Thermomyces Lanuginosus Lipase Research Articles

On the interaction between adsorbed layers of monoolein and the lipase action on the formed layers

We used the Surface Force Apparatus (SFA) and ellipsometry techniques to study the interaction forces and the adsorption behavior of monoolein (MO), respectively. MO was adsorbed from water to a hydrophobised mica or silica surface. In addition the effect of added lipase, Thermomyces ( Humicula) lanuginosa lipase (TLL), to an adsorbed layer of MO was investigated. The force versus distance curves between two MO covered surfaces feature a strong repulsive interaction beneath 400 Å. The range of the repulsive force decreases, however, with the number of approaches. No adhesion was observed, provided that the surfaces were not taken to hydrophobic contact. The surface separation at MO–MO contact was determined to about 55 Å. This means a layer thickness of about 27 Å, which is comparable to the thickness (25 Å) determined by ellipsometry. The repulsive force may arise from compression of a cubic phase of MO. This phase are suggested to form between the surfaces when they approach close contact due to capillary induced phase separation (CIPS) from the saturated MO solution. The repulsive force changes significantly with time after addition of TLL (concentration of about 1×10 −8 M). In contrast to the force curves recorded before adding TLL, the surfaces do not seem to be completely covered with MO as we always observed an attractive force (inward jump) of similar range as was observed between pure OTE surfaces. Ellipsometry measurement of TLL action on MO covered hydrophobic surface reveals a significant and sharp decrease of the amounts adsorbed. Furthermore, the rate of decrease and reduction in adsorbed amount increased with TLL concentration.

Orientation and Conformation of a Lipase at an Interface Studied by Molecular Dynamics Simulations

Electron density profiles calculated from molecular dynamics trajectories are used to deduce the orientation and conformation of Thermomyces lanuginosa lipase and a mutant adsorbed at an air-water interface. It is demonstrated that the profiles display distinct fine structures, which uniquely characterize enzyme orientation and conformation. The density profiles are, on the nanosecond timescale, determined by the average enzyme conformation. We outline a computational scheme that from a single molecular dynamics trajectory allows for extraction of electron density profiles referring to different orientations of the lipase relative to an implicit interface. Profiles calculated for the inactive and active conformations of the lipase are compared with experimental electron density profiles measured by x-ray reflectivity for the lipase adsorbed at an air-water interface. The experimental profiles contain less fine structural information than the calculated profiles because the resolution of the experiment is limited by the intrinsic surface roughness of water. Least squares fits of the calculated profiles to the experimental profiles provide areas per adsorbed enzyme and suggest that Thermomyces lanuginosa lipase adsorbs to the air-water interface in a semiopen conformation with the lid oriented away from the interface.

Selective reduction and chemical modification of oxidized lipase cysteine mutants

Thirteen single-cysteine mutants of the 33 kDa fungal triacylglycerol lipase Thermomyces (formerly Humicola) lanuginosa lipase (TLL, EC 3.1.1.3) were produced and characterized for the purpose of site-directed chemical modification with spectroscopic reporter groups. All cysteine mutants were found to be predominantly blocked by oxidation to disulfides with endogenous cysteine during production. The fraction of lipase molecules with free sulfhydryl groups was analyzed by labeling with N-biotinylaminoethyl methanethiosulfonate, followed by a novel dot-blot method based on biotin-streptavidin interactions. A non-invasive method for the reduction of the introduced cysteine was elaborated for this protein containing three native disulfide bridges. The site-specifically reduced TLL mutants were then labeled with the sulfhydryl-specific reagents 2-(5-dimethylaminonaphth-1-ylsulfonamido)ethyl methanethiosulfonate or (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl) methanethiosulfonate, and studied by fluorescence and electron spin resonance (ESR) spectroscopy.Key words: lipase, cysteine mutant, selective reduction, chemical modification, methanethiosulfonate.

Production of structured lipids in a packed‐bed reactor with thermomyces lanuginosa lipase

AbstractLipase‐catalyzed interesterification between fish oil and medium‐chain TAG has been investigated in a packedbed reactor with a commercially immobilized enzyme. The enzyme, a Thermomyces lanuginosa lipase immobilized on silica by granulation (lipozyme TL IM; Novozymes A/S, Bagsvaerd, Denmark), has recently been developed for fat modification. This study focuses on the new characteristics of the lipase in a packed‐bed reactor when applied to interesterification of TAG. The degree of reaction was strongly related to the flow rate (residence time) and temperature, whereas formation of hydrolysis by‐products (DAG and FFA) were only slightly affected by reaction conditions. The degree of reaction reached equilibrium at 30–40 min residence time, and the most suitable temperature was 60°C or higher with respect to the maximal degree of reaction. The lipase was stable in a 2‐wk continuous operation without adjustment of water content or activity of the column and the substrate mixture.

Lipase action on a monoolein/sodium oleate aqueous cubic liquid crystalline phase—a NMR and X-ray diffraction study

The effect of adding Thermomyces (formerly Humicola) lanuginosa lipase (TLL) to a monoolein (MO)/sodium oleate (NaO) aqueous cubic liquid crystalline phase has been studied. 1H-NMR, 13C-NMR, 1H-PGSE (Pulsed-magnetic field Gradient Spin-Echo) self-diffusion measurements, and Small Angle X-ray Diffraction were used to follow the degradation of the cubic phase. The reaction sequence in terms of phase transitions follows the order bicontinuous cubic→reverse hexagonal→micellar cubic→micellar phase and corresponds to the previously determined phase diagrams. These changes correlate with changes in the lipid composition observed by 13C-NMR and confirmed by HPLC analysis. The initial decrease of the diffusion coefficients of water and lipid can be related to the transformation of the bicontinuous cubic phase to a reverse hexagonal structure.

Effects of betaine on the structural dynamics of Thermomyces ( Humicola) lanuginosa lipase

The impact of betaine on the structural dynamics of a soluble protein, Thermomyces ( Humicola) lanuginosa lipase (HLL) was studied using differential scanning calorimetry (DSC), surface tension measurements, and both steady-state and time-resolved fluorescence spectroscopy. Betaine increased the thermal stability of wild type HLL and protected this enzyme from unfolding by the chaotrope, guanidine hydrochloride. Time-resolved fluorescence anisotropy measurements revealed betaine to decrease the mean hydrodynamic volume of the single Trp HLL mutant, W89m, and to reduce the movements of the α-helical segment constituting the so-called ‘lid’. In the presence of betaine, the average environment of Trp89 became more hydrophobic with decreased accessibility to this residue for the water soluble quencher I −, the bimolecular collisional rate constant, k q decreasing from 6.86×10 8 to 5.06×10 8 M −1 s −1 in 1.0 M betaine. The above results are in keeping with an augmented packing of the lipase in the presence of betaine. Betaine increases surface tension ( γ) of water and enhances the denaturation of HLL into the air–water interface. The increased stability of HLL and increased γ by betaine suggest low or no binding of betaine to the surface of protein. The effects of betaine on the structural dynamics of HLL are readily explained by the increased surface tension of water caused by this solute.

Glycosylation of Thermomyces lanuginosa lipase enhances surface binding towards phospholipids, but does not significantly influence the catalytic activity

Binding properties of the native Thermomyces lanuginosa lipase (Tll), the inactive mutant of Tll (S146A; active Ser146 mutated to Ala), and the non-glycosylated mutant of Tll (N33Q) were determined using fluorescence spectroscopy. Tll, S146A mutant and N33Q mutant show significant different binding behavior to phosphatidylcholine (PC) and phosphatidylglycerol (PG) liposomes. Generally, weaker association of lipase molecules is observed to PC liposomes than to PG liposomes. Strong lipase–lipid interactions are observed for the S146A mutant, which is less pronounced for Tll and the N33Q variant. Addition of fatty acid to PG liposomes reduces significantly the binding affinity of the lipases. This effect is less pronounced in fatty acid/PC liposomes. Although the catalytic activity of the N33Q mutant is comparable to Tll, the non-glycosylated variant shows generally lower binding affinity to PC or PG matrix than Tll. Addition of the substrate analog benzene boronic acid (BBA) increases the binding affinity of the S146A and N33Q mutants, while only small changes are observed for Tll suggesting that the dynamics of the active site lid influences the binding affinity and that the flexibility of the loop region 33–48 might contribute to the activation of the lipase.

Binding of Thermomyces (Humicola) lanuginosa lipase to the mixed micelles of cis-parinaric acid/NaTDC.

The binding of Thermomyces lanuginosa lipase and its mutants [TLL(S146A), TLL(W89L), TLL(W117F, W221H, W260H)] to the mixed micelles of cis-parinaric acid/sodium taurodeoxycholate at pH 5.0 led to the quenching of the intrinsic tryptophan fluorescence emission (300-380 nm) and to a simultaneous increase in the cis-parinaric acid fluorescence emission (380-500 nm). These findings were used to characterize the Thermomyces lanuginosa lipase/cis-parinaric acid interactions occurring in the presence of sodium taurodeoxycholate. The fluorescence resonance energy transfer and Stern-Volmer quenching constant values obtained were correlated with the accessibility of the tryptophan residues to the cis-parinaric acid and with the lid opening ability of Thermomyces lanuginosa lipase (and its mutants). TLL(S146A) was found to have the highest fluorescence resonance energy transfer. In addition, a TLL(S146A)/oleic acid complex was crystallised and its three-dimensional structure was solved. Surprisingly, two possible binding modes (sn-1 and antisn1) were found to exist between oleic acid and the catalytic cleft of the open conformation of TLL(S146A). Both binding modes involved an interaction with tryptophan 89 of the lipase lid, in agreement with fluorescence resonance energy transfer experiments. As a consequence, we concluded that TLL(S146A) mutant is not an appropriate substitute for the wild-type Thermomyces lanuginosa lipase for mimicking the interaction between the wild-type enzyme and lipids.

Studies on ferulic acid esterase activity in fungal lipases and cutinases

In this study we have tested a number of lipases, lipase variants and cutinases for ferulic acid esterase activity, using ferulic acid ethyl ester as substrate. It was shown that Thermomyces lanuginosa lipase (TLL), Candida antartica lipase A, Candida antartica lipase B, Rhizomucor miehei lipase and Fusarium oxysporum lipase have no significant ferulic acid esterase activity. Thirteen variants of TLL were constructed based on a model of Aspergillus niger ferulic acid esterase A (FAE-A). Activity assay using ferulic acid ethyl ester as substrate gave, for FAE-A, 112 U/mg=112 μmol ferulic acid released per min per mg enzyme. Two of the variants of TLL had significant ferulic acid esterase activity, TLLv1 (7 U/mg) and TLLv10 (20 U/mg). Both these variants contain the mutation F113Y that seems to be essential for ferulic acid esterase activity. In addition to lipase activity, three cutinases showed ferulic acid esterase activity, Aspergillus oryzae cutinase (5 U/mg), Fusarium solani pisi cutinase (13 U/mg), Humicola insolence cutinase (20 U/mg).

Effects of i-propanol on the structural dynamics of Thermomyces lanuginosa lipase revealed by tryptophan fluorescence.

Influence of isopropanol (iPrOH) on the structural dynamics of Thermomyces lanuginosa lipase (TLL) was studied by steady-state, time-resolved, and stopped-flow fluorescence spectroscopy, monitoring the intrinsic emission of Trp residues. The fluorescence of the four Trps of the wild-type enzyme report on the global changes of the whole lipase molecule. To monitor the conformational changes in the so-called "lid," an alpha-helical surface loop, the single Trp mutant W89m (W117F, W221H, W260H) was employed. Circular dichroism (CD) spectra revealed that iPrOH does not cause major alterations in the secondary structures of the wild-type TLL and W89m. With increasing [iPrOH], judged by the ratio of emission intensities at 350 nm and 330 nm, the average microenvironment of the Trps in the wild-type TLL became more hydrophobic, whereas Trp89 of W89m moved into a more hydrophilic microenvironment. Time-resolved fluorescence measurements revealed no major changes to be induced by iPrOH neither in the shorter fluorescence lifetime component (tau(1) = 0.5--1.2 ns) for the wild-type TLL nor in the longer fluorescence lifetime component (tau(2) = 4.8--6.0 ns) in the wild-type TLL and the W89m mutant. Instead, for W89m on increasing iPrOH from 25% to 50% the value for tau(1) increased significantly, from 0.43 to 1.5 ns. The shorter correlation time phi(1) of W89m had a minimum of 0.08 ns in 25% iPrOH. Judged from the residual anisotropy r(infinity) the amplitude of the local motion of Trp89 increased upon increasing [iPrOH] 10%. Stopped-flow fluorescence spectroscopy measurements suggested the lid to open within approximately 2 ms upon transfer of W89m into 25% iPrOH. Steady-state anisotropies and longer correlation times revealed increasing concentrations of iPrOH to result also in the formation of dimers as well as possibly also higher oligomers by TLL.

Production of margarine fats by enzymatic interesterification with silica‐granulated Thermomyces lanuginosa lipase in a large‐scale study

AbstractInteresterification of a blend of palm stearin and coconut oil (75∶25, w/w), catalyzed by an immobilized Thermomyces lanuginosa lipase by silica granulation, Lipozyme TL IM, was studied for production of margarine fats in a 1‐ or 300‐kg pilot‐scale batch‐stirred tank reactor. Parameters and reusability were investigated. The comparison was carried out between enzymatic and chemical interesterified products. Experimentally, Lipozyme TL IM had similar activity to Lipozyme IM for the interesterification of the blend. Within the range of 55–80°C, temperature had little influence on the degree of interesterification for 6‐h reaction, but it had slight impact on the content of free fatty acids (FFA). Drying of Lipozyme TL IM from water content 6 to 3% did not affect its activity, whereas it greatly reduced FFA and diacylglycerol contents in the products. Lipozyme TL IM was stable in the 1‐kg scale reactor at least for 11 batches and the 300‐kg pilot‐scale reactor at least for nine batches. Due to regiospecificity of the lipase (sn‐1,3 specific), enzymatically interesterified products had different fatty acid distribution at sn‐2 position from the chemically randomized products, implying the potential nutritional benefits of the new technology.

Structural origins of the interfacial activation in Thermomyces (Humicola) lanuginosa lipase.

The already known X-ray structures of lipases provide little evidence about initial, discrete structural steps occurring in the first phases of their activation in the presence of lipids (process referred to as interfacial activation). To address this problem, five new Thermomyces (formerly Humicola) lanuginosa lipase (TlL) crystal structures have been solved and compared with four previously reported structures of this enzyme. The bias coming from different crystallization media has been minimized by the growth of all crystals under the same crystallization conditions, in the presence of detergent/lipid analogues, with low or high ionic strength as the only main variable. Resulting structures and their characteristic features allowed the identification of three structurally distinct species of this enzyme: low activity form (LA), activated form (A), and fully Active (FA) form. The isomerization of the Cys268-Cys22 disulfide, synchronized with the formation of a new, short alpha(0) helix and flipping of the Arg84 (Arginine switch) located in the lid's proximal hinge, have been postulated as the key, structural factors of the initial transitions between LA and A forms. The experimental results were supplemented by theoretical calculations. The magnitude of the activation barrier between LA (ground state) and A (end state) forms of TlL (10.6 kcal/mol) is comparable to the enthalpic barriers typical for ring flips and disulfide isomerizations at ambient temperatures. This suggests that the sequence of the structural changes, as exemplified in various TlL crystal structures, mirror those that may occur during interfacial activation.

Using DNA-Tagged Mutagenesis to Improve Heterologous Protein Production in Aspergillus oryzae

Using DNA-tagged mutagenesis to improve heterologous protein production in Aspergillus oryzae.Fungal Genetics and Biology29, 28–37. Restriction enzyme-mediated integration (REMI) has been employed as a mutagen to generate two insertion libraries in an Aspergillus oryzae strain expressing a Thermomyces lanuginosus lipase. The REMI libraries were created using linearized plasmid containing the A. oryzaepyrG and either BamHI or EcoRI enzyme. The libraries were screened for lipase production, and mutants with increased production were isolated. The genomic DNA flanking the integration event was cloned from one of the mutants with increased lipase titers (DEBY10.3). Nucleotide sequence of the flanking DNA revealed similarity to the Aspergillus nidulans palB gene. Disruption of the palB gene in a strain producing lipase resulted in increased lipase expression. Additionally, complementation of the palB phenotype of DEBY10.3 led to a decrease in lipase production. These lines of evidence demonstrate that the increase in lipase yield in DEBY10.3 is linked to the palB phenotype generated by the integration of the pyrG gene into the palB gene. The results also demonstrated that tagged mutagenesis with REMI can be used to identify genes that influence expression of heterologous proteins.

Kinetics of mRNA and protein synthesis of genes controlled by the 1,4-beta-endoxylanase A promoter in controlled fermentations of Aspergillus awamori.

In this study, induction and repression kinetics of the expression of the Aspergillus awamori 1,4-beta-endoxylanase A (exlA) gene under defined physiological conditions was analyzed at the mRNA and the protein levels. Induction was analyzed by pulsing D-xylose to a sucrose-limited continuous culture of an A. awamori 1,4-beta-endoxylanase A (EXLA)-overproducing strain. Directly after the D-xylose pulse, exIA mRNA was synthesized, and it reached a constant maximal level after 45 to 60 min. This level was maintained as long as D-xylose was present. The kinetics of mRNA synthesis of the genes encoding Thermomyces lanuginosa lipase (lplA) and Escherichia coli beta-glucuronidase (uidA), which were also under the control of the exlA promoter, were similar to those observed for exlA mRNA. The repression of exlA expression was analyzed by pulsing D-glucose to a D-xylose-limited continuous culture. Immediately after the glucose pulse, the exlA mRNA level declined rapidly, with a half-life of approximately 20 to 30 min, and it reached a minimal level after 60 to 90 min. The time span between mRNA synthesis and the secretion of proteins was determined for EXLA and lipase. In both cases, mRNA became visible after approximately 7.5 min. After 1 h, both proteins became detectable in the medium but the rate of secretion of EXLA was faster than that of lipase.