Gene_locus Report for: humla-1lipa

The function of most lipases is controlled by the lid, which undergoes conformational changes at a water-lipid interface to expose the active site, thus activating catalysis. Understanding how lid mutations affect lipases' function is important for designing improved variants. Lipases' function has been found to correlate with their diffusion on the substrate surface. Here, we used single-particle tracking (SPT), a powerful tool for deciphering enzymes' diffusional behavior, to study Thermomyces lanuginosus lipase (TLL) variants with different lid structures in a laundry-like application condition. Thousands of parallelized recorded trajectories and hidden Markov modeling (HMM) analysis allowed us to extract three interconverting diffusional states and quantify their abundance, microscopic transition rates, and the energy barriers for sampling them. Combining those findings with ensemble measurements, we determined that the overall activity variation in the application condition is dependent on surface binding and lipase mobility when bound. Specifically, the L4 variant with a TLL-like lid and wild-type (WT) TLL displayed similar ensemble activity, but WT bound stronger to the surface than L4, while L4 had a higher diffusion coefficient and thus activity when bound to the surface. These mechanistic elements can only be de-convoluted by our combined assays. Our findings offer fresh perspectives on the development of the next iteration of enzyme-based detergent.

Lipases are remarkable biocatalysts and are broadly applied in many industry fields because of their versatile catalytic capabilities. Considering the harsh biotechnological treatment of industrial processes, the activities of lipase products are required to be maintained under extreme conditions. In our current study, Gibbs free energy calculations were performed to predict potent thermostable Thermomyces lanuginosus lipase (TLL) variants by Rosetta design programs. The calculating results suggest that engineering on R209 may greatly influence TLL thermostability. Accordingly, ten TLL mutants substituted R209 were generated and verified. We demonstrate that three out of ten mutants (R209H, R209M, and R209I) exhibit increased optimum reaction temperatures, melting temperatures, and thermal tolerances. Based on molecular dynamics simulation analysis, we show that the stable hydrogen bonding interaction between H198 and N247 stabilizes the local configuration of the 250-loop in the three R209 mutants, which may further contribute to higher rigidity and improved enzymatic thermostability. Our study provides novel insights into a single residue, R209, and the 250-loop, which were reported for the first time in modulating the thermostability of TLL. Additionally, the resultant R209 variants generated in this study might be promising candidates for future-industrial applications.

The autoimmobilization of enzymes via cross-linked enzyme crystals (CLECs) has regained interest in recent years, boosted by the extensive knowledge gained in protein crystallization, the decrease of cost and laboriousness of the process, and the development of potential applications. In this work, we present the crystallization and preparative-scale production of reinforced cross-linked lipase crystals (RCLLCs) using a commercial detergent additive as a raw material. Bulk crystallization was carried out in 500 mL of agarose media using the batch technique. Agarose facilitates the homogeneous production of crystals, their cross-linking treatment, and their extraction. RCLLCs were active in an aqueous solution and in hexane, as shown by the hydrolysis of p-nitrophenol butyrate and alpha-methylbenzyl acetate, respectively. RCLLCs presented both high thermal and robust operational stability, allowing the preparation of a packed-bed chromatographic column to work in a continuous flow. Finally, we determined the three-dimensional (3D) models of this commercial lipase crystallized with and without phosphate at 2.0 and 1.7 A resolutions, respectively.

The function of most lipases is controlled by the lid, which undergoes conformational changes at a water-lipid interface to expose the active site, thus activating catalysis. Understanding how lid mutations affect lipases' function is important for designing improved variants. Lipases' function has been found to correlate with their diffusion on the substrate surface. Here, we used single-particle tracking (SPT), a powerful tool for deciphering enzymes' diffusional behavior, to study Thermomyces lanuginosus lipase (TLL) variants with different lid structures in a laundry-like application condition. Thousands of parallelized recorded trajectories and hidden Markov modeling (HMM) analysis allowed us to extract three interconverting diffusional states and quantify their abundance, microscopic transition rates, and the energy barriers for sampling them. Combining those findings with ensemble measurements, we determined that the overall activity variation in the application condition is dependent on surface binding and lipase mobility when bound. Specifically, the L4 variant with a TLL-like lid and wild-type (WT) TLL displayed similar ensemble activity, but WT bound stronger to the surface than L4, while L4 had a higher diffusion coefficient and thus activity when bound to the surface. These mechanistic elements can only be de-convoluted by our combined assays. Our findings offer fresh perspectives on the development of the next iteration of enzyme-based detergent.

Lipases are remarkable biocatalysts and are broadly applied in many industry fields because of their versatile catalytic capabilities. Considering the harsh biotechnological treatment of industrial processes, the activities of lipase products are required to be maintained under extreme conditions. In our current study, Gibbs free energy calculations were performed to predict potent thermostable Thermomyces lanuginosus lipase (TLL) variants by Rosetta design programs. The calculating results suggest that engineering on R209 may greatly influence TLL thermostability. Accordingly, ten TLL mutants substituted R209 were generated and verified. We demonstrate that three out of ten mutants (R209H, R209M, and R209I) exhibit increased optimum reaction temperatures, melting temperatures, and thermal tolerances. Based on molecular dynamics simulation analysis, we show that the stable hydrogen bonding interaction between H198 and N247 stabilizes the local configuration of the 250-loop in the three R209 mutants, which may further contribute to higher rigidity and improved enzymatic thermostability. Our study provides novel insights into a single residue, R209, and the 250-loop, which were reported for the first time in modulating the thermostability of TLL. Additionally, the resultant R209 variants generated in this study might be promising candidates for future-industrial applications.

The autoimmobilization of enzymes via cross-linked enzyme crystals (CLECs) has regained interest in recent years, boosted by the extensive knowledge gained in protein crystallization, the decrease of cost and laboriousness of the process, and the development of potential applications. In this work, we present the crystallization and preparative-scale production of reinforced cross-linked lipase crystals (RCLLCs) using a commercial detergent additive as a raw material. Bulk crystallization was carried out in 500 mL of agarose media using the batch technique. Agarose facilitates the homogeneous production of crystals, their cross-linking treatment, and their extraction. RCLLCs were active in an aqueous solution and in hexane, as shown by the hydrolysis of p-nitrophenol butyrate and alpha-methylbenzyl acetate, respectively. RCLLCs presented both high thermal and robust operational stability, allowing the preparation of a packed-bed chromatographic column to work in a continuous flow. Finally, we determined the three-dimensional (3D) models of this commercial lipase crystallized with and without phosphate at 2.0 and 1.7 A resolutions, respectively.

Aim: To understand the details of the action of fungal lipase and the mechanism for its observed interfacial activation. Background: Fungal lipase, crucial to biotechnology, functions at the lipid - water interface where it undergoes a poorly understood interfacial activation. Biochemical factors influencing its activation and inhibition are also poorly understood. This study provides a basis for its activity and a plausible mechanism for interfacial activation. Objective: To determine the structures of fungal lipase in different crystal forms in complex with their enzymatic reactants and inhibitors. Methods: X-ray crystallography. Results: Thermomyces lanuginosa lipase was visualized in three crystal forms, of space groups H32, P21 and I222 at 1.3 to 1.45 A resolution. Rhombohedral crystals have one molecule, lacking segment 241 to 252, as an asymmetric unit, with molecules organized as two trimers. Monoclinic crystals asymmetric unit is six intact molecules organized as two, nearly identical trimers, each exhibiting an NCS threefold axis. The -Ylid helix was consistently closed. Oligomerization into trimers creates an internal hydrophobic cavity where catalysis occurs. In monoclinic and orthorhombic crystals, active site serines were esterified to fatty acids. Lipase had bound within their trimeric, hydrophobic cavities 1,3-diacylglycerols with fatty acid chain lengths of about 18 carbons. Conclusion: Results suggest trimers are likely the active form of the enzyme at the lipid - water interface. Formation of trimers may provide an explanation for 'interfacial activation'.

The specific activity and enantioselectivity of immobilized cutinases from Aspergillus oryzae (AoC) and Humicola insolens (HiC) were compared with those of lipases from Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML) and Lipase B from Candida antarctica (CALB) for menthol and its analogs that include isopulegol, trans-2-tert-butylcyclohexanol (2TBC), and dihydrocarveol (DHC). Common features of these alcohols are two bulky substituents: a cyclohexyl ring and an alkyl substituent. Dissimilarities are that the alkyl group reside at different positions or have dissimilar structures. The aim was to develop an understanding at a molecular level of similarities and differences in the catalytic behavior of the selected cutinases and lipases as a function of substrate structural elements. The experimental results reflect the (-)-enantioselectivity for AoC, HiC, TLL, and RML, while CALB is only active on DHC with (+)-enantioselectivity. In most cases, AoC has the highest activity while HiC is significantly more active than other enzymes on 2TBC. The E values of AoC, HiC, TLL, and RML for menthol are 27.8, 16.5, 155, and 125, respectively. HiC has a higher activity (>10-fold) on (-)-2TBC than AoC while they exhibit similar activities on menthol. Docking results reveal that the bulky group adjacent to the hydroxyl group determines the enantioselectivity of AoC, HiC, TLL, and RML. Amino acid residues that dominate the enantioselectivity of these enzymes are AoC's Phe195 aromatic ring; HiC's hydrophobic Leu 174 and Ile 169 groups; TLL's ring structures of Trp89, His258 and Tyr21; and Trp88 for RML. Results of this study highlight that cutinases can provide important advantages relative to lipases for enantioselective transformation, most notably with bulky and sterically hindered substrates.

An immobilization method for binding cross-linked enzyme aggregates of Lipozyme TL 100L on macroporous resin NKA (CLEA-TLL@NKA) was developed in this study. The esterification activity of CLEA-TLL@NKA reached 6.4 U/mg. The surface structure of immobilized lipase was characterized by scanning electron microscopy. Methyl esterification reaction of soybean oil deodorizer distillate (SODD) was catalyzed by CLEA-TLL@NKA, which the conversion rate reached 98% and its activity retained over 90% after 20 batches of reaction. Compared with the commercial enzyme Lipozyme TLIM, half-life (t 1/2) of CLEA-TLL@NKA increased by 25 times and the catalytic activity increased by approximate 10 times. Thus, CLEA-TLL@NKA had high catalytic activity, good operational stability, and potential industrial application in the field of oil processing.

Lipases are interfacially activated enzymes that catalyze the hydrolysis of ester bonds and constitute prime candidates for industrial and biotechnological applications ranging from detergent industry, to chiral organic synthesis. As a result, there is an incentive to understand the mechanisms underlying lipase activity at the molecular level, so as to be able to design new lipase variants with tailor-made functionalities. Our understanding of lipase function primarily relies on bulk assay averaging the behavior of a high number of enzymes masking structural dynamics and functional heterogeneities. Recent advances in single molecule techniques based on fluorogenic substrate analogues revealed the existence of lipase functional states, and furthermore so how they are remodeled by regulatory cues. Single particle studies of lipases on the other hand directly observed diffusional heterogeneities and suggested lipases to operate in two different modes. Here to decipher how mutations in the lid region controls Thermomyces lanuginosus lipase (TLL) diffusion and function we employed a Single Particle Tracking (SPT) assay to directly observe the spatiotemporal localization of TLL and rationally designed mutants on native substrate surfaces. Parallel imaging of thousands of individual TLL enzymes and HMM analysis allowed us to observe and quantify the diffusion, abundance and microscopic transition rates between three linearly interconverting diffusional states for each lipase. We proposed a model that correlate diffusion with function that allowed us to predict that lipase regulation, via mutations in lid region or product inhibition, primarily operates via biasing transitions to the active states.

Background: Lipolytic enzymes of hyperthermophilic archaea generally prefer small carbon chain fatty acid esters (C2-C12) and are categorized as esterases. However, a few have shown activity with long-chain fatty acid esters, but none of them have been classified as a true lipase except a lipolytic enzyme AFL from Archaeglobus fulgidus. Thus, our main objective is to engineer an archaeal esterase into a true thermostable lipase for industrial applications. Lipases which hydrolyze long-chain fatty acid esters display an interfacial activation mediated by the lid domain which lies over active site and switches to open conformation at the oil-water interface. Lid domains modulate enzyme activities, substrate specificities, and stabilities which have been shown by protein engineering and mutational analyses. Here, we report engineering of an uncharacterized monoacylglycerol lipase (TON-LPL) from an archaeon Thermococcus onnurineus (strain NA1) into a triacylglycerol lipase (rc-TGL) by replacing its 61 N-terminus amino acid residues with 118 residues carrying lid domain of a thermophilic fungal lipase-Thermomyces lanuginosus (TLIP). Results: TON-LPL and rc-TGL were cloned and overexpressed in E. coli, and the proteins were purified by Ni-NTA affinity chromatography for biochemical studies. Both enzymes were capable of hydrolyzing various monoglycerides and shared the same optimum pH of 7.0. However, rc-TGL showed a significant decrease of 10 degrees C in its optimum temperature (Topt). The far UV-CD spectrums were consistent with a well-folded alpha/beta-hydrolase fold for both proteins, but gel filtration chromatography revealed a change in quaternary structure from trimer (TON-LPL) to monomer (rc-TGL). Seemingly, the difference in the oligomeric state of rc-TGL may be linked to a decrease in temperature optimum. Nonetheless, rc-TGL hydrolyzed triglycerides and castor oil, while TON-LPL was not active with these substrates. Conclusions: Here, we have confirmed the predicted esterase activity of TON-LPL and also performed the lid engineering on TON-LPL which effectively expanded its substrate specificity from monoglycerides to triglycerides. This approach provides a way to engineer other hyperthermophilic esterases into industrially suitable lipases by employing N-terminal domain replacement. The immobilized preparation of rc-TGL has shown significant activity with castor oil and has a potential application in castor oil biorefinery to obtain value-added chemicals.

Determining optimal conditions for the production of well diffracting crystals is a key step in every biocrystallography project. Here, a microfluidic device is described that enables the production of crystals by counter-diffusion and their direct on-chip analysis by serial crystallography at room temperature. Nine 'non-model' and diverse biomacromolecules, including seven soluble proteins, a membrane protein and an RNA duplex, were crystallized and treated on-chip with a variety of standard techniques including micro-seeding, crystal soaking with ligands and crystal detection by fluorescence. Furthermore, the crystal structures of four proteins and an RNA were determined based on serial data collected on four synchrotron beamlines, demonstrating the general applicability of this multipurpose chip concept.

In the present work, monoacylglycerol derivatives, i.e., 1-monomyristin, 2-monomyristin, and 2-monopalmitin were successfully prepared from commercially available myristic acid and palmitic acid. The 1-monomyristin compound was prepared through a transesterification reaction between ethyl myristate and 1,2-O-isopropylidene glycerol, which was obtained from the protection of glycerol with acetone, then followed by deprotection using Amberlyst-15. On the other hand, 2-monoacylglycerol derivatives were prepared through enzymatic hydrolysis of triglycerides in the presence of Thermomyces lanuginosa lipase enzymes. The synthesized products were analyzed using fourier transform infrared (FTIR) spectrophotometer, gas or liquid chromatography-mass spectrometer (GC-MS or LC-MS), and proton and carbon nuclear magnetic resonance ((1)H- and (13)C-NMR) spectrometers. It was found that monomyristin showed high antibacterial and antifungal activities, while 2-monopalmitin did not show any activity at all. The 1-monomyristin compound showed higher antibacterial activity against Staphylococcus aureus and Aggregatibacter actinomycetemcomitans and also higher antifungal activity against Candida albicans compared to the positive control. Meanwhile, 2-monomyristin showed high antibacterial activity against Escherichia coli. The effect of the acyl position and carbon chains towards antibacterial and antifungal activities was discussed.

A lipase from Thermomyces lanuginosus (Lipozyme TL IM) exhibited high enantioselectivity for kinetic resolution of (+/-)-menthol in organic solvent. The various reaction parameters affecting the conversion and enantioselectivity were studied. The optimum reaction conditions for the transesterification reaction were found with vinyl acetate in the solvent of methyl tert-butyl ether with a vinyl acetate:(+/-)-menthol molar ratio of 5:1 and an enzyme concentration of 200 g/L at 30 degreeC. In these conditions, (-)-menthyl acetate with 99.3% enantiomeric excess was obtained, whereas the conversion was 34.7% with the reaction time of 12 H at the substrate concentration of 0.5 M. In addition, the enzyme allowed the substrate loading to be increased up to 1.5 M without the decrease of the enantioselectivity. These results indicated that Lipozyme TL IM was a promising biocatalyst in the resolution of (+/-)-menthol.

Thermomyces lanuginosus lipase (TlL) and related lipases become activated in low-polarity environments that exist at the water-lipid interface where a structural change of the "lid" region occurs. In this work, we have investigated the activation of TlL (Lipase_W89) and certain lid mutants, containing either a single positive charge mutation, E87K (Lipase_K87_W89), within the lid region or a lid residue composition of both lipase and esterase character (Hybrid_W89) as a function of solvent polarity. Activation differences between the variants and TlL were studied by a combination of biophysical and theoretical methods. To investigate the structural changes taking place in the lid region upon lipase activation, we used a fluorescence-based method measuring the efficiency of Trp89 in the lid to quench the fluorescence of a bimane molecule attached in front (C255) and behind (C61) the lid. These structural changes were compared to the enzymatic activity of each variant at the water-substrate interface and to theoretical calculations of the energies associated with lid opening as a function of the dielectric constant (epsilon) of the environment. Our results show that the lid in Lipase_K87_W89 undergoes a pronounced structural transition toward an open conformation around epsilon = 50, whereas only small changes are detected for Lipase_W89 ascribed to the stabilizing effect of the positive charge mutation on the open lid conformation. Interestingly, Hybrid_W89, with the same charge as Lipase_W89, shows a stabilization of the open lid even more pronounced at high solvent polarities than that of Lipase_K87_W89, allowing activation at epsilon < 80. This is further indicated by measurement of the lipase activity for each variant showing that Hybrid_W89 is more quickly activated at the water-lipid interface of a true, natural substrate. Combined, we show that a correlation exists between structural changes and enzymatic activities detected on one hand and theoretical calculations on lid opening energies on the other. These results highlight the key role that the lid plays in determining the polarity-dependent activation of lipases.

Here, we present a lipase mutant containing a biochemical switch allowing a controlled opening and closing of the lid independent of the environment. The closed form of the TlL mutant shows low binding to hydrophobic surfaces compared to the binding observed after activating the controlled switch inducing lid-opening. We directly show that lipid binding of this mutant is connected to an open lid conformation demonstrating the impact of the exposed amino acid residues and their participation in binding at the water-lipid interface. The switch was created by introducing two cysteine residues into the protein backbone at sites 86 and 255. The crystal structure of the mutant shows the successful formation of a disulfide bond between C86 and C255 which causes strained closure of the lid-domain. Control of enzymatic activity and binding was demonstrated on substrate emulsions and natural lipid layers. The locked form displayed low enzymatic activity (~10%) compared to wild-type. Upon release of the lock, enzymatic activity was fully restored. Only 10% binding to natural lipid substrates was observed for the locked lipase compared to wild-type, but binding was restored upon adding reducing agent. QCM-D measurements revealed a seven-fold increase in binding rate for the unlocked lipase. The TlL_locked mutant shows structural changes across the protein important for understanding the mechanism of lid-opening and closing. Our experimental results reveal sites of interest for future mutagenesis studies aimed at altering the activation mechanism of TlL and create perspectives for generating tunable lipases that activate under controlled conditions.

Background The lipase enzyme converts long chain acyltriglycerides into di- and monoglycerides, glycerol and fatty acids. The catalytic site in lipase is situated deep inside the molecule. It is connected through a tunnel to the surface of the molecule. In the unbound state under aqueous conditions, the tunnel remains closed. The tunnel can be opened when the enzyme is exposed to a lipid bilayer or a detergent or many hydrophobic/hydrophilic surfaces. Results In the present study, the lipase was subjected to three-phase partitioning (TPP) which consisted of mixing in tert-butanol and ammonium sulphate to the solution of lipase in the aqueous buffer. The enzyme formed an interfacial precipitate between the tert-butanol rich and water rich phases. The stability of the enzyme subjected to TPP was found to be higher (Tm of 80 C) than the untreated enzyme (Tm of 77 C). The activity of the enzyme subjected to TPP (3.3 U/mg) was nearly half of that of the untreated one (5.8 U/mg). However, the activity of the treated enzyme was higher (17.8 U/mg) than the untreated one (8.6 U/mg) when a detergent was incorporated in the assay buffer. Conclusions The structure determination showed that the substrate binding site in the treated enzyme was more tightly closed than that of the untreated protein.

We have used the crystal structure of Thermomyces lanuginosus lipase (TlL) to identify and strengthen potential protein-protein interaction sites in solution. As wildtype we used a deglycosylated mutant of TlL (N33Q). We designed a number of TlL mutants to promote interactions via interfaces detected in the crystal-lattice structure, through strengthening of hydrophobic, polar or electrostatic contacts or truncation of sterically blocking residues. We identify a mutant predicted to lead to increased interfacial hydrophobic contacts (N92F) that shows markedly increased self-association properties on native gradient gels. While wildtype TlL mainly forms monomer and <5% dimers, N92F forms stable trimers and dimers according to Size-Exclusion Chromatography and Small-Angle X-ray Scattering. These oligomers account for ~25% of the population and their enzymatic activity is comparable to that of the monomer. Self-association stabilizes TlL against thermal denaturation. Furthermore, the trimer is stable to dilution and requires high concentrations (>2M) of urea to dissociate. We conclude that crystal lattice contacts are a good starting point for design strategies to promote protein self-association.

Enantioenriched promethazine and ethopropazine were synthesized through a simple and straightforward four-step chemoenzymatic route. The central chiral building block, 1-(10H-phenothiazin-10-yl)propan-2-ol, was obtained via a lipase-mediated kinetic resolution protocol, which furnished both enantiomeric forms, with superb enantioselectivity (up to E = 844), from the racemate. Novozym 435 and Lipozyme TL IM have been found as ideal biocatalysts for preparation of highly enantioenriched phenothiazolic alcohols (up to >99% ee), which absolute configurations were assigned by Mosher's methodology and unambiguously confirmed by XRD analysis. Thus obtained key-intermediates were further transformed into bromide derivatives by means of PBr3, and subsequently reacted with appropriate amine providing desired pharmacologically valuable (R)- and (S)-stereoisomers of title drugs in an ee range of 84-98%, respectively. The modular amination procedure is based on a solvent-dependent stereodivergent transformation of the bromo derivative, which conducted in toluene gives mainly the product of single inversion, whereas carried out in methanol it provides exclusively the product of net retention. Enantiomeric excess of optically active promethazine and ethopropazine were established by HPLC measurements with chiral columns.

The variant Lip-T (S88T/A99N/V116D) of lipase Lip from Thermomyces lanuginosus has been proved to be a potential biocatalyst for kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE) to produce valuable chiral intermediate of Pregabalin. In this study, random, site-directed and site-saturation mutagenesis were performed to further enhance the activity of Lip-T, and the key residues responsible for catalytic efficiency were revealed. A mutant S63L/D232A with improved activity toward CNDE was obtained after screening of approximately 2500 clones from random-mutant libraries. Site-directed mutagenesis at site 63 and 232 demonstrated that the single-point mutants S63L and D232A showed opposite effect on activity. S63L exhibited a significant improvement on activity, whereas D232A exerted a slight inhibitory effect. Then a mutant S63M with a 4.5-fold higher catalytic efficiency than Lip-T was obtained by site-saturation mutagenesis. Structural changes resulting from the mutations were analyzed and the mechanisms responsible for the enhanced activity were discussed. Moreover, the engineered lipase catalyzed enantioselective hydrolysis of CNDE at a very high substrate loading (765g/l). As only 5% (w/v) resting cells were used, the bioprocess is much more cost-effective than Pfizer's process using 8% (w/v) commercially available lipase Lipolase(R). These results provide not only new insights into lipase structure-function relationships but also a novel robust biocatalyst for the production of Pregabalin.

A novel esterase encoding gene, tle, was cloned from the thermophilic fungus Thermomyces lanuginosus DSM 10635. The tle had an open reading frame of 945bp encoding TLE of 314 amino acids with a theoretical molecular mass of 34.5kDa. The putative catalytic triad of TLE was consisted of Ser151, His279, and Asp249. TLE was heterologously expressed in Escherichia coli in biologically active form and purified to homogeneity. Several biochemical properties of TLE were studied: Among the tested p-nitrophenol esters, TLE showed the highest hydrolytic activity with p-nitrophenyl butyrate (C4) and exhibited the maximum activity at 60 degrees C and pH 8.5. The enzyme was stable at temperatures below 60 degrees C and retained 53% of the maximum activity after treatment at 70 degrees C for 60min. Esterase activity was notably enhanced by addition of Ca(2+) and Ba(2+), respectively. Furthermore, TLE showed high enantioselectivity (E=95) in the kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE), which produce a valuable chiral intermediate-(3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid for Pregabalin. These unique properties of the esterase indicate that TLE is a potential candidate for industrial application.

It is shown by rational site-directed mutagenesis of the lid region in Thermomyces lanuginosus lipase that it is possible to generate lipase variants with attractive features, e.g., high lipase activity, fast activation at the lipid interface, ability to act on water-soluble substrates, and enhanced calcium independence. The rational design was based on the lid residue composition in Aspergillus niger ferulic acid esterase (FAEA). Five constructs included lipase variants containing the full FAEA lid, a FAEA-like lid, an intermediate lid of FAEA and TlL character, and the entire lid region from Aspergillus terreus lipase (AtL). To investigate an altered activation mechanism for each variant compared to that of TlL, a combination of activity- and spectroscopic-based measurements were applied. The engineered variant with a lid from AtL displayed interfacial activation comparable to that of TlL, whereas variants with FAEA lid character showed interfacial activation independence with pronounced activity toward pNP-acetate and pNP-butyrate below the critical micelle concentration. For variants with lipase and esterase character, lipase activity measurements further indicated a faster activation at the lipid interface. Relative to their activity toward pNP-ester substrates in calcium-rich buffer, all lid variants retained between 15 and 100% activity in buffer containing 5 mM EDTA whereas TlL activity was reduced to less than 2%, demonstrating the lid's central role in governing calcium dependency. For FAEA-like lid variants, accessible hydrophobic surface area measurements showed an approximate 10-fold increase in the level of binding of extrinsic fluorophores to the protein surface relative to that of TlL accompanied by a blue shift in emission indicative of an open lid in aqueous solution. Together, these studies report on the successful alteration of the activation mechanism in TlL by rational design creating novel lipases with new, intriguing functionalities.

A lipase may catalyze either one or more of the three reactions of 11-acetyl-prostaglandin E2 in methanol-containing reaction medium: esterification, deacetylation, and/or elimination. The catalytic performance depends on the lipase and on the methanol content. An increase in the methanol concentration in benzene from 5% to 95% leads to the exclusive switch of reactions from esterification to elimination catalyzed by Thermomyces lanuginosus lipase (TLL). To explain the switch, molecular dynamics simulations of solvation of TLL in benzene and in methanol were performed. Solvation in methanol leads to the closing of the lid. The repositioning of the oxyanion hole towards the catalytic triad blocks the catalysis of ester synthesis whereas enabling TLL to act as an acetyl-beta-ketol eliminase. In benzene the lid is open, allowing esterification to occur. Docking analysis of 11-acetyl-prostaglandin E2 into the active site of the solvated TLL structures suggested the occurrence of reactions in accordance with the experiment.

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 degC; 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.

Two immobilized preparations from Thermomyces lanuginosus lipase (TLL) were compared in the synthesis of butyl butyrate. The commercial Lipozyme TL-IM, and TLL immobilized on styrene-divinylbenzene beads (MCI-TLL) were tested in the esterification reaction using n-hexane as solvent. The variables temperature (30-60 degrees C), substrate molar ratio (1:1 to 5:1), added water (0-1%), and biocatalyst content (3-40%) were evaluated in terms of initial reaction rate for each biocatalyst. SDS-PAGE analysis revealed that MCI-TLL had an immobilized enzymatic load twice as high as Lipozyme TL-IM, but with an activity 3-fold higher. MCI-TLL presented high initial reaction rates up to 1.0 M butyric acid, while Lipozyme TL-IM showed a decrease in its activity above 0.5 M. Moreover, MCI-TLL allowed a productivity of 14.5 mmol g(-1) h(-1), while Lipozyme TL-IM 3.2 mmol g(-1) h(-1), both by mass of biocatalyst.

Lipases with high kinetic stability and enzymatic efficiency in the human gastro-intestinal tract may help against exocrine pancreatic insufficiency. Here we mimic gastric conditions to study how bile salts and pH affect the stability and activity of Thermomyces lanuginosus lipase (TlL) and its stabler variant StL using spectroscopy, calorimetry and gel electrophoresis. Both enzymes resist trypsin digestion with and without bile salts. Bile salts activate native TlL and StL equally well, bind weakly to denatured TlL and StL at lower pH and precipitate native TlL and StL at pH 4. StL refolds more efficiently than TlL from gastric pH in bile salts, regaining activity when refolding from pH as low as 1.8 and above while TlL cannot go below pH 2.6. StL also unfolds 10-40 fold more slowly in the denaturant guanidinium chloride and the anionic surfactant SDS. We ascribe StL's superior performance to general alterations in its electrostatic potential which makes it more acid-resistant. These superior properties make StL a good candidate for pancreatic enzyme replacement therapy.

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 10mM 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.

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%.

The process of biodiesel production by the hydroesterification route that is proposed here involves a first step consisting of triacylglyceride hydrolysis catalyzed by lipase from Thermomyces lanuginosus (TL 100L) to generate free fatty acids (FFAs). This step is followed by esterification of the FFAs with alcohol, catalyzed by niobic acid in pellets or without a catalyst. The best result for the enzyme-catalyzed hydrolysis was obtained under reaction conditions of 50% (v/v) soybean oil and 2.3% (v/v) lipase (25 U/mL of reaction medium) in distilled water and at 60 degrees C; an 89% conversion rate to FFAs was obtained after 48 hours of reaction. For the esterification reaction, the best result was with an FFA/methanol molar ratio of 1:3, niobic acid catalyst at a concentration of 20% (w/w FFA), and 200 degrees C, which yielded 92% conversion of FFAs to soy methyl esters after 1 hour of reaction. This study is exceptional because both the hydrolysis and the esterification use a simple reaction medium with high substrate concentrations.

In the pharmaceutical industry, protein drugs are modified by, for instance, glycosylation in order to obtain protein drugs with improved delivery profiles and/or increased stability. The effect of glycosylation on protein adsorption behaviour is one of the stability aspects that must be evaluated during development of glycosylated protein drug products. We have studied the effect of glycosylation on the adsorption behaviour of Thermomyces lanuginosus lipase to hydrophobic and hydrophilic surfaces using total internal reflection fluorescence, surface plasmon resonance, far-UV circular dichroism and fluorescence. Three glyco-variants were used, namely the mono-glycosylated wildtype T. lanuginosus lipase, a non-glycosylated variant and a penta-glycosylated variant, the latter two containing one and nine amino acid substitutions, respectively. All the glycosylations were N-linked and contained no charged sugar residues. Glycosylation did not affect the adsorption of wildtype T. lanuginosus lipase to the hydrophobic surfaces. The number of molecules adsorbing per unit surface area, the structural changes occurring upon adsorption, and the orientation upon adsorption were found to be unaffected by the varying glycosylation. However, the interaction with a hydrophilic surface was different between the three glyco-variants. The penta-glycosylated T. lanuginosus lipase adsorbed, in contrast to the two other glyco-variants. In conclusion, adsorption of T. lanuginosus lipase to hydrophobic surfaces was not affected by N-linked glycosylation. Only penta-glycosylated T. lanuginosus lipase adsorbed to the hydrophilic surface, apparently due to its increased net charge of +3 caused by amino acid substitutions in the primary sequence.

Lipase from Thermomyces lanuginosus has been immobilized within particulate mesoporous silica carriers, with either hydrophilic or hydrophobic supporting surfaces, produced by the newly developed emulsion and solvent evaporation (ESE) method. The Michaelis-Menten model was used to calculate the parameters related to the enzymatic activity of lipase i.e. the turnover number, k(cat), and the specific activity. The specific activity was improved by immobilization of lipase onto the hydrophobic support, compared to lipase immobilized onto the hydrophilic support and lipase free in solution. The enhanced enzymatic activity of lipase onto a hydrophobic support was attributed to interfacial activation of the Thermomyces lanuginosus lipase when it is attached to a hydrophobic surface and a reduced denaturation. Confocal scanning laser microscopy (CLSM) studies, of fluorescently tagged lipase, showed that leakage of the lipase from the mesoporous particles was limited to an initial period of only a few hours. Both the rate and the amount of lipase leached were reduced when the lipase was immobilized onto the hydrophobic support.

With the aim of determining the fatty acid (FA) selectivity of lipases, a mixture of oleic acid and monoacid triacylglycerols (TAGs) including tricaproin (T6), tricaprylin (T8), tricaprin (T10), trilaurin (T12), trimyristin (T14), tripalmitin (T16) and tristearin (T18) was used as the substrate in acidolysis performed in hexane. Three immobilized lipases, namely, Lipozyme TL IM from Thermomyces lanoginosus, Lipozyme RM IM from Rhizomucor miehei and Novozym 435 from Candida antarctica, were used as biocatalyst. The effects of operating variables including the mole ratio of oleic acid to monoacid TAG, temperature, enzyme dosage and reaction time on incorporation were also investigated. Significantly different incorporation rates were obtained for different TAGs used (P < 0.05). Incorporation of oleic acid into TAGs except tricaproin and tricaprylin was higher for all the TAGs with Lipozyme TL IM catalyzed reactions than those of other two enzymes tested. Incorporation of oleic acid decreased as the acyl chain length of FA in the TAG increased with Novozyme 435 catalyzed acidolysis. Compared to the other substrate mixtures, the highest incorporation was observed for oleic acid and tricaproin mixture with three lipases tested. It was shown that the FA selectivity of the lipases is strongly dependent on the acyl chain length of FA in a TAG.

Substrate recognition of Thermomyces lanuginosus lipase in the acylation of nucleosides was revealed through rational substrate engineering for the first time. T. lanuginosus lipase displayed higher catalytic activities and excellent 5'-regioselectivities (94->99%) in the acylation of ribonucleosides 1f-1j as compared to those in the acylation of 2'-deoxynucleosides 1a-1e. The higher reaction rates and excellent 5'-regioselectivities might derive from a favorable hydrogen bonding between the 2'-hydroxyl group of 1f-1j and phenolic hydroxyl group of Tyr21 present in the hydrophilic region of the lipase.

Immobilized-stabilized aminated lipase from Thermomyces lanuginosus (TLL-A) is not easily reactivated after inactivation by incubation in the presence of organic solvents or chaotropic reagents. To improve the recovered activity of this biocatalyst, immobilized TLL-A has been submitted to different modifications. The best results were obtained when the enzyme was coated with a very hydrophilic and inert polymer: dextran modified with glycine (Dx-Gly). This modification did not reduce enzymatic activity while it increased the stability of this already very stable preparation, in thermal and organic solvent induced inactivation (by a 4-fold factor). Simple incubation in aqueous medium at pH 7 and 25 degrees C permitted to fully recover the activity of the immobilized and modified TLL-A enzyme inactivated by incubation in organic solvents or saturated guanidine during 3 cycles, while the non-modified enzyme only recover some activity. When the inactivation was caused by exposition at high temperatures, the reactivation was higher using the modified biocatalyst, but was far for complete (40% after 3 inactivation-reactivation cycles). The determination of the TLL-A activity in the presence of detergents (that helps the opening of active site of the lipase) allowed, in this case, to significantly improve the results, now near to 90% of the initial activity was recovered (using the non-modified enzyme the recovered activity was around 60%). This very hydrophilic and inert polymer, coating the enzyme surface, seems to help the correct positioning of the hydrophilic and hydrophobic groups of the enzyme, and that way improve both the stability and possibility of reactivation of the enzyme.

The influence of Thermomyces lanuginosus lipase (TLL) on the phase behaviour of liquid-crystalline phases of aqueous phytantriol as well as conformational changes of TLL entrapped in the cubic Q230 phase have been studied by small angle X-ray diffraction (SAXD), FT-Raman, and FT-IR techniques. It was found that the lipidic Q230 phase is able to accommodate up to 10 wt.% of TLL, and the temperature of phase transition to the inverted hexagonal phase H(II) increases indicating stabilizing effect of the protein. FT-Raman analysis of Trp amino acid marker band W3 revealed that the average rotation angle around the C3-Cbeta bond of four Trp residues of TLL in the Q230 phase increases. Reasoning from available TLL crystallographic data, this result is explained by structural transition of entrapped protein to so-called "open" and more related to the enzymatically-active conformation. TLL secondary structure analysis by amide I and amide III vibrational bands showed that content of alpha-helixes does not change, while a part of beta-sheet structures transforms to less ordered elements upon incorporation of protein into the Q230 phase of aqueous phytantriol.

Lipases are activated at interfaces between aqueous and hydrophobic phases, where they typically undergo conformational changes leading to significant activity increase. Here I use a quartz crystal microbalance with dissipation (QCM-D) to study changes in layer thickness and viscosity during the adsorption of variants of the Thermomyces lanuginosus lipase (TlL) onto a methyl-terminated hydrophobic surface. Unlike wildtype TlL, the variant Mut1, which shows improved performance under certain test conditions, shows a large dissipation increase during the binding process, leading to a significantly thicker layer. This altered adsorption behaviour may be linked to Mut1's changes in secondary structure. This is corroborated by the fact that four other TlL mutants with unaltered secondary structure showed wildtype-like absorption behaviour. Unlike wildtype TlL and the other variants, Mut1 contains several consecutive basic residues introduced into the C-terminal region which is close in space to the N-terminal part of the protein, which also contains several basic residues. Electrostatic repulsion between these two regions leading to local structural flexibility may facilitate altered adsorption behaviour and ultimately to improved enzymatic performance on a solid surface. QCM-D thus provides a good approach to screen protein variants for their adsorption properties on hydrophobic surfaces.

The adsorption and activity of Thermomyces lanuginosus lipase (TLL) was measured with dual polarization interferometry (DPI) and confocal microscopy at a hydrophilic and hydrophobic surface. In the adsorption isotherms, it was evident that TLL both had higher affinity for the hydrophobic surface and adsorbed to a higher adsorbed amount (1.90 mg/m(2)) compared to the hydrophilic surface (1.40-1.50mg/m(2)). The thickness of the adsorbed layer was constant (approximately 3.5 nm) on both surfaces at an adsorbed amount >1.0mg/m(2), but decreased on the hydrophilic surface at lower surface coverage, which might be explained by partially unfolding of the TLL structure. However, a linear dependence of the refractive index of the adsorbed layer on adsorbed amount of TLL on C18 surfaces indicated that the structure of TLL was similar at low and high surface coverage. The activity of adsorbed TLL was measured towards carboxyfluorescein diacetate (CFDA) in solution, which upon lipase activity formed a fluorescent product. The surface fluorescence intensity increase was measured in a confocal microscope as a function of time after lipase adsorption. It was evident that TLL was more active on the hydrophilic surface, which suggested that a larger fraction of adsorbed TLL molecules were oriented with the active site facing the solution compared to the hydrophobic surface. Moreover, most of the activity remained when the TLL surface coverage decreased. Earlier reports on TLL surface mobility on the same surfaces have found that the lateral diffusion was highest on hydrophilic surfaces and at low surface coverage of TLL. Hence, a high lateral mobility might lead to a longer exposure time of the active site towards solution, thereby increasing the activity against a water-soluble substrate.

The process of biodiesel production from corn oil catalyzed by lipozyme TL IM, an inexpensive 1,3-position specific lipase from Thermomyces lanuginosus was optimized by response surface methodology (RSM) and a central composite rotatable design (CCRD) was used to study the effects of enzyme dosage, ratio of t-butanol to oil (v/v) and ratio of methanol to oil (mol/mol) on the methyl esters (ME) yield of the methanolysis. The optimum combinations for the reaction were 25.9U/goil of enzyme, 0.58 volume ratio of t-butanol to oil and 0.5, 0.5, 2.8 molar equivalent of methanol to oil added at the reaction time of 0, 2, and 4h, respectively, by which a ME yield of 85.6%, which was very close to the predicted value of 85.0%, could be obtained after reaction for 12h. Waste oil was found to be more suitable feedstock, and could give 93.7% ME yield under the optimum conditions described above. Adding triethylamine (TEA), an acyl migration enhancer, could efficiently improve the ME yield of the methanolysis of corn oil, giving a ME yield of 92.0%.

The effects of various detergents and pH on the interfacial binding and activity of two fungal lipases from Yarrowia lipolytica (YLLIP2) and Thermomyces lanuginosus (TLL) were investigated using trioctanoin emulsions as well as monomolecular films spread at the air-water interface. Contrary to TLL, YLLIP2 was found to be more sensitive than TLL to interfacial denaturation but it was protected by detergent monomers and lowering the temperature. At pH 7.0, both the interfacial binding and the activities on trioctanoin of YLLIP2 and TLL were inhibited by sodium taurodeoxycholate (NaTDC). At pH 6.0, however, YLLIP2 remained active on trioctanoin in the presence of NaTDC, whereas TLL did not. YLLIP2 activity on trioctanoin was associated with strong interfacial binding of the enzyme to trioctanoin emulsion, whereas TLL was mostly detected in the water phase. The combined effects of bile salts and pH on lipase activity were therefore enzyme-dependent. YLLIP2 binds more strongly than TLL at oil-water interfaces at low pH when detergents are present. These findings are particularly important for lipase applications, in particular for enzyme replacement therapy in patients with pancreatic enzyme insufficiency since high detergent concentrations and highly variable pH values can be encountered in the GI tract.

We have studied the mobility of active and inactive Thermomyces lanuginosus lipase (TLL) on a spin-coated trimyristin substrate surface using fluorescence recovery after photobleaching (FRAP) in a confocal microscopy setup. By photobleaching a circular spot of fluorescently labeled TLL adsorbed on a smooth trimyristin surface, both the diffusion coefficient D and the mobile fraction f could be quantified. FRAP was performed on surfaces with different surface density of lipase and as a function of time after adsorption. The data showed that the mobility of TLL was significantly higher on the trimyristin substrate surfaces compared to our previous studies on hydrophobic model surfaces. For both lipase variants, the diffusion decreased to similar rates at high relative surface density of lipase, suggesting that crowding effects are dominant with higher adsorbed amount of lipase. However, the diffusion coefficient at extrapolated infinite surface dilution, D0, was higher for the active TLL compared to the inactive (D0 = 17.9 x 10(-11) cm2/s vs D0 = 4.1 x 10(-11) cm2/s, data for the first time interval after adsorption). Moreover, the diffusion decreased with time after adsorption, most evident for the active TLL. We explain the results by product inhibition, i.e., that the accumulation of negatively charged fatty acid products decreased the diffusion rate of active lipases with time. This was supported by sequential adsorption experiments, where the adsorbed amount under flow conditions was studied as a function of time after adsorption. A second injection of lipase led to a significantly lower increase in adsorbed amount when the trimyristin surface was pretreated with active TLL compared to pretreatment of inactive TLL.

The triglyceride lipase (EC 3.1.1.3) Thermomyces lanuginosus lipase (TLL) binds with high affinity to unilamellar phospholipid vesicles that serve as a diluent interface for both lipase and substrate, but it displays interfacial activation on only small and negatively charged such vesicles [Cajal, Y., et al. (2000) Biochemistry 39, 413-423]. The productive-mode binding orientation of TLL at the lipid-water interface of small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) was previously determined using electron spin resonance (ESR) spectroscopy in combination with site-directed spin-labeling [Hedin, E. M. K., et al. (2002) Biochemistry 41, 14185-14196]. In our investigation, we have studied the interfacial orientation of TLL when bound to large unilamellar vesicles (LUV) consisting of POPG, and bound to SUV consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC). Eleven single-cysteine TLL mutants were spin-labeled as previously described, and studied upon membrane binding using the water soluble spin-relaxation agent chromium(III) oxalate (Crox). Furthermore, dansyl-labeled vesicles revealed the intermolecular fluorescence quenching efficiency between each spin-label positioned on TLL, and the lipid membrane. ESR exposure and fluorescence quenching data show that TLL associates closer to the negatively charged PG surface than the zwitterionic PC surface, and binds to both POPG LUV and POPC SUV predominantly through the concave backside of TLL opposite the active site, as revealed by the contact residues K74C-SL, R209C-SL, and T192C-SL. This orientation is significantly different compared to that on the POPG SUV, and might explain the differences in activation of the lipase. Evidently, both the charge and accessibility (curvature) of the vesicle surface determine the TLL orientation at the phospholipid interface.

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.

The binding orientation of the interfacially activated Thermomyces lanuginosa lipase (TLL, EC 3.1.1.3) on phospholipid vesicles was investigated using site-directed spin labeling and electron spin resonance (ESR) relaxation spectroscopy. Eleven TLL single-cysteine mutants, each with the mutation positioned at the surface of the enzyme, were selectively spin labeled with the nitroxide reagent (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl) methanethiosulfonate. These were studied together with small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), to which TLL has previously been shown to bind in a catalytically active form [Cajal, Y., et al. (2000) Biochemistry 39, 413-423]. The orientation of TLL with respect to the lipid membrane was investigated using a water-soluble spin relaxation agent, chromium(III) oxalate (Crox), and a recently developed ESR relaxation technique [Lin, Y., et al. (1998) Science 279, 1925-1929], here modified to low microwave amplitude (<0.36 G). The exposure to Crox for the spin label at the different positions on the surface of TLL was determined in the absence and presence of vesicles. The spin label at positions Gly61-Cys and Thr267-Cys, closest to the active site nucleophile Ser146 of the positions analyzed, displayed the lowest exposure factors to the membrane-impermeable spin relaxant, indicating the proximity to the vesicle surface. As an independent technique, fluorescence spectroscopy was employed to measure fluorescence quenching of dansyl-labeled POPG vesicles as exerted by the protein-bound spin labels. The resulting Stern-Volmer quenching constants showed excellent agreement with the ESR exposure factors. An interfacial orientation of TLL is proposed on the basis of the obtained results.

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.

Thermal stability of wild type Humicola lanuginosa lipase (wt HLL) and its two mutants, W89L and the single Trp mutant W89m (W117F, W221H, and W260H), were compared. Differential scanning calorimetry revealed unfolding of HLL at T(d)=74.4 degrees C whereas for W89L and W89m this endotherm was decreased to 68.6 and 62 degrees C, respectively, demonstrating significant contribution of the above Trp residues to the structural stability of HLL. Fluorescence emission spectra revealed the average microenvironment of Trps of wt HLL and W89L to become more hydrophilic at elevated temperatures whereas the opposite was true for W89m. These changes in steady-state emission were sharp, with midpoints (T(m)) at approx. 70.5, 61.0, and 65.5 degrees C for wt HLL, W89L, and W89m, respectively. Both steady-state and time resolved fluorescence spectroscopy further indicated that upon increasing temperature, the local movements of tryptophan(s) in these lipases were first attenuated. However, faster mobilities became evident when the unfolding temperatures (T(m)) were exceeded, and the lipases became less compact as indicated by the increased hydrodynamic radii. Even at high temperatures (up to 85 degrees C) a significant extent of tertiary and secondary structure was revealed by circular dichroism. Activity measurements are in agreement with increased amplitudes of conformational fluctuations of HLL with temperature. Our results also indicate that the thermal unfolding of these lipases is not a two-state process but involves intermediate states. Interestingly, a heating and cooling cycle enhanced the activity of the lipases, suggesting the protein to be trapped in an intermediate, higher energy state. The present data show that the mutations, especially W89L in the lid, contribute significantly to the stability, structure and activity of HLL.

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.

Lipase from the fungi Thermomyces (formerly Humicola) lanuginosa (TlL) is widely used in industry. This interfacial enzyme is inactive under aqueous conditions, but catalytic activation is induced on binding to a lipid-water interface. In order for protein engineering to design more efficient mutants of TlL for specific applications, it is important to characterize its interfacial catalysis. A complete analysis of steady-state kinetics for the hydrolysis of a soluble substrate by TlL has been developed using an interface different from the substrate. Small vesicles of 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG) or other anionic phospholipids are a neutral diluent interface for the partitioning of substrate and enzyme. TlL binds to these interfaces in an active or open form, thus implying a displacement of the helical lid away from the active site. A study of the influence of substrate and diluent concentration dependence of the rate of hydrolysis provides a basis for the determination of the primary interfacial catalytic parameters. The interfacial activation is not supported by zwitterionic vesicles or by large anionic vesicles of 100 nm diameter, although TlL binds to these interfaces. Using a combination of fluorescence-based techniques applied to several mutants of TlL with different tryptophan residues we have shown that TlL binds to phospholipid vesicles in different forms rendering different catalytic activities, and that the open lid conformation is achieved and stabilized by a combination of electrostatic and hydrophobic interactions between the enzyme's lipid-binding face and the interface.

Small unilamelar vesicles of anionic phospholipids (SUV), such as 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG), provide an interface where Thermomyces lanuginosa triglyceride lipase (TlL) binds and adopts a catalytically active conformation for the hydrolysis of substrate partitioned in the interface, such as tributyrin or p-nitrophenylbutyrate, with an increase in catalytic rate of more than 100-fold for the same concentration of substrate [Berg et al. (1998) Biochemistry 37, 6615-6627.]. This interfacial activation is not seen with large unilamelar vesicles (LUV) of the same composition, or with vesicles of zwitterionic phospholipids such as 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine (POPC), independently of the vesicle size. Tryptophan fluorescence experiments show that lipase binds to all those types of vesicles with similar affinity, but it adopts different forms that can be correlated with the enzyme catalytic activity. The spectral change on binding to anionic SUV corresponds to the catalytically active, or "open" form of the enzyme, and it is not modified in the presence of substrate partitioned in the vesicles, as demonstrated with inactive mutants. This indicates that the displacement of the lid characteristic of lipase interfacial activation is induced by the anionic phospholipid interface without blocking the accessibility of the active site to the substrate. Experiments with a mutant containing only Trp89 in the lid show that most of the spectral changes on binding to POPG-SUVs take place in the lid region that covers the active site; an increase in Trp anisotropy indicates that the lid becomes less flexible in the active form, and quenching experiments show that it is significantly buried from the aqueous phase. On the other hand, results with a mutant where Trp89 is changed to Leu show that the environment of the structural tryptophans in positions 117, 221, and 260 is somehow altered on binding, although their mobility and solvent accessibility remains the same as in the inactive form in solution. The form of TlL bound to POPC-SUV or -LUV vesicles as well as to LUV vesicles of POPG has the same spectral signatures and corresponds to an inactive or "closed" form of the enzyme. In these interfaces, the lid is highly flexible, and Trp89 remains accessible to solvent. Resonance energy transfer experiments show that the orientation of TlL in the interface is different in the active and inactive forms. A model of interaction consistent with these data and the available X-ray structures is proposed. This is a unique system where the composition and physical properties of the lipid interface control the enzyme activity.

Detergent (pentaoxyethylene octyl ether, C(8)E(5))-induced conformational changes of Humicola lanuginosa lipase (HLL) were investigated by stationary and time-resolved fluorescence intensity and anisotropy measurements. Activation of HLL is characterized by opening of a surface loop (the "lid") residing directly over the enzyme active site. The interaction of HLL with C(8)E(5) increases fluorescence intensities, prolongs fluorescence lifetimes, and decreases the values of steady-state anisotropy, residual anisotropy, and the short rotational correlation time. Based on these data, we propose the following model. Already below critical micellar concentration (CMC) the detergent can intercalate into the active site accommodating cleft, while the lid remains closed. Occupation of the cleft by C(8)E(5) also blocks the entry of the monomeric substrate, and inhibition of catalytic activity at [C(8)E(5)] less than or equal to CMC is evident. At a threshold concentration close to CMC the cooperativity of the hydrophobicity-driven binding of C(8)E(5) to the lipase increases because of an increase in the number of C(8)E(5) molecules present in the premicellar nucleates on the hydrophobic surface of HLL. These aggregates contacting the lipase should have long enough residence times to allow the lid to open completely and expose the hydrophobic cleft. Concomitantly, the cleft becomes filled with C(8)E(5) and the "open" conformation of HLL becomes stable.

We have investigated the binding properties of and dynamics in Humicola lanuginosa lipase (Hll) and the inactive mutant S146A (active Ser146 substituted with Ala) using fluorescence spectroscopy and molecular dynamics simulations, respectively. Hll and S146A show significantly different binding behavior for phosphatidylcholine (PC) and phosphatidylglycerol (PG) liposomes. Generally, higher binding affinity is observed for Hll than the S146A mutant. Furthermore, depending on the matrix, the addition of the transition state analogue benzene boronic acid increases the binding affinity of S146A, whereas only small changes are observed for Hll suggesting that the active site lid in the latter opens more easily and hence more lipase molecules are bound to the liposomes. These observations are in agreement with molecular dynamics simulations and subsequent essential dynamics analyses. The results reveal that the hinges of the active site lid are more flexible in the wild-type Hll than in S146A. In contrast, larger fluctuations are observed in the middle region of the active site loop in S146A than in Hll. These findings reveal that the single mutation (S146A) of the active site serine leads to substantial conformational alterations in the H. lanuginosa lipase and different binding affinities.

The importance of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase for the hydrolytic activity at the water/lipid interface was investigated by site-directed mutagenesis. It was found that the effect on the hydrolytic activity upon the replacement of Trp89 with Phe, Leu, Gly or Glu was substrate dependent. The Trp89 mutants displayed an altered chain length specificity towards triglycerides, with a higher relative activity towards triacetin and trioctanoin compared with tributyrin. Trp89 was shown to be less important in the hydrolysis of vinyl esters compared with ethyl esters and triglycerides. An exclusive effect on the acylation reaction rate by the mutation of Trp89 was consistent with the data. It is suggested that Trp89 is important in the process of binding the acyl chain of the substrate into the active site for optimal acylation reaction rate. The Trp89Phe mutation resulted in an increased hydrolytic activity towards 2-alkylalkanoic acid esters. This is suggested to be due to reduction of unfavourable van der Waals contacts between Trp89 and the 2-substituent of the substrate. Thus, in contrast to natural substrates, Trp89 has a negative impact on the catalytic efficiency when substrates with bulky acyl chains are used. In contrast to the Trp89 mutations, the effect on the hydrolytic activity of the Glu87Ala mutation was almost substrate independent, 35-70% activity of wild-type lipase. A reduction of both the acylation and deacylation reaction was consistent with the data.

To reveal the functional role of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase, site-directed mutagenesis at Glu87 and Trp89 was carried out. The catalytic performance of wild-type and mutated lipases was studied in transesterification reactions in cyclohexane at a controlled water activity. Two different acyl donors were used in the investigation: tributyrin, a natural substrate for a lipase, and vinyl butyrate, an activated ester suitable for fast and efficient lipase-catalyzed transformations in preparative organic synthesis. As acyl acceptor 1-heptanol was used. The Glu87Ala mutation decreased the Vmax,app value with tributyrin and vinyl butyrate by a factor of 1.5 and 2, respectively. The Km,app for tributyrin was not affected by the Glu87Ala mutation, but the Km,app for vinyl butyrate increased twofold compared to the wild-type lipase. Changing Trp89 into a Phe residue afforded an enzyme with a 2.7- and 2-fold decreased Vmax,app with the substrates tributyrin and vinyl butyrate, respectively, compared to the wild-type lipase. No significant effects on the Km,app values for tributyrin or vinyl butyrate were seen as a result of the Trp89Phe mutation. However, the introduction of a Glu residue at position 89 in the lid increased the Km,app for tributyrin and vinyl butyrate by a factor of > 5 and 2, respectively. The Trp89Glu mutated lipase could not be saturated with tributyrin within the experimental conditions (0-680 mM) studied here. With vinyl butyrate as a substrate the Vmax,app was only 6% of that obtained with wild-type enzyme.

The antigenicity of 36 Humicola lanuginosa lipase (HL) variants, generated by site directed mutagenesis, was compared with that of the unchanged enzyme. Polyclonal antibodies raised against variant lipases were investigated and compared with the antibodies raised against the wild type lipase in an ELISA competition assay. The results showed that exchange of charged amino acids with polar residues in surface epitopes of HL, results in a tighter binding of the antibody to the epitope. Four amino acids (Trp at position 89, Asp at positions 96 and 254 and Phe at position 211) were found to be essential for antibody binding in each their epitope of the wild type enzyme.

Considerable controversy exists regarding the exact nature of the molecular mechanism of interfacial activation, a process by which most lipases achieve maximum catalytic activity upon adsorption to an oil water interface. X-ray crystallographic studies show that lipases contain buried active centers and that displacements of entire secondary structure elements, or "lids," take place when the enzymes assume active conformations [Derewenda, U., A. M. Brzozowski, D. M. Lawson, and Z. S. Derewenda. 1992. Biochemistry: 31: 1532-1541; van Tilbeurgh, H., M-P. Egloff, C. Martinez, N. Rugani, R. Verger, and C. Cambillau. 1993. Nature: 362: 814-820; Grochulski, P., L. Yunge, J. D. Schrag, F. Bouthillier, P. Smith, D. Harrison, B. Rubin, and M. Cygler. 1993. J. Biol. Chem. 268: 12843-12847]. A simple two-state model inferred from these results implies that the "closed" conformation is stable in an aqueous medium, rendering the active centers inaccessible to water soluble substrates. We now report that in crystals of the Humicola lanuginosa lipase the "lid" is significantly disordered irrespective of the ionic strength of the medium, while in a related enzyme from Rhizopus delemar, crystallized in the presence of a detergent, the two molecules that form the asymmetric unit show different "lid" conformations. These new results call into question the simplicity of the "enzyme theory" of interfacial activation.

The stability of globular proteins arises largely from the burial of non-polar amino acids in their interior. These residues are efficiently packed to eliminate energetically unfavorable cavities. Contrary to these observations, high resolution X-ray crystallographic analyses of four homologous lipases from filamentous fungi reveal an alpha/beta fold which contains a buried conserved constellation of charged and polar side chains with associated cavities containing ordered water molecules. It is possible that this structural arrangement plays an important role in interfacial catalysis.

Lipases from filamentous fungi have been studied extensively over many years. They exhibit properties attractive for industrial applications, e.g. in laundry detergents, tanning and paper industries and stereospecific organic synthesis. Enzymes from the fungi Rhizomucor miehei and Geotrichum candidum have been among the first neutral lipases to be characterized structurally by X-ray diffraction methods. In this paper we report a preliminary account of crystallographic studies of three other fungal lipases homologous to that from R. miehei and obtained from Humicola lanuginosa, Penicillium camembertii and Rhizopus delemar. These newly characterized structures have important implications for our understanding of structure-function relationships in lipases in general and the molecular basis of interfacial activation.

To determine whether Trp89 located in the lid of the lipase (EC 3.1.1.3) from Humicola lanuginosa is important for the catalytic property of the enzyme, site-directed mutagenesis at Trp89 was carried out. The kinetic properties of wild type and mutated enzymes were studied with tributyrin as substrate. Lipase variants in which Trp89 was changed to Phe, Leu, Gly or Glu all showed less than 14% of the activity compared to that of the wild type lipase. The Trp89Glu mutant was the least active with only 1% of the activity seen with the wild type enzyme. All Trp mutants had the same binding affinity to the tributyrin substrate interface as did the wild type enzyme. Wild type lipase showed saturation kinetics against tributyrin when activities were measured with mixed emulsions containing different proportions of tributyrin and the nonionic alkyl polyoxyethylene ether surfactant, Triton DF-16. Wild type enzyme showed a Vmax = 6000 +/- 300 mmol.min-1.g-1 and an apparent Km = 16 +/- 2% (vol/vol) for tributyrin in Triton DF-16, while the mutants did not show saturation kinetics in an identical assay. The apparent Km for tributyrin in Triton DF-16 was increased as the result of replacing Trp89 with other residues (Phe, Leu, Gly or Glu). The activities of all mutants were more sensitive to the presence of Triton DF-16 in the tributyrin substrate than was wild type lipase. The activity of the Trp89Glu mutant was decreased to 50% in the presence of 2 vol% Triton DF-16 compared to the activity seen with pure tributyrin as substrate.(ABSTRACT TRUNCATED AT 250 WORDS)