydrolysis of fish oils by these hyperactivated enzyme derivatives was sevenfold faster than by covalently immobilized derivatives and three and a half times faster than by the enzyme hyperactivated on octyl‐Sepharose. The open structure of the hyperactivated lipase is fairly exposed to the medium, and no steric hindrance should interfere with the hydrolysis of large substrates. These new hyperactivated derivatives seem to be more suitable for hydrolysis of oils by RML immobilized inside porous supports. In addition, the hyperactivated derivatives are fairly stable against heat and organic cosolvents. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Synthesis of 2-ethyl hexanol fatty acid esters in a packed bed bioreactor using a lipase immobilized on a textile membrane

An enzymatic process using a packed bed bioreactor with recirculation was developed for the scale-up synthesis of 2-ethylhexyl palmitate with a lipase from Candida sp. 99–125 immobilized on a fabric membrane by natural attachment to the membrane surface. Esterification was effectively performed by circulating the reaction mixture between a packed bed column and a substrate container. A maximum esterification yield of 98% was obtained. Adding molecular sieves and drying the immobilized lipase both decreased the water content at the reactor outlet and around the enzyme, which led to an increase in the rate of esterification. The long-term stability of the reactor was tested by continuing the reaction for 30 batches (over 300 h) with an average esterification yield of about 95%. This immobilized lipase bioreactor is scalable and is thus suitable for industrial production of 2-ethylhexyl palmitate.     

Immobilization of lipase into mesoporous silica particles by physical adsorption

Mesoporous silica particles for immobilization of lipase from Candida rugosa were prepared by precipitation and aggregation of primary particles from highly basic sodium silicate solution but without addition of templates. The average pore size of the material was 15.8 nm, which allowed enzyme adsorption inside the pores and high enzyme loading. Specific surface area of the material was found to be 359 m²g^?1. A loading of 100 mglipasegdrysilica^?1 was obtained at initial enzyme concentration of 1.8 mgmL^?1 by physical adsorption. The FTIR spectrum showed the structural conformation of lipase to be retained after adsorption into the mesoporous silica support. Although the efficiency of the mesoporous biocatalyst was shown to be lower than that of the free enzyme, the immobilized enzyme showed enhanced thermal stability and could be desorbed with Triton X-100, indicating the hydrophobic nature of the adsorption.