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     

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%.     

Reactivation of covalently immobilized lipase from Thermomyces lanuginosus

Lipase from Thermomyces lanuginosus (TLL) immobilized on cyanogen bromide agarose (CNBr) may be fully inactivated when incubated in saturated solutions of guanidine. When this inactivated enzyme is re-incubated in aqueous medium, 20% of the activity may be recovered for several cycles. However, if the activity was determined in the presence of a detergent (CTAB, an activator of this enzyme), 100% of the initial activity in the presence of detergent was recovered. The enzyme was also inactivated in the presence of organic solvents and at high temperatures. Inactivations were more rapid when the activity was determined in absence of detergent. In both cases, some activity could be recovered just by incubation under mild conditions, and this increase was higher if the activity measurements were performed in the presence of CTAB. These results suggested that the opening of the lipase could be a critical step in the inactivation or reactivation of immobilized TLL. In inactivations in the presence of solvents, 100% of activity could be recovered during several cycles, while in thermal inactivations, the recovered activity decreased in each inactivation–reactivation cycle. The incubation of the enzyme inactivated by temperature in guanidine improved the results, but still 100% could not be achieved during several cycles even measured in the presence of CTAB.Thus, the simple incubation of the partially or fully inactivated enzyme under mild conditions permitted to recover some activity (enhancing the half life of the biocatalysts), even in thermal inactivations.     

Ionic strength-dependent denaturation of Thermomyces lanuginosus lipase induced by SDS

Triglyceride lipase from Thermomyces lanuginosus (TlL) has been reported to be resistant to denaturation by sodium dodecyl sulfate (SDS). We have found that at neutral pH, structural integrity is strongly dependent on ionic strength. In 10 mM phosphate buffer and SDS, the lipase exhibits a far-UV CD spectrum similar to other proteins denatured in this surfactant while the near-UV CD spectrum shows a complete loss of tertiary structure, observations supported by steady state fluorescence spectroscopy. However, when increasing the ionic strength by the addition of NaCl, the lipase was rendered resistant towards SDS denaturation, as observed by all techniques employed. The effect of salt on the critical micelle concentration (CMC) of SDS was observed to correlate with the effect on the degree of SDS-induced denaturation. This finding is compatible with the notion that the concentration of SDS monomers is a crucial factor for SDS–lipase interactions. The presented results are important for the understanding and improvement of protein stability in surfactant systems.     

Immobilization of Candida rugosa lipase on electrospun cellulose nanofiber membrane

A biocatalyst with high activity retention of lipase was fabricated by the covalent immobilization of Candida rugosa lipase on a cellulose nanofiber membrane. This nanofiber membrane was composed of nonwoven fibers with 200 nm nominal fiber diameter. It was prepared by electrospinning of cellulose acetate (CA) and then modified with alkaline hydrolysis to convert the nanofiber surface into regenerated cellulose (RC). The nanofiber membrane was further oxidized by NaIO₄. Aldehyde groups were simultaneously generated on the nanofiber surface for coupling with lipase. Response surface methodology (RSM) was applied to model and optimize the modification conditions, namely NaIO₄ content (2–10 mg/mL), reaction time (2–10 h), reaction temperature (25–35 °C) and reaction pH (5.5–6.5). Well-correlating models were established for the residual activity of the immobilized enzyme (R² = 0.9228 and 0.8950). We found an enzymatic activity of 29.6 U/g of the biocatalyst was obtained with optimum operational conditions. The immobilized lipase exhibited significantly higher thermal stability and durability than equivalent free enzyme.     

Surface charge engineering of Thermomyces lanuginosus lipase improves enzymatic activity and biodiesel synthesis

Objectives

This study was aimed at engineering charged residues on the surface of Thermomyces lanuginosus lipase (TLL) to obtain TLL variant with elevated performance for industrial applications.

Results

Site-directed mutagenesis of eight charged amino acids on the TLL surface were conducted and substitutions on the negatively charged residues D111, D158, D165, and E239 were identified with elevated specific activities and biodiesel yields. Synergistic effect was not discovered in the double mutants, D111E/D165E and D165E/E239R, when compared with the corresponding single mutants. One TLL mutant, D165E, was identified with increased specific activity (456.60 U/mg), catalytic efficiency (k_cat/K_m: 44.14 s^?1 mM^?1), the highest biodiesel conversion yield (93.56%), and comparable thermostability with that of the TLL.

Conclusions

Our study highlighted the importance of surface charge engineering in improving TLL activity and biodiesel production, and the resulting TLL mutant, D165E, is a promising candidate for biodiesel industry.

     

Activation, inhibition, and destabilization of Thermomyces lanuginosus lipase by detergents

Lipases catalyze the hydrolysis of triglycerides and are activated at the water-lipid interface. Thus, their interaction with amphiphiles such as detergents is relevant for an understanding of their enzymatic mechanism. In this study, we have characterized the effect of nonionic, anionic, cationic, and zwitterionic detergents on the enzymatic activity and thermal stability of Thermomyces lanuginosus lipase (TlL). For all detergents, low concentrations enhance the activity of TlL toward p-nitrophenyl butyrate by more than an order of magnitude; at higher detergent concentrations, the activity declines, leveling off close to the value measured in the absence of detergent. Surprisingly, these phenomena mainly involve monomeric detergent, as activation and inhibition occur well below the cmc for the nonionic and zwitterionic detergents. For anionic and cationic detergents, activation straddles the monomer-micelle transition. The data can be fitted to a three state interaction model, comprising free TlL in the absence of detergent, an activated complex with TlL at low detergent concentrations, and an enzyme-inhibiting complex at higher concentrations. For detergents with the same headgroup, there is an excellent correspondence between carbon chain length and ability to activate and inhibit TlL. However, the headgroup and number of chains also modulate these effects, dividing the detergents overall into three broad groups with rising activation and inhibition ability, namely, anionic and cationic detergents, nonionic and single-chain zwitterionic detergents, and double-chain zwitterionic detergents. As expected, only anionic and cationic detergents lead to a significant decrease in lipase thermal stability. Since nonionic detergents activate TlL without destabilizing the protein, activation/inhibition and destabilization must be independent processes. We conclude that lipase-detergent interactions occur at many independent levels and are governed by a combination of general and structurally specific interactions. Furthermore, activation of TlL by detergents apparently does not involve the classical interfacial activation phenomenon as monomeric detergent molecules are in most cases responsible for the observed increase in activity.     

Effect of solid-phase chemical modification on the features of the lipase from Thermomyces lanuginosus

The lipase from Thermomyces lanuginosus (TLL) was immobilized on octyl Sepharose and further modified with ethylenediamine (EDA) after activation of the carboxylic groups with carbodiimide. Different degrees of modification of the carboxyl groups were carried out by controlling the concentration of carbodiimide (10%, 50% or 100%). Subsequently, the effect of incubation of the modified preparations on hydroxylamine to recover the modified tyrosine was also studied. The modified enzymes exhibited a mobility in native electrophoresis quite different from that of the unmodified lipase (as expected by the changes in charge), and required higher concentrations of cationic detergent to become desorbed from the support. Interestingly, the chemical modification of the immobilized TLL produced an improvement in its activity, proportional to the amination degree. This increase in activity was much more significant at pH 10, where the fully modified preparation increased the activity by a factor of 10 as compared to the unmodified preparation. Moreover, the incubation of the chemically aminated preparations in a hydroxylamine solution improved the activity by an additional factor of 1.2. The fully aminated and incubated in hydroxylamine preparation exhibited a thermostability higher than that of the unmodified preparation, mainly at pH 5 (almost a 30 fold factor). In the presence of tetrahydrofurane, some stabilization was observed at pH 7, while at pH 9 the stability of the modified enzyme decreased (under all the assayed amination degrees) when compared to that of the unmodified enzyme. Thus, this simple protocol may be a rapid and efficient way of preparing a TLL biocatalyst with higher activity and stability, although this will depend on the inactivation conditions.     

Improving reuse cycles of Thermomyces lanuginosus lipase (NS-40116) by immobilization in flexible polyurethane

Enzyme immobilization using a low-cost support that allows increasing operational stability and reutilization arise as a great economic advantage for the industry. In this work, it was explored different methods of Thermomyces lanuginosus lipase (NS-40116) immobilization in flexible polyurethane foam (PU). PU polymer was synthesized using polyether and toluene diisocyanate as monomers. PU-NS-40116 immobilized was evaluated in terms of stability in a range of pH (7.0 and 9.0), temperature (24, 50 and 60?°C) for 24?h, and storage stability (room temperature and 4?°C) for 30?days. The results showed that after 30?days of storage immobilized enzyme kept 80% of initial enzyme activity. PU support before and after immobilization process was characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. Free and immobilized enzymes were compared in terms of hydrolysis of soybean oil. Immobilized enzyme by entrapment was evaluated in successive cycles of reuse showing catalytic activity above 50% even after 5 successive cycles of reuse, confirming the efficiency of immobilization process.     

Influence of water activity on the synthesis of triolein catalyzed by immobilized Mucor miehei lipase

The influence of the thermodynamic activity of water (a(w))on the synthesis of triolein catalyzed by Mucor miehei lipase was investigated. Its effect on the equilibrium and on the rates of the different reactions present, esteification and mono- and diglyceride isomerization, was evaluated through measurements made in controlled water activity atmosphere. The apparent equilibrium constants were measured from the concentration of the different species as a function of the intial glycerol-to oleic-acid ratio using all the values at once with a multi-response nonlinear regression technique. Rate constants were determined from kinetic measurements and non-linear regression uning the variation of the concentration of all significant species in the system. Except for the synthesis of diolein from monoolein, which shows a maximum for a(w) approximately 0.5, the apparent rate constants of the various reactions are not significantly affected by the value of the water activity. The equilibrium is shifted to-ward the synthesis of triolein for low values of a(w), indicating that in the design of a process for triglyceride synthesis, using M. miehei lipase as a catalyst, the water activity can be lowered to extreme values to favor the synthesis, without any sacrifice on the productivity of the process. (c) 1995 John Wiley & Sons, Inc.     

Enzymatic synthesis of isoamyl acetate using immobilized lipase from Rhizomucor miehei

The effects of important reaction parameters for enhancing isoamyl acetate formation through lipase-catalyzed esterification of isoamyl alcohol were investigated in this study. Increase in substrate (acid) concentration led to decrease in conversions. A critical enzyme concentration of 3 g l(-1) was detected for a substrate concentration of 0.06 M (each of alcohol and acid). Solvents with partition coefficient higher than 1000 (log P>3.0) supported enzyme activity to give high conversions. Acetic acid at higher concentrations could not be esterified easily probably owing to its role in lowering the microaqueous pH of the enzyme. Extraneous water/buffer addition decreased the isoamyl acetate yields slightly ( approximately 10%) at 0.005-0.01% v/v of the reaction mixture and drastically (>40%) at above 0.01% v/v. Buffer saturation of the organic solvent employed improved esterification (upto two-fold), particularly at moderately higher substrate concentrations (>0.18 M). Employing acetic anhydride instead of acetic acid resulted in a two-fold increase in the yields (at 0.25 M substrate). Use of excess nucleophile (alcohol) concentration by increasing the alcohol/acid molar ratio resulted in higher conversions in shorter duration (upto eight-fold even at 1.5 M acetic acid). Yields above 80% were achieved with substrate concentrations as high as 1.5 M and more than 150 g l(-1) isoamyl acetate concentrations were obtained employing a relatively low enzyme concentration of 10 g l(-1). The operational stability of lipase was also observed to be reasonably high enabling ten reuses of the biocatalyst.     

Kinetic modeling of interesterification reactions catalyzed by immobilized lipase

Kinetic data for lipase-catalyzed interesterification reactions between free fatty acids and triglycerides were collected and the dynamics of the interesterification reactions were successfully modeled using tow rate experssions requiring a total of five adjustable parameters. One rate expression describes the disappearance of the free fatty acid (octanoic or linolenic acid), and the second describes the rate of release of fatty acid residues from the triglycerides (olive oil or milkfat). This model is able to account for the effects of the concentration of all chemical species participating in interesterification throughout the entire reaction. When the data for both milkfat and olive oil were subjected to nonlinear regression analyses using the same mathematical model, the parameter estimates for both systems were comparable. In addition to reproducing the tendencies observed experimentally, simulations of the interesterification system under a variety of initial conditions provided insight into the effects of several reaction variables which could not be examined experimentally. Among the most significant findings of the simulation work are (1) there is a limit beyond which increasing the initial concentration of water produces no further increase in the initial rate of the interesterification reaction; (2) an increase in the initial concentration of lower glycerides produces a concomitant increase in the rate of the interesterification reaction; (3) the free fatty acids inhibit the rate of hydrolysis of the fatty acid residues of the triglycerides; (4) there is a limit beyond which increasing the initial concentration of triglycerides produces no significant increase in the rate of either the hydrolysis reaction or the interesterification reaction. (c) 1994 John Wiley & Sons, Inc.     

Ectoine improves yield of biodiesel catalyzed by immobilized lipase

To improve the production of biodiesel by enzymatic conversion of triglycerides in cottonseed oil, compatible solutes were added to the solvent-free methanolysis system to prevent competitive methanol inhibition on the immobilized lipase (Novozym^® 435). The results indicated that the addition of ectoine increased biodiesel synthesis using a three-step methanol addition process. The concentration of methyl ester (ME) reached a maximum of 95.0% in the presence of 1.1 mmol/l ectoine, an increase of 20.9% compared to that in the absence of ectoine. On the other hand, excess ectoine decreased the ME concentration. Ectoine was also shown to enhance reuse of the immobilized lipase, significantly improving ME concentrations in each recycling test. Total concentrations of ME with added ectoine were about 1.5 times that without ectoine during five recycling tests (molar ratio of cottonseed oil to methanol, 1:4). Enzymatic reaction kinetics showed, in the concentration ranges of 0.8–1.14 mol/l and 0.03–8 mol/l for triglyceride and methanol, respectively, that ectoine had no effect on the initial reaction rates when methanol concentrations were below 0.5 mol/l. When methanol concentration exceeded 0.5 mol/l, the addition of 0.8 mmol/l ectoine increased the initial reaction rates, and the lipase exhibited a lower affinity for methanol and higher affinity for triglyceride (kinetic parameters of K_mA increase, K_mTG decrease). However, the initial reaction rates decreased significantly when 8 mmol/l ectoine was added, with the lipase having higher affinity for methanol and lower affinity for triglyceride (K_mA decrease, K_mTG increase). The supplementation of ectoine provided a new method for the purpose of improving yield of biodiesel catalyzed by enzyme.     

Kinetics of solvent-free geranyl acetate synthesis by Rhizopus oligosporus NRRL 5905 lipase immobilized on to cross-linked silica

The objective of the present work was to study the kinetics of the solvent-free synthesis of geranyl acetate by a novel lipase (activity 60 U g^?1) made by immobilization of lipase from Rhizopus oligosporous NRRL 5905 on to cross-linked silica gel. Transesterification was performed with vinyl acetate as the acyl donor. Vinyl acetate was used in large excess compared to geraniol, which made the reaction pseudo first order with respect to geraniol and the reaction rate followed Michaelis–Menten kinetics for a single substrate. To obtain the highest yield for geranyl acetate, various relevant physical parameters such as shaking speed, reaction time, enzyme concentration, initial water amount and reaction temperature that influence the activity of lipase were investigated. A maximum molar conversion of 67% was achieved after 48 h of reaction at 30°C, at an enzyme concentration of 25% w/v of reaction mixture. Substrate conversion remained constant for five successive cycles; thereafter the conversion dropped by only 11%. Using a pseudo first-order kinetic model for geranyl acetate synthesis in the absence of organic solvents, apparent K_m and V_max values were evaluated as 60 mM and 141 µmol g^?1 h^?1, respectively.     

Preparation of a biocatalyst via physical adsorption of lipase from Thermomyces lanuginosus on hydrophobic support to catalyze biolubricant synthesis by esterification reaction in a solvent-free system

Lipase from Thermomyces lanuginosus (TLL) was immobilized on mesoporous hydrophobic poly-methacrylate (PMA) particles via physical adsorption (interfacial activation of the enzyme on the support). The influence of initial protein loading (5–200 mg/g of support) on the catalytic properties of the biocatalysts was determined in the hydrolysis of olive oil emulsion and synthesis of isoamyl oleate (biolubricant) by esterification reaction. Maximum adsorbed protein loading and hydrolytic activity were respectively ≈100 mg/g and ≈650 IU/g using protein loading of 150 mg/g of support. The adsorption process followed the Langmuir isotherm model (R² = 0.9743). Maximum ester conversion around 85% was reached after 30 min of reaction under continuous agitation (200 rpm) using 2500 mM of each reactant in a solvent-free system, 45 °C, 20% m/v of the biocatalyst prepared using 100 mg of protein/g of support. Apparent thermodynamic parameters of the esterification reaction were also determined. Under optimal experimental conditions, reusability tests of the biocatalyst (TLL-PMA) after thirty successive cycles of reaction were performed. TLL-PMA fully retained its initial activity up to twenty two cycles of reaction, followed by a slight decrease around 8.6%. The nature of the product (isoamyl oleate) was confirmed by attenuated total reflection Fourier transform infrared (ATR-FTIR), proton (¹H NMR) and carbon (¹³C NMR) nuclear magnetic resonance spectroscopy analyses.     

Ester synthesis from trimethylammonium alcohols in dry organic media catalyzed by immobilized Candida antarctica lipase B

Twenty-one different organic solvents were assayed as possible reaction media for the synthesis of butyryl esters from trimethylammonium alcohols in dry conditions catalyzed by immobilized Candida antarctica lipase B. The reactions were carried out following a transesterification kinetic approach, using choline and L-carnitine as primary and secondary trimethylammonium alcohols, respectively, and vinyl butyrate as acyl donor. The synthetic activity of the enzyme was strictly dependent on the water content, the position of the hydroxyl group in the trimethylammonium molecule, and the Log P parameter of the assayed solvent. Anhydrous conditions and a high excess of vinyl butyrate over L-carnitine were necessary to synthesize butyryl-L-carnitine. The synthetic reaction rates of butyryl choline were practically 100-fold those of butyryl-L-carnitine with all the assayed solvents. In both cases, the synthetic activity of the enzyme was dependent on the hydrophobicity of the solvent, with the optimal reaction media showing a Log P parameter of between -0.5 and 0.5. In all cases, 2-methyl-2-propanol and 2-methyl-2-butanol were shown to be the best solvents for both their high synthetic activity and negligible loss of enzyme activity after 6 days.