Humicola Lanuginosa Research Articles

Enhancement of lipase stability and productivity through chemical modification and its application to latex-based polymer emulsions

Abstract The primary focus of this research was to employ amino-group specific chemical modifications for improving the productivity and stability of two commercially produced lipases, Lipase-A from Candida antarctica (CAL UM ) and Greasex from Humicola lanuginosa (HLL UM ), for application in a latex-based paint formulation. The modified lipases showed higher percentage increase (benzoic anhydride-modified, HLL BA , 150%; PEG-modified, HLL PEG ,162% at 75 °C) as well as higher absolute productivities 41, 50, 52 and 53 μmole substrate mg −1 lipase for unmodified, CAL PEG , HLL PEG and HLL BA , respectively at 37 °C. The half-lives of thermal inactivation for all modified variants were improved from 40 to 166% at 50, 60 and 70 °C relative to unmodified lipases. The higher thermal stability and catalytic efficiency (k cat /K m ) with concomitant lower activity (k cat ) indicates that enhanced productivity is likely to be due to the modified enzymes being better able to resist thermal denaturation over the time course of the productivity experiments. Importantly, both lipases, CAL BA (60%) and HLL BA (55%) retained the highest activity in paint compared with CAL UM (36%) and HLL UM (39%) after 20 weeks incubation at 25 °C. The long term stability of the modified lipases illustrates their potential value for commercial paint and other industrial applications.

A contribution to an investigation of nitrogen metabolism of some thermophilic and thermophilous soil fungi

The author has examined the assimilation of nitrogen on the basis of different sources of nitrogen and the secretion of nitrite to the substrate by some soil fungi. The following species of thermophilic and thermophilous fungi have been investigated: <i>Malbranchea pulchella</i> Saccardo et Penzig var. <i>sulfurea</i> Miehe, <i>Humicola lanuginosa</i> (Griffon et Maublanc) Bunce. <i>Sporotrichum thermophile</i> Apinis, <i>Thielavia sepedonium</i> Emmons, <i>Paecilomyces varioti</i> Bainier, <i>Aspergillus fumigatus</i> Fresenius, A. terreus Thom.

Characterization of a β-xylosidase produced by a mutant derivative of Humicola lanuginosa in solid state fermentation

The production of extracellular β-xylosidase by a newly isolated mutant derivative of Humicola lanuginosa M7D on lignocellulosic substrates and xylan was maximized in solid state fermentation by adopting a search technique varying one parameter at a time. The H. lanuginosa mutant achieved maximum production of β-xylosidase (728 IU g−1 substrate, YP/S) when grown on Vogel’s medium containing xylan, followed by medium containing corncobs (669 IU g−1) supplemented with corn steep liquor (initial pH 6.5, moisture level 75%) at 45°C. Purified mutant- and parent-derived enzyme exhibited Km values of 1.8 and 2.0 mM, respectively. Both enzymes were optimally active at pH 8.5 and a temperature of 60°C. Both enzymes displayed high thermostability, with a half-life of 2.9 and 0.9 min, enthalpy of denaturation (ΔH*) of 102.1 and 110.10 kJ mol−1, entropy of denaturation (ΔS*) of −38.5 and −4.5 J/mol K, and free energy of denaturation (ΔG*) of 115.7 and 111.7 kJ/mol at 80°C for the mutant- and parent organism-derived enzymes, respectively. The kinetic and thermodynamic properties suggest that the β-xylosidases from both strains are superior to several previously reported enzymes from other thermophilic species, and may have potential applications in various industrial fields.

Optimization of immobilization conditions of Thermomyces lanuginosus lipase on styrene–divinylbenzene copolymer using response surface methodology

Microbial lipase from Thermomyces lanuginosus (formerly Humicola lanuginosa) was immobilized by covalent binding on a novel microporous styrene–divinylbenzene polyglutaraldehyde copolymer (STY–DVB–PGA). The response surface methodology (RSM) was used to optimize the conditions for the maximum activity and to understand the significance and interaction of the factors affecting the specific activity of immobilized lipase. The central composite design was employed to evaluate the effects of enzyme concentration (4–16%, v/v), pH (6.0–8.0), buffer concentration (20–100 mM) and immobilization time (8–40 h) on the specific activity. The results indicated that enzyme concentration, pH and buffer concentration were the significant factors on the specific activity of immobilized lipase and quadratic polynomial equation was obtained for specific activity. The predicted specific activity was 8.78 μmol p-NP/mg enzyme min under the optimal conditions and the subsequent verification experiment with the specific activity of 8.41 μmol p-NP/mg enzyme min confirmed the validity of the predicted model. The lipase loading capacity was obtained as 5.71 mg/g support at the optimum conditions. Operational stability was determined with immobilized lipase and it indicated that a small enzyme deactivation (12%) occurred after being used repeatedly for 10 consecutive batches with each of 24 h. The effect of methanol and tert-butanol on the specific activity of immobilized lipase was investigated. The immobilized lipase was almost stable in tert-butanol (92%) whereas it lost most of its activity in methanol (80%) after 15 min incubation.

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.

Kinetics of high-Level of ß-glucosidase production by a 2-deoxyglucose-resistant mutant of Humicola lanuginosa in submerged fermentation

Um mutante de Hemicola lanuginosa resistente a 2-deoxiglucose(M7) foi obtido atraves de exposicao de conidios a raios γ, permitindo a expressao em caldo contendo 0,6% de 2-deoxiglucose (DG) e celobiose (1%) antes da semeadura em agar DG esculina citrato de ferro amoniacal, da qual foram selecionadas as colonias com halo negro. Os parâmetros cineticos para producao aumentada de s-glucosidase (BGL) foram obtidos empregando-se sabugo de milho como fonte de carbono. A combinacao de espiga de milho com agua de maceracao de milho foi a que forneceu os valores mais altos nos parâmetros cineticos de formacao de todos os produtos. O efeito da temperatura na cinetica e atributos termodinâmicos da producao de BGL pelas cepas selvagem e M7 foi avaliado empregando-se processo de batelada em oito temperaturas diferentes in frascos em agitacao. O melhor desempenho foi observado a 45oC e 20g.l-1 de espiga de milho em 64h. Tanto a multiplicacao quanto a formacao do produto foram muito altas a 45oC e ambas estavam ligadas em condicoes otimas de trabalho. O rendimento de BGL produzido pelo mutante M7 (1556 U.g-1 de espiga seca) foi significativamente superior aos valores reportados para todos os sistemas fungicos e bacterianos. A mutacao influenciou a termoestabilizacao no microrganismo, sendo que o mutante necessitou de energia de ativacao mais baixa para multiplicacao e valores mais baixos de entalpia e entropia para a formacao do produto quando comparado a cepa selvagem e a outros microrganismos mesofilicos e termotolerantes. Na fase de inativacao, os microrganismos necessitaram valores mais baixos de energia de ativacao, entalpia e entropia para o equilibrio da formacao de produto, confirmando a natureza termofilica da maquina metabolica do mutante.

Kinetics of high-Level of ß-glucosidase production by a 2-deoxyglucose-resistant mutant of Humicola lanuginosa in submerged fermentation.

A 2-deoxyglucose-resistant mutant (M7) of Humicola lanuginosa was obtained by exposing conidia to γ-rays and permitting expression in broth containing 0.6% 2-deoxyglucose (DG) and cellobiose (1%) before plating on DG esculin-ferric ammonium citrate agar medium from which colonies showing faster and bigger blackening zones were selected. Kinetic parameters for enhanced ß-glucosidase (BGL) synthesis by M7 were achieved when corncobs acted as the carbon source. The combination between corncobs and corn steep liquor was the best to support higher values of all product formation kinetic parameters. Effect of temperature on the kinetic and thermodynamic attributes of BGL production equilibrium in the wild organism and M7 was studied using batch process at eight different temperatures in shake-flask studies. The best performance was found at 45°C and 20 g L−1 corncobs in 64 h. Both growth and product formation (17.93 U mL−1) were remarkably high at 45°C and both were coupled under optimum working conditions. Product yield of BGL from the mutant M7 (1556.5 U g−1 dry corncobs) was significantly higher than the values reported on all fungal and bacterial systems. Mutation had thermo-stabilization influence on the organism and mutant required lower activation energy for growth and lower magnitudes of enthalpy and entropy for product formation than those demanded by the wild organism, other mesophilic and thermo-tolerant organisms. In the inactivation phase, the organisms needed lower values of activation energy, enthalpy and entropy for product formation equilibrium, confirming thermophilic nature of metabolic network possessed by the mutant organism.

Action of Humicola lanuginosa lipase on mixed monomolecular films of tricaprylin and polyethylene glycol stearate

The hydrolysis catalyzed by Humicola lanuginosa lipase (HLL) of pure tricaprylin (TC) or stearate of polyethylene glycol 1500 (PEG-St) as well as their mixtures spread as monomolecular films were studied. The catalytic transformation of the two substrates TC or PEG-St into their respective reaction products was detected by measuring simultaneously the decrease in the film area and the surface potential using the “zero order” trough at constant surface pressure. A kinetic model describing the enzymatic hydrolysis was developed. The surface concentrations of the two substrates and their respective reaction products as well as the values of the global kinetic constants of hydrolysis were determined. The experimentally obtained global kinetic constants of the catalytic action of HLL against TC and PEG-St present in mixed monolayers of TC/PEG-St are approximately the same as in the case of pure monolayers. These obtained results give some indications that the activity of enzyme is not significantly affected by the different molecular environments in the mixed monolayers.

Atomic force microscope visualization of lipid bilayer degradation due to action of phospholipase A 2 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.

Immobilization of lipases for non-aqueous synthesis

Immobilization of lipases involves many levels of complications relating to the structure of the active site and its interactions with the immobilization support. Interaction of the so called hydrophobic ‘lid’ with the support has been reported to affect synthetic activity of an immobilized lipase. In this work we evaluate and compare the synthetic activity of lipases from different sources immobilized on different kinds of supports with varying hydrophobicity. Humicola lanuginosa lipase, Candida antarctica lipase B and Rhizomucor miehei lipase were physically adsorbed onto two types of hydrophobic carriers, namely hydrophilic carriers with conjugated hydrophobic ligands, and supports with base matrix hydrophobicity. The prepared immobilized enzymes were used for acylation of n-butanol with oleic acid as acyl donor in iso-octane with variable water content (0–2.8%, v/v) as reaction medium. Enzyme activity and effect of water on the activity of the immobilized derivatives were compared with those of respective soluble lipases and a commercial immobilized lipase Novozyme 435. Both R. miehei and H. lanuginosa immobilized lipases showed maximum activity at 1.39% (v/v) added water concentration. Sepabeads, a methacrylate based hydrophilic support with conjugated octadecyl chain showed highest immobilized esterification (synthetic) activity for all three enzymes, and of the three R. miehei lipase displayed maximum esterification activity comparable to the commercial enzyme.

Kinetics of Enhanced Substrate Consumption and Endo-β-xylanase Production by a Mutant Derivative of Humicola lanuginosa in Solid-state Fermentation

Industrial byproducts namely canola meal, rice bran, sunflower meal, and wheat straw were used as substrates for endo-xylanase production by Humicola lanuginosemutant TH1 through solid substrate fermentation. The enzyme was secreted extracellularly by both wild and mutant cultures. Rice bran supported the maximum production of endo-xylanase followed by wheat straw, canola meal and sunflower meal. The highest activity was achieved after 72 h of culture and the highest yields from the above substrates were 842, 840, 610 and 608 IU per g substrate consumed respectively. The highest productivity (281 IU flask−1 h−1 corresponding to 5620 l−1 h-1) of endo-xylanase by the mutant of H. lanuginosa was 1.6-fold more than that produced by the parental organism in solid-state fermentation of rice bran at 45 °C. Maximum specific activity (180 IU mg−1 protein) and substrate consumption rates were significantly more than those reported by previous researchers on Humicola sp. The mutant possessed markedly low accompanying cellulase activity. Thermodynamic studies revealed that the mutant required significantly lower activation energy for enzyme production and higher for thermal inactivation which signified that the endogenous metabolic machinery of mutant cells exerted more protection against thermal inactivation during product formation than that needed by its parental cultures.

Quantization of pH: Evidence for Acidic Activity of Triglyceride Lipases

Here we present a study of lipolytic activity of lipases from Fusarium solani pisi (cutinase), Rhizomucor miehei, Pseudomonas cepacia, and Humicola lanuginosa. Their activities toward triolein provide clear evidence for considerable enzymatic activity under acidic conditions. The activity was followed using Fourier transform infrared attenuated total reflection (FTIR-ATR) and nuclear magnetic resonance (NMR). Using these approaches, all the lipases that were studied exhibited lipolytic activity down to pH 4. The common model for the catalytic activity of the F. solani pisi cutinase, and lipases in general, requires the deprotonation of the active site histidine. Measurements using (13)C NMR spectroscopy showed a pK(a) value in the absence of substrate that is not consistent with the detected acid activity. We propose a novel model for the electrostatics in the active site of cutinase that could explain the observed acidic activity. The active site is essentially covered with the lipid surface during catalysis, thus preventing chemical communication between the active site and the bulk solvent. We propose that the classical definition of pH in bulk solution is not applicable to the active site environment of a lipase when the active site is inaccessible to solvent. In small restricted volumes, the pH must be quantized, and since much of the biological world is dependent on compartmentalization of processes in small volumes, it becomes relevant to investigate when this mechanism comes into play. We have made a quantitative assessment of how large the restricted volume can be and still lead to quantization of pH.

Discrimination between closed and open forms of lipases using electrophoretic techniques

The enhanced catalytic activity of lipases is often associated with structural changes. The three-dimensional (3D) structures showed that the covalently inhibited lipases exist under their open conformations, in contrast to their native closed forms. We studied the inhibition of various lipases—human and dog gastric lipases, human pancreatic lipase, and Humicola lanuginosa lipase—by the octyl-undecyl phosphonate inhibitor, and we measured the subsequent modifications of their respective electrophoretic mobility. Furthermore, the experimental values of the isoelectric points found for the native (closed) and inhibited (open) lipases are in agreement with theoretical calculations based on the electrostatic potential. We concluded that there is a significant difference in the isoelectric points between the closed (native) and open (inhibited) conformations of the four lipases investigated. Thus, analysis of the electrophoretic pattern is proposed as an easy experimental tool to differentiate between a closed and an open form of a given lipase.

Fluorescence spectroscopic characterization of Humicola lanuginosa lipase dissolved in its substrate

The conformational dynamics of Humicola lanuginosa lipases (HLL) and its three mutants were investigated by steady state and time-resolved fluorescence spectroscopy in two different media, aqueous buffer and the substrate triacetin. The fluorescence of the four Trps of the wild-type HLL (wt) reports on the global changes of the whole lipase molecule. In order to monitor conformational changes specifically in the α-helical surface loop, the so-called ‘lid’ of HLL comprised of residues 86–93, the single Trp mutant W89m (W117F, W221H, W260H) was employed. Mutants W89L and W89mN33Q (W117F, W221H, W260H, N33Q) were used to survey the impact of Trp89 and mannose residues, respectively. Based on the data obtained, the following conclusions can be drawn. (i) HLL adapts the ‘open’ conformation in triacetin, with the α-helical surface loop moving so as to expose the active site. (ii) Trp89 contained in the lid plays an unprecedently important role in the structural stability of HLL. (iii) In triacetin, but not in the buffer, the motion of the Trp89 side chain becomes distinguishable from the motion of the lid. (iv) The carbohydrate moiety at Asn33 has only minor effects on the dynamics of Trp89 in the lid as judged from the fluorescence characteristics of the latter residue.

Isolation of high-affinity human IgE and IgG antibodies recognising Bet v 1 and Humicola lanuginosa lipase from combinatorial phage libraries

Allergen-specific Fab fragments isolated from combinatorial IgE and IgG libraries are useful tools for studying allergen–antibody interactions. To characterise the interaction between different allergens and antibodies we have created recombinant human phage antibody libraries in the Fab format. Human IgE and IgG libraries have been created from patients allergic to birch pollen or lipase. These libraries have been used to select binders recognising the major birch pollen allergen Bet v 1 and Humicola lanuginosa lipase. A panel of allergen-specific IgE and IgG antibodies were identified; these were further characterised by allergen binding studies using Biacore and competition studies using human sera and antibodies purified from human sera. Affinities in the nM range were recorded and a competition with human sera for allergen binding was observed.

Use of immobilized lipases for lipase purification via specific lipase–lipase interactions

Lipase from Pseudomonas fluorescens (PFL), an enzyme with a great tendency to yield bimolecular aggregates, was immobilized via multipoint covalent attachment on glyoxyl-agarose in the presence of Triton X-100. This strategy permitted to obtain the enzyme with the active center oriented towards the reaction medium. This immobilized enzyme presents the capacity of specifically adsorbing PFL molecules, that can be easily desorbed by the use of detergents. More interesting, the enzyme was also able to adsorb other lipases. That is, the lipase from Bacillus thermocatenulatus (BTL2) cloned in Escherichia coli was selectively adsorbed on this immobilized enzyme, enabling a very simple purification strategy. Similar results were achieved with some other lipases (those from Rhizomucor miehei (RML), Rhizopus oryzae (ROL), and Humicola Lanuginosa (HLL)). In all cases, the enzyme could be easily desorbed by incubation with Triton X-100. The matrix could be used several cycles without any detrimental effect on the adsorption capacity.

Xylanase Production by the Thermophilic Mold <I>Humicola lanuginosa</I> in Solid-State Fermentation

Among the lignocellulosic substrates tested, wheat bran supported a high xylanase (EC 3.2.1.8) secretion by Humicola lanuginosa in solid-state fermentation (SSF). Enzyme production reached a peak in 72 h followed by a decline thereafter. Enzyme production was very high (7832 U/g of dry moldy bran) when wheat bran was moistened with tap water at a substrate--to--moistening agent ratio of 1:2.5 (w/v) and an inoculum level of 3 x 106 spores/10 g of wheat bran at a water activity (aw) of 0.95. Cultivation of the mold in large enamel trays yielded a xylanase titer comparable with that in flasks. Parametric optimization resulted in a 31% increase in enzyme production in SSF. Xylanase production was approx 23-fold higher in SSF than in submerged fermentation (SmF). A threshold constitutive level of xylanase was secreted by H. lanuginosa in a medium containing glucose as the sole carbon source. The enzyme was induced by xylose and xylan. Enzyme synthesis was repressed beyond 1.0% (w/v) xylose in SmF, whereas it was unaffected up to 3.0% (w/w) in SSF, suggesting a minimization of catabolite repression in SSF.

Action of Humicola lanuginosa lipase on long-chain lipid substrates : 2. Hydrolysis of diolein monolayers

The hydrolysis of 1,2-diolein (DO) monomolecular films by Humicola lanuginosa lipase (HLL) was studied by simultaneous measuring the decrease in the film area and the changes in the surface potential in the “zero-order trough” at constant surface pressure and in the presence of β-cyclodextrin (β-CD). The decrease with time in the film area reflects both the reduction in the area per molecule due to the transformation of substrate DO molecules into the products molecules of monoolein (MO) and oleic acid (OA) and the desorption of the soluble inclusion complexes β-CD–MO and β-CD–OA. The surface potential data were interpreted as an accumulation at the interface of negatively charged products of OA and insoluble β-CD–DO complexes. In the proposed kinetic model, the product solubilization rates in the presence of β-CD and the flux supplied progressively by the moving barrier from the reservoir to the reaction compartment in order to keep the constant surface pressure were taken into account. The surface concentrations of MO and OA transiently present at the interface were determined. The values of the global kinetic constant Q m ′ of hydrolysis of DO to MO were obtained. Comparison with the values of the global kinetic constant of hydrolysis of monoglyceride MO to OA shows that the rates of hydrolysis of diglyceride and monoglyceride by HLL are of the same order of magnitude.

Molecular characterization of a xylanase-producing thermophilic fungus isolated from Japanese soil.

We describe the molecular characteristics of Scytalidium thermophilum isolated from Japanese soil. The S. thermophilum isolates produced higher xylanase activity than Humicola lanuginosa isolated from Japanese soil. A G/11 family xylanase-encoding gene was detected in the S. thermophilum genome by using the polymerase chain reaction technique. The S. thermophilum AF101-3 strain, which was one of the isolates in this study, grew well at 37 degrees C and 50 degrees C, and contained the maximum xylanase activity detected among the isolates. Phylogenetic analysis revealed that the S. thermophilum strains isolated from Japanese soil were clustered in a different group from the S. thermophilum strains reported by Lyons et al. [Mycol Res 104:1431, 2000], suggesting that the S. thermophilum strains isolated in this study are genetically new isolates. Therefore, the genetic diversity of S. thermophilum might be higher than that of H. lanuginosa. Moreover, this is the first report about detection of a xylanase-encoding gene in S. thermophilum.

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)/d t= k P c HLL. We estimate the specific rate of MOG hydrolysis by HLL to be 2.5×10 4 MOG molecules/(minute·molecule of HLL).