Impact of the tryptophan residues of Humicola lanuginosa lipase on its thermal stability.

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.     

Enantioselective resolution of 3-phenylthio-2-propanol with Humicola lanuginosa lipase

The acetylation of 3-phenylthio-2-propanol (168 mg) was performed with vinyl acetate (1 ml) using different lipases from 15°C to 51°C. As a result, the (R)-enantiomer was selectively acetylated and the (S)-enantiomer was non-reactive in all the cases. An appropriate choice of conditions can be made to isolate both (R)-alcohol (ee 99%, 36 h, conversion 46%, sub/enz: 1/2) and (S)-alcohol (ee 93%, 38 h, conversion 46%, THF, sub/enz: 1 l^–1) using Humicola lanuginosalipase (Lipolase). Increasing the amount of enzyme increased the ee.     

Detergent-induced conformational changes of Humicola lanuginosa lipase studied by fluorescence spectroscopy

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.     

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.     

Humicola lanuginosa lipase hydrolysis of mono-oleoyl-rac-glycerol at the lipid-water interface observed by atomic force microscopy

A new type of planar lipid substrate for Humicola lanuginosa lipase (HLL) has been prepared by depositing a monolayer of 1-mono-oleoyl-rac-glycerol (MOG) on top of a monolayer of dipalmitoyl-phosphatidylcholine (DPPC) on mica by the Langmuir-Blodgett (LB) technique. The bilayer was subsequently exposed to HLL in a liquid cell of an atomic force microscope (AFM) allowing the time course of the lipolytic degradation to be observed. By analysing a series of AFM images, we find that enzymes are preferentially activated at the edge of nano-scale defects present in the bilayer prior to enzyme injection, while defect-free areas of the substrate are surprisingly stable towards enzyme degradation. The initial rate of hydrolysis is found to be proportional to the perimeter length, P, of the initial nano-scale defects as well as the bulk enzyme concentration, c(HLL); d(lipid)/dt=k P c(HLL). We estimate the specific rate of MOG hydrolysis by HLL to be 2.5x10(4) MOG molecules/(minute x molecule of HLL).     

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 the Lipase of Thermophilic Fungus Humicola lanuginosa

A protease occurring in the endosperm fraction of germinating corn was purified by means of (NH₄)₂SO₄ fractionation, CM-celluIose chromatography, DEAE-cellulose chromatography, Sephadex G-100 gel filtration and preparative polyacrylamide gel electrophoresis. The purified protease was found to have a molecular weight of about 21,000 and an isoelectric point of pH 2.3 or lower. The optimum pH was found to lie at 3.0 when measured with denatured hemoglobin as substrate. The protease was generally activated by thiol compounds and completely inhibited by p-chloromercuribenzoic acid. Neither diisopropylphosphofluoridate nor diazoacetyl-dl-norleucine methyl ester affected the protease activity. Antipain greatly inhibited the protease action whereas pepstatin had no significant effect. These data indicate, in conclusion, that the protease possesses a unique property to be a sulfhydryl enzyme most active in an acidic region around pH 3.     

Atomic force microscope visualization of lipid bilayer degradation due to action of phospholipase A2 and Humicola lanuginosa lipase

An important application of liquid cell Atomic Force Microscopy (AFM) is the study of enzyme structure and behaviour in organized molecular media that mimic in-vivo systems. In this study we demonstrate the use of AFM as a tool to study the kinetics of lipolytic enzyme reactions occurring at the surface of a supported lipid bilayer. In particular, the time course of the degradation of lipid bilayers by Phospholipase A(2) (PLA(2)) and Humicola Lanuginosa Lipase (HLL) has been investigated. Contact mode imaging allows visualization of enzyme activity on the substrate with high lateral resolution. Lipid bilayers were prepared by the Langmuir-Blodgett technique and transferred to an AFM liquid cell. Following injection of the enzyme into the liquid cell, a sequence of images was acquired at regular time intervals to allow the identification of substrate structure, preferred sites of enzyme activation, and enzyme reaction rates.     

Computer modeling of substrate binding to lipases from Rhizomucor miehei, Humicola lanuginosa, and Candida rugosa.

The substrate-binding sites of the triacyl glyceride lipases from Rhizomucor miehei, Humicola lanuginosa, and Candida rugosa were studied by means of computer modeling methods. The space around the active site was mapped by different probes. These calculations suggested 2 separate regions within the binding site. One region showed high affinity for aliphatic groups, whereas the other region was hydrophilic. The aliphatic site should be a binding cavity for fatty acid chains. Water molecules are required for the hydrolysis of the acyl enzyme, but are probably not readily accessible in the hydrophobic interface, in which lipases are acting. Therefore, the hydrophilic site should be important for the hydrolytic activity of the enzyme. Lipases from R. miehei and H. lanuginosa are excellent catalysts for enantioselective resolutions of many secondary alcohols. We used molecular mechanics and dynamics calculations of enzyme-substrate transition-state complexes, which provided information about molecular interactions important for the enantioselectivities of these reactions.     

Steady state and time resolved effects of guanidine hydrochloride on the structure of Humicola lanuginosa lipase revealed by fluorescence spectroscopy

Effects of guanidine hydrochloride (GdnHCl) on the structure and dynamics of wild-type Humicola lanuginosa lipase (HLL) and its two mutants were studied. The latter were S146A (with the active site Ser replaced by Ala) and the single Trp mutant W89m, with substitutions W117F, W221H, and W260H. Steady-state, stopped-flow, and time-resolved laser-induced fluorescence spectroscopy were carried out as a function of [GdnHCl]. The maximum emission wavelength and fluorescence lifetimes revealed the microenvironment of the tryptophan(s) in these lipases to become more polar upon increasing [GdnHCl]. However, significant extent of tertiary structure in GdnHCl is suggested by the observation that both wild-type HLL and W89m remain catalytically active at rather high GdnHCl concentrations of >6 and 4.0 M, respectively. Changes in steady-state emission anisotropy, as well as variation in rotational correlation times and residual anisotropy values, demonstrate that upon increasing [GdnHCl] the structure of the lipases became more loose, with increasing amplitude of structural fluctuations. Finally, intermediate states in the course of exposure of the proteins to GdnHCl were revealed by stopped-flow fluorescence measurements.     

Purification and properties of trehalase from the thermophilic fungus Humicola lanuginosa.

Trehalase (alpha,alpha-Trehalose glucohydrolase, EC 3.2.1.28) was partially solubilized from the thermophilic fungus Humicola lanuginosa RM-B, and purified 184-fold. The purified enzyme was optimally active at 50 degrees C in acetate buffer at pH 5.5. It was highly specific for alpha,alpha-trehalose and had an apparent Km = 0.4 mM at 50 degrees C. None of the other disaccharides tested either inhibited or activated the enzyme. The molecular weight of the enzyme was around 170 000. Trehalase from mycelium grown at 40 and 50 degrees C had similar properties. The purified enzyme, in contrast to that in the crude-cell free extract, was less stable. At low concentration, purified trehalase was afforded protection against heat-inactivation by "protection against heat-inactivation by "protective factor(s)" present in mycelial extracts. The "protective factor(s)" was sensitive to proteolytic digestion. It was not diffusible and was stable to boiling for at least 30 min. Bovine serum albumin and casein also protected the enzyme from heat-inactivation.     

Interfacial control of lid opening in Thermomyces lanuginosa lipase

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.     

Solvent-free glycerolysis of palm and palm kernel oils catalyzed by commercial 1,3-specific lipase from Humicola lanuginosa and composition of glycerolysis products

Glycerolysis of palm and palm kernel oils were conducted using a commercial 1,3-specific lipase from Humicola lanuginosa (trade name: SP 398) as catalyst (500 units lipase g^–1 oil) at 40°C and oil:glycerol (1:2 mol mol^–1) in a solvent-free system. After 24 h, the glycerolysis products of palm and palm kernel oils consisted of 23% triacylglycerols, 18% monoacylglycerols, 38% diacylglycerols and 18% triacylglycerols, 31% monoacylglycerols, 42% diacylglycerols, respectively. The monoacylglycerol fraction of the glycerolysis product of palm oil was enriched in oleic acid. Palmitic acid content of the monoacylglycerol fraction of the same product was less than that of the original oil. Under the same conditions, monacylglycerol fraction of the palm kernel oil glycerolysis product was enriched in palmitic, stearic and oleic acids.     

Lipase production of a new thermophilic fungus,Humicola lanuginosa var. catenulata

We isolated a new thermophilic fungus from soil and identified it as Humicola lagunisoa var. catenulata, a new variety of Humicola lanuginosa. We cultured the fungus and found that these were two kinds of lipases in the culture filtrate.     

Conformational changes and orientation of Humicola lanuginosa lipase on a solid hydrophobic surface: an in situ interface Fourier transform infrared-attenuated total reflection study

This study was done to better understand how lipases are activated at an interface. We investigated the conformational and solvation changes occurring during the adsorption of Humicola lanuginosa lipase (HLL) onto a hydrophobic surface using Fourier transform infrared-attenuated total reflection spectroscopy. The hydrophobic surfaces were obtained by coating silicon attenuated total reflection crystal with octadecyltrichlorosilane. Analysis of vibrational spectra was used to compare the conformation of HLL adsorbed at the aqueous-solid interface with its conformation in solution. X-ray crystallography has shown that HLL exists in two conformations, the closed and open forms. The conformational changes in HLL caused by adsorption onto the surface were compared with those occurring in three reference proteins, bovine serum albumin, lysozyme, and alpha-chymotrypsin. Adsorbed protein layers were prepared using proteins solutions of 0.005 to 0.5 mg/mL. The adsorptions of bovine serum albumin, lysozyme, and alpha-chymotrypsin to the hydrophobic support were accompanied by large unfoldings of ordered structures. In contrast, HLL underwent no secondary structure changes at first stage of adsorption, but there was a slight folding of beta-structures as the lipase monolayer became complete. Solvation studies using deuterated buffer showed an unusual hydrogen/deuterium exchange of the peptide CONH groups of the adsorbed HLL molecules. This exchange is consistent with the lipase being in the native open conformation at the water/hydrophobic interface.     

Neurospora crassa and Humicola lanuginosa cytochromes c: more homology in the heme region

Neurospora crassa and Humicola lanuginosa cytochromes c were submitted to an automatic Edman degradation. It was found that residue 16 is a glutamine, as we had predicted (1) and not a glutamic acid, as published for both proteins (4,7). Moreover, residues 19 to 26 were found to have been placed in a wrong order in both cases. The corrected order shows more homology with other cytochromes c in this area.     

Fluorescence spectroscopic characterization of dissolved organic matter in the waters of Lake Fuxian and adjacent rivers in Yunnan,China

The fluorescence properties of dissolved organic matter (DOM) in the water of Lake Fuxian and its adjacent rivers on the Yunnan Plateau, southwestern China, were studied to specify the characterization of DOM in the lake and river waters. The fluorescence properties with the excitation–emission matrix in the water of Lake Fuxian are different from those in the river water. The differences in these properties between the lake and river water could arise not only from their sources but also from the reactivity of the photobleaching of DOM. In the lake, the supplying of allochthonous fluorescent materials from inflowing rivers to the fluorescent DOM is less significant than the photobleaching of fluorescent substances.     

Cultural Conditions and Some Properties of the Lipase of Humicola lanuginosa S-38

The cultural conditions for the production of thermostable lipase by a thermophilic fungus Humicola lanuginosa S-38 were investigated. The optimal cultural conditions to obtain the maximum yield of thermostable lipase with a 600-liter stainless steel fermentor were as follows: optimal medium- 2.0% soluble starch, 5.0% corn steep liquor, 0.2% K₂HPO₄, 0.1% MgSO₄·7H₂O, 0.5% CaCO₃, 0.5% soybean oil, 0.005% deforming agent (Adecanol LG-109); optimal fermentation conditions- temperature 45°C; rate of agitation 300 rpm; initial pH 7.0; rate of aeration 1/1 volume per volume of medium per minute. The optimal pH of the crude lipase preparation for the hydrolysis of the polyvinyl alcohol-emulsified olive oil was 8.0 and the optimal temperature was 60°C. It retained 100% of activity with the heat treatment at 60°C for 2 hr, but at 70°C for 20 min only 35% activity retained.     

Effect of Various Inhibitors on Lipase Action of Thermophilic Fungus Humicola lanuginosa S-38

The hydrolysis of olive oil by the Humicola lipase was inhibited by the addition of n-alcohols, fatty acids and surface active agents. The inhibition of n-alcohols was overcomed by the addition of more substrate but not by the addition of more enzyme. The inhibition of fatty acids and bile salts was eliminated by adding calcium ion. It was concluded that the inhibition of the Humicola lipase by n-alcohols, fatty acids and bile salts was not due to inactivation of the enzyme directly but due to the displacing of the substrate from the oil/water interface, thus blocking the enzyme from the substrate.     

Stabilisation of some of the protein synthesis components in the thermophilic fungus,Humicola lanuginosa

The thermal stabilities of tRNA from the thermophilic fungus,Humicola lanuginose were compared with that from the mesophilic yeast,Candida utilis, by measuring the increase in the optical density with temperature. tRNAs from both the species were stable in the presence of millimolar quantities of magnesium chloride upto 50°C, the optimum growth temperature of the fungus. Aminoacyl tRNA synthetases were maximally active at 40°C under thein vitro assay conditions. They were fractionated and one species of valine tRNA synthetase was purified to homogeneity. The purified enzyme was protected against inactivation to varying degrees when preincubated with the substrates valine, tRNA and ATP as well as spermine. Protein turnover studies showed that the rate of turnover was higher at higher temperatures. It was concluded from these results that the protein synthesizing machinery of this fungus has no intrinsic stability but it is stabilised by intracellular factors. Higher rate of protein turnover also plays a role for growth at higher temperature.