The Lipase From Thermomyces Lanuginosus Research Articles

Insights on the role of blocking agent on the properties of the lipase from Thermomyces lanuginosus immobilized on heterofunctional support for hydroesterification reactions

Here, we report a study of the effect of the blocking agent on the properties of the lipase from Thermomyces lanuginosus (TLL) immobilized on a heterofunctional support (Purolite C18-ethylnediamina (EDA)- vinyl sulfone (VS)-TLL-blocking agent) in different reactions. The performance of the biocatalysts was compared to those immobilized on standard hydrophobic support (Purolite C18-TLL) and the commercial one (TLL-IM). The nature of the blocking agent (Cys, Gly and Asp) altered the enzyme features. TLL-IM always gave a comparatively worse performance, with its specificity for the oil being very different to the Purolite biocatalysts. Under optimized conditions, Purolite C18-TLL yielded 97 % of hydrolysis conversion after 4 h using a water/waste cooking soybean oil (WCSO) mass ratio of 4.3, biocatalyst load of 6.5 wt% and a temperature of 44.2 °C (without buffer or emulsification agent). In esterification reactions of the purified free fatty acids (FFAs) obtained from WCSO, the best TLL biocatalysts depended on the utilized alcohol: linear amyl alcohol was preferred by Purolite C18-TLL and Purolite C18-EDA-VS-TLL-Gly, while higher activity was achieved utilizing isoamyl alcohol as nucleophile by Purolite C18-EDA-VS-TLL-Cys, Purolite C18-EDA-VS-TLL-Asp and IM-TLL as catalysts. All the results indicate the influence of the blocking step on the final biocatalyst features.

The nature of the buffer alters the effects of the chemical modification on the stability of immobilized lipases

The objective of this paper was to analyze whether an interaction between the effects of the buffer nature and chemical modification on enzyme stability exists. For this, the lipase B from Candida antarctica (CALB) and the lipase from Thermomyces lanuginosus (TLL) were immobilized on octyl agarose beads and modified with picryl sulfonic acid (TNBS) and ethylenediamine via the carbodiimide route. The obtained biocatalysts were them inactivated in different buffers (2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris)-HCl, N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) or phosphate). A significant interaction was found between the chemical modification of the enzyme surfaces and their stabilities in these different buffers. While phosphate was the buffer where the lowest enzyme stabilities were found for both unmodified immobilized enzymes, the differences on enzyme stabilities become much smaller after some chemical modifications. In many instances, chemical modification improves enzyme stability when using a buffer and was negative when using the other one (e.g., TNBS modification of TLL was positive using Tris-HCl or negative using the other buffers, amination of CALB decreased its stability when inactivated in Tris-HCl or HEPES while it almost had no effect in phosphate). Thus, clear co-interactions of the effects on enzyme stability between chemical modification and buffer nature were established.

Chemical amination of immobilized enzymes for enzyme coimmobilization: Reuse of the most stable immobilized and modified enzyme

Although Lecitase and the lipase from Thermomyces lanuginosus (TLL) could be coimmobilized on octyl-agarose, the stability of Lecitase was lower than that of TLL causing the user to discard active immobilized TLL when Lecitase was inactivated. Here, we propose the chemical amination of immobilized TLL to ionically exchange Lecitase on immobilized TLL, which should be released to the medium after its inactivation by incubation at high ionic strength. Using conditions where Lecitase was only adsorbed on immobilized TLL after its amination, the combibiocatalyst was produced. Unfortunately, the release of Lecitase was not possible using just high ionic strength solutions, and if detergent was added, TLL was also released from the support. This occurred when using 0.25 M ammonium sulfate, Lecitase did not immobilize on aminated TLL. That makes the use octyl-vinylsulfone supports necessary to irreversibly immobilize TLL, and after blocking with ethylendiamine, the immobilized TLL was aminated. Lecitase immobilized and released from this biocatalyst using 0.25 M ammonium sulfate and 0.1% Triton X-100. That way, a coimmobilized TLL and Lecitase biocatalyst could be produced, and after Lecitase inactivation, it could be released and the immobilized, aminated, and fully active TLL could be utilized to build a new combibiocatalyst.

Integrated Green and Enzymatic Process to Produce Omega‐3 Acylglycerols from Echium plantagineum Using Immobilized Lipases

AbstractThis work presents an original approach to develop an integrated process to improve the nutritional characteristics of natural oils, starting with the extraction from raw material by ecofriendly methods, followed by the production of novel acylglycerols using immobilized lipases. Specifically, 2‐monoacylglycerols (2‐MAG) enriched in omega‐3 stearidonic acid (SDA) were synthesized by selective ethanolysis of extracted Echium plantagineum oil using the lipase from Thermomyces lanuginosus (TLL). Different reaction conditions were investigated to minimize acyl migration and to ensure the purity of final products. The biocatalyst produced in our laboratory by the immobilization of TLL on Sepabeads C‐18 reached the maximum theoretical amount of 2‐MAG (33%) in only 2 hour at mild reaction conditions (25 °C), achieving a product enriched in omega‐3 SDA (up to 25%). Moreover, the produced biocatalyst exhibited higher stability than commercial lipases. The average activity after five cycles was 71%, allowing several reutilization cycles and developing a feasible enzymatic process. Finally, 2‐MAG were used as starting material to synthesize structured triacyclglycerols (STAG) through transesterification with caprylic acid ethyl esters using the lipase from Rhizomucor miehei (RML) in solvent‐free systems. The use of molecular sieves in combination with RML immobilized on Sepabeads C‐18 was demonstrated to be an extraordinarily fast strategy to produce pure STAG (100% in 1 hour), four times higher than the activity showed by commercial biocatalyst. Thus, the enzymatic processes developed in this study open a range of possibilities to synthesize omega‐3 acylglycerols with improved characteristics, proving the usefulness of immobilized lipases to produce novel lipids.

Improved immobilization of lipase from Thermomyces lanuginosus on a new chitosan-based heterofunctional support: Mixed ion exchange plus hydrophobic interactions

In this study, a new mixed heterofunctional support (Chit–GA–Gly) has been prepared by sequential activation of chitosan hydrogel (Chit) with glutaraldehyde (GA) and further functionalization with glycine (Gly). The immobilization of the lipase from Thermomyces lanuginosus (TLL) on this support was compared with that on GA-activated Chit hydrogel (Chit–GA). The supports have been characterized by FT–IR, zeta potential and TG analyses. A similar maximum lipase loading of 53–55 mg per gram of support has been obtained for both supports. Both biocatalysts retained ≈40% of their initial activity after 48 h of incubation at 50 °C in heptane, toluene or iso-octane. The immobilization of TLL on Chit–GA proceeded via preferential covalent attachment (95%) and a combined ion exchange (cationic and anionic) and hydrophobic adsorption was observed using Chit–GA–Gly. TLL immobilized on Chit–GA–Gly was ≈4-times more active than when immobilized on Chit–GA in both olive oil emulsion hydrolysis and alkyl palmitate synthesis via esterification. Isoamyl palmitate synthesis in iso-octane at 50 °C using this new biocatalyst gave a maximum acid conversion of 85% after 90 min of reaction. After nine consecutive esterification batches, the biocatalyst retained around 40% of its initial activity.

Immobilized Biocatalysts of Eversa® Transform 2.0 and Lipase from Thermomyces Lanuginosus: Comparison of Some Properties and Performance in Biodiesel Production

Eversa® Transform (ET), and the lipase from Thermomyces lanuginosus (TLL), liquid commercial lipases formulations, have been immobilized on octyl agarose beads and their stabilities were compared. Immobilized and free ET forms were more thermostable than TLL formulations at pH 5.0, 7.0, and 9.0, and the ET immobilized form was more stable in the presence of 90% methanol or dioxane at 25 °C and pH 7. Specific activity versus p-nitrophenyl butyrate was higher for ET than for TLL. However, after immobilization the differences almost disappeared because TLL was very hyperactivated (2.5-fold) and ET increased the activity only by 1.6 times. The enzymes were also immobilized in octadecyl methacrylate beads. In both cases, the loading was around 20 mg/g. In this instance, activity was similar for immobilized TLL and ET using triacetin, while the activity of immobilized ET was lower using (S)-methyl mandelate. When the immobilized enzymes were used to produce biodiesel from sunflower oil and methanol in tert-butanol medium, their performance was fairly similar.

Modulating the properties of the lipase from Thermomyces lanuginosus immobilized on octyl agarose beads by altering the immobilization conditions

The lipase from Thermomyces lanuginosus (TLL) has been immobilized on octyl-agarose beads via interfacial activation under 16 different conditions (changing the immobilization pH, the ionic strength, the presence of additives like calcium, phosphate or glycerol) and using a low loading (1 mg/g support). Then, the properties of the different biocatalysts have been evaluated: stability at pH 7.0 and 70 °C and activity versus p-nitro phenyl propionate, triacetin and R- and S- methyl mandelate. Results clearly indicate that the immobilization conditions determine the final enzyme properties, altering enzyme stability (by 10 folds), activity (by 8 folds using R- methyl mandelate) and specificity (VR/VS changed from 0.7 to 2.3 using mandelate esters). For instance, the enzymes immobilized at pH 7.0 using 5 mM buffer were the most stable preparations, while the presence of 250 mM sodium phosphate greatly decreased the final enzyme stability. The biocatalyst stability of TLL increased with increasing NaCl in the immobilization buffer at pH 5. Fluorescence studies confirmed that the conformation of the different immobilized enzymes were different, despite being a physical and reversible immobilization method. Thus, the immobilization of TLL on octyl agarose beads under different conditions produced biocatalysts with different properties, the optimal condition depends on the studied reaction and condition.

Glycolipid Biosurfactants Activate, Dimerize, and Stabilize Thermomyces lanuginosus Lipase in a pH-Dependent Fashion.

We present a study of the interactions between the lipase from Thermomyces lanuginosus (TlL) and the two microbially produced biosurfactants (BSs), rhamnolipid (RL) and sophorolipid (SL). Both RL and SL are glycolipids; however, RL is anionic, while SL is a mixture of anionic and non-ionic species. We investigate the interactions of RL and SL with TlL at pH 6 and 8 and observe different effects at the two pH values. At pH 8, neither RL nor SL had any major effect on TlL stability or activity. At pH 6, in contrast, both surfactants increase TlL's thermal stability and fluorescence and activity measurements indicate interfacial activation of TlL, resulting in 3- and 6-fold improved activity in SL and RL, respectively. Nevertheless, isothermal titration calorimetry reveals binding of only a few BS molecules per lipase. Size-exclusion chromatography and small-angle X-ray scattering suggest formation of TlL dimers with binding of small amounts of either RL or SL at the dimeric interface, forming an elongated complex. We conclude that RL and SL are compatible with TlL and constitute promising green alternatives to traditional surfactants.

Coimmobilization of enzymes in bilayers using pei as a glue to reuse the most stable enzyme: Preventing pei release during inactivated enzyme desorption

Coimmobilization of enzymes using polyethylenimine (PEI) as glue permits to reuse the most stable enzyme, immobilized on the support, after its incubation at high ionic strength to desorb the other enzyme, immobilized via ion exchange. However, this produced PEI desorption. Now, we solve this problem via covalent immobilization of the PEI on the immobilized first and more stable enzyme and the glyoxyl support. The phospholipase Lecitase ultra (LU) and the lipase from Thermomyces lanuginosus (TLL) have been immobilized in octyl (OC) and OC-glyoxyl (OC-GLX) agarose beads, coated with PEI and used to immobilize β-galactosidase from Aspergillus oryze. The treatment of immobilized lipases with glutaraldehyde and the use of glyoxyl-octyl allowed preventing PEI release, even in the presence of 6 M NaCl. As covalently immobilized LU and TLL were more stable than β-galactosidase, 3 cycles of β-galactosidase adsorption, thermal inactivation, desorption by incubation in 6 M NaCl and reloading of a fresh batch of galactosidase batch were performed. During these treatments, LU retained around 90% of the activity and TLL more than 80%, and the PEI bound to these biocatalysts was maintained. This new strategy probed to be useful to allowing reusing the most stable enzyme without any further treatment.

Design of a lipase-nano particle biocatalysts and its use in the kinetic resolution of medicament precursors

Superparamagnetic iron oxide nanoparticles (Fe3O4) were prepared by the co-precipitation method and functionalized with 3-amino-propyltriethoxysilane (APTES) or branched-polyethylenimine (PEI). After that, two parallels methods to immobilize the lipase from Thermomyces lanuginosus (TLL) were performed: the first one by ionic exchange and the second one by covalent attachment after the functionalization of the support with glutaraldehyde (GA). X-ray powder diffraction, magnetometry and infrared spectroscopy analysis were used to characterize the TLL preparations. These analyses showed that all samples presented superparamagnetic properties even after the immobilization procedure. The SPMN (superparamagnetic nanoparticle) @APTES covalent preparation had around 450min of half-life time at pH 7.0 and 70°C while that of the free enzyme was 46min. These biocatalysts were evaluated in the kinetic resolution of rac-1-methyl-2-(2,6-dimethylphenoxy)ethyl acetate in different co-solvents (acetonitrile, isopropanol, ethyl ether and tetrahydrofuran). The best results were for the enzyme/substrate ratio of 2:1, in the presence of the ethyl ether or THF (20% v/v both) at 30°C during 24h with the SPMN@PEI-TLL biocatalyst. The conversion attained was 50% and the enantiomeric excess of the product was 99%. The new SPMN support are an excellent strategy to easy recovery of the biocatalyst by applying a magnetic field.

When Enzymes and Green Surfactants Meet

Surfactants and proteins coexist in many different industries, ranging from detergents to pharmaceutics. Surfactant-protein interactions are multi-faceted and can range from limited interactions, as typically observed with nonionic surfactants, to complete denaturation of the protein, more typical of anionic surfactants such as sodium dodecyl sulphate (SDS). Denaturation of proteins and enzymes can be driven by both monomeric and micellar surfactant interactions [1].Biosurfactants are produced by microorganisms and have promising properties. They have strong interfacial properties [2] that are promising for industrial use. Despite growing interest, their impact on protein and enzyme stability remains at an early stage of investigation. Studies with model proteins suggest a relatively mild interaction with slow inactivation kinetics [3]. Membrane proteins can be refolded in their presence [4].Here we report interactions between industrial/detergent enzymes and two glycolipid-biosurfactants: Anionic rhamnolipid (RL) produced in Pseudomonas aeruginosa and mixed anionic/nonionic sophorolipid (SL) produced in Candida bombicola. RL and SL both act as mild biosurfactants that despite their anionic charge are compatible with industrial enzymes. The lipase from Thermomyces lanuginosus (TlL) shows pH dependent interactions. At pH 8 we do not detect interactions between TlL and RL or SL, with only minor perturbations to stability and activity. However, lowering pH from 8 to 6 favor stronger interactions between TlL and RL/SL that strongly stabilizes TlL and opens the active site with a 3- to 6-fold boost to activity. We use SAXS to qualitatively determine formation of complexes between TlL and SL and RL micelles at pH 6.1.Otzen, D.E., P. Sehgal, and P. Westh. J Colloid Interface Sci, 2009.329(2): p.273-83.2.Nguyen, T.T. and D.A. Sabatini. Int J Mol Sci, 2011.12(2): p.1232-44.3.Andersen, K.K. and D.E. Otzen. Biochim Biophys Acta, 2014.1844: p.2338-2345.4.Andersen, K.K. and D.E. Otzen. FEBS Lett, 2014.588: p.1955-60.

Hydrolases on silica surfaces: Coverage-activity–molecular property relationships revealed

A set of recommendations to maintain high activity of immobilized enzymes is developed based on direct observation via AFM. This helps to close knowledge gaps that often lead to poor performance of nanobiocatalysts for chemical synthesis. Molecule-level height and volume distribution analyses from high-resolution AFM images were applied to Candida antarctica Lipase B (CALB), subtilisin Carlsberg, and the Lipase from Thermomyces lanuginosus (TLL) deposited on model silica surfaces. Ensembles of flexible or “soft” enzymes appear separated when interactions with the surface are considerable at low surface coverage but form highly entangled structures of increased conformational stability at high surface coverage. By contrast, ensembles of rigid or “hard” enzymes appear to maintain stable aggregates even under strong interaction with the surface. The more rigid the enzyme the higher its tendency to remain in a densely packed state that is able to withstand surface-induced conformational transitions detrimental to catalysis. Weakening of surface-protein interactions for “soft” enzymes will prevent single-molecule immobilization, which reduces catalytic competency through structural changes. Multi-layer coverage in enzyme immobilization should generally be avoided due to mass transfer limitations.

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.

Conformational changes and catalytic competency of hydrolases adsorbing on fumed silica nanoparticles: II. Secondary structure

Secondary conformational analysis via Circular Dichroism (CD) and Amide-I FTIR was applied to preparations of Candida antarctica Lipase B (CALB), subtilisin Carlsberg, and the Lipase from Thermomyces lanuginosus (TLL) on fumed silica to confirm that the “hardness” and packing density of the enzymes on the solid fumed silica nanoparticle surface can be used to rationalize the variable enzyme-dependent changes of catalytic competency with surface coverage. “Soft” enzymes should be immobilized at a surface coverage where enzyme–enzyme interactions predominate thereby preventing detrimental structural changes caused by enzyme-support interactions, while “hard” enzymes can be immobilized at low to intermediate surface coverage with good catalytic performance. Multi-layered coverage reduces the superficial average catalytic performance in all cases due to mass transfer limitations.

Conformational changes and catalytic competency of hydrolases adsorbing on fumed silica nanoparticles: I. Tertiary structure

We have recently introduced an immobilization protocol for preparations of enzymes on fumed silica for catalysis in organic solvents. The observation of a maximum in apparent catalytic activity at intermediate surface coverage for one enzyme while another enzyme showed continuously increasing apparent catalytic activity with decreasing surface coverage led to speculation on the impact of surface coverage on apparent catalytic activity through different relative surface–protein and protein–protein interactions, combined with different “hardness” or resistance towards unfolding by the enzymes. The kinetics of tertiary unfolding of Candida antarctica Lipase B (CALB), subtilisin Carlsberg, and the Lipase from Thermomyces lanuginosus (TLL) adsorbing on fumed silica nanoparticles were inferred here from tryptophan fluorescence for 2 to 1250%SC, 0.5–4.70 mg/mL enzyme concentration in aqueous buffer solution, and in the presence of the structural modifiers 2,2,2-trifluoroethanol (TFE) and dithiothreitol (DTT). The results shown here confirm the earlier speculation that “hard” enzymes can perform well at low and intermediate surface coverage of the solid fumed silica particles until multi-layer packing imposes mass-transfer limitations, while “soft” enzymes unfold at low surface coverage and therefore show a maximum in catalytic competency at intermediate surface coverage before declining apparent activity is caused by multi-layer packing.

Immobilization–stabilization of the lipase from Thermomyces lanuginosus: Critical role of chemical amination

This paper describes the immobilization and stabilization of the lipase from Thermomyces lanuginosus (TLL) on glyoxyl agarose. Enzymes attach to this support only by the reaction between several aldehyde groups of the support and several Lys residues on the external surface of the enzyme molecules at pH 10. However, this standard immobilization procedure is unsuitable for TLL lipase due to the low stability of TLL at pH 10 and its low content on Lys groups that makes that the immobilization process was quite slow. The chemical amination of TLL, after reversible immobilization on hydrophobic supports, has been shown to be a simple and efficient way to improve the multipoint covalent attachment of this enzyme. The modification enriches the enzyme surface in primary amino groups with low p Kb, thus allowing the immobilization of the enzyme at lower pH values. The aminated enzyme was rapidly immobilized at pH 9 and 10, with activities recovery of approximately 70%. The immobilization of the chemically modified enzyme improved its stability by 5-fold when compared to the non-modified enzyme during thermal inactivation and by hundreds of times when the enzyme was inactivated in the presence of organic solvents, being both glyoxyl preparations more stable than the enzyme immobilized on bromocyanogen.

A comparison between dual polarization interferometry (DPI) and surface plasmon resonance (SPR) for protein adsorption studies

This work was performed with the aim of comparing protein adsorption results obtained from the recently developed dual polarization interferometry (DPI) with the well-established surface plasmon resonance (SPR) technique. Both techniques use an evanescent field as the sensing element but completely different methods to calculate the adsorbed mass. As a test system we used adsorption of the lipase from Thermomyces lanuginosus (TLL) on C18 surfaces. The adsorbed amount calculated with both techniques is in good agreement, with both adsorption isotherms saturating at 1.30–1.35 mg/m 2 at TLL concentrations of 1000 nM and above. Therefore, this supports the use of both SPR and DPI as tools for studying protein adsorption, which is very important when comparing adsorption data obtained from the use different techniques. Due to the spot sensing in SPR, this technique is recommended for initial kinetic studies, whereas DPI is more accurate when the refractive index and thickness of the adsorbed layer is of more interest.