Immobilization of Lipase from Thermomyces Lanuginosus and Its Glycerolysis Ability in Diacylglycerol Preparation.

In the glycerolysis process for diacylglycerol (DAG) preparation, free lipases suffer from poor stability and the inability to be reused. To address this, a cost-effective immobilized lipase preparation was developed by cross-linking macroporous resin with poly (ethylene glycol) diglycidyl ether (PEGDGE) followed by lipase adsorption. The selected immobilization conditions were identified as pH 7.0, 35 °C, cross-linking agent concentration 2.0%, cross-linking time 4 h, lipase amount 5 mg/g of support, and adsorption time 4 h. Enzymatic properties of the immobilized lipase were analyzed, revealing enhanced pH stability, thermal stability, storage stability, and operational stability post-immobilization. The conditions for immobilized enzyme-catalyzed glycerolysis to produce DAG were selected, demonstrating the broad applicability of the immobilized lipase. The immobilized lipase catalyzed glycerolysis reactions using various oils as substrates, with DAG content in the products ranging between 35 and 45%, demonstrating broad applicability. Additionally, the changes during the repeated use of the immobilized lipase were characterized, showing that mechanical damage, lipase leakage, and alterations in the secondary structure of the lipase protein contributed to the decline in catalytic activity over time. These findings provide valuable insights for the industrial application of lipase.

A Study on the Regioselective Acetylation of Flavonoid Aglycons Catalyzed by Immobilized Lipases.

This study aimed to explore the capacity of immobilized lipases on the acetylation of six aglycon flavonoids, namely myricetin, quercetin, luteolin, naringenin, fisetin and morin. For this purpose, lipase B from Candida antarctica (CaLB) and lipase from Thermomyces lanuginosus (TLL) were immobilized onto the surface of ZnOFe nanoparticles derived from an aqueous olive leaf extract. Various factors affecting the conversion of substrates and the formation of monoesterified and diesterified products, such as the amount of biocatalyst and the molar ratio of the substrates and reaction solvents were investigated. Both CaLB and TLL-ZnOFe achieved 100% conversion yield of naringenin to naringenin acetate after 72 h of reaction time, while TLL-ZnOFe achieved higher conversion yields of quercetin, morin and fisetin (73, 85 and 72% respectively). Notably, CaLB-ZnOFe displayed significantly lower conversion yields for morin compared with TLL-ZnOFe. Molecular docking analysis was used to elucidate this discrepancy, and it was revealed that the position of the hydroxyl groups of the B ring on morin introduced hindrances on the active site of CaLB. Finally, selected flavonoid esters showed significantly higher antimicrobial activity compared with the original compound. This work indicated that these lipase-based nanobiocatalysts can be successfully applied to produce lipophilic derivatives of aglycon flavonoids with improved antimicrobial activity.

Investigating Lipase/Stain Interactions: Determining Interfacial Protein Conformation with Surface Spectroscopy.

Conventional bulk protein structure determination methods are not suitable for understanding the distinct and diverse interactions of proteins with interfaces. Notably, interfacial activation is a feature common to many lipases involving movement of a helical "lid" region upon contact with a hydrophobic surface to expose the catalytic site. Here we use the surface specificity of vibrational sum frequency generation spectroscopy (VSFG) spectroscopy to directly probe the conformation of Thermomyces lanuginosus lipase (TLL) at hydrophobic interfaces. The TLL-catalyzed reaction at the air/water interface is monitored by VSFG spectroscopy, showing loss of ester carbonyl modes and appearance of carboxylate stretching modes of the fatty acid products. Furthermore, comparison of experimental and calculated VSFG spectra of the amide I band of TLL allows us to discern the subtle structural changes involved with lid-opening at a hydrophobic surface. Finally, we report a likely orientation of this lid-open state, which interacts with the surface through a loop region away from the lid and active site. This experimental framework for probing protein structure and function at interfaces addresses a significant problem in protein science that is not only impeding the design of better enzymes for biotechnology applications but also drug discovery targeting membrane associated proteins.

Water-mediated active conformational transitions of lipase on organic solvent interfaces.

When it comes to enzyme stability and their application in organic solvents, enzyme biocatalysis has emerged as a popular substitute for conventional chemical processes. However, the demand for enzymes exhibiting improved stability remains a persistent challenge. Organic solvents can significantly impacts enzyme properties, thereby limiting their practical application. This study focuses on Lipase Thermomyces lanuginose, through molecular dynamics simulations and experiments, we quantified the effect of different solvent-lipase interfaces on the interfacial activation of lipase. Revealed molecular views of the complex solvation processes through the minimum distance distribution function. Solvent-protein interactions were used to interpret the factors influencing changes in lipase conformation and enzyme activity. We found that water content is crucial for enzyme stability, and the optimum water content for lipase activity was 35 % in the presence of benzene-water interface, which is closely related to the increase of its interfacial activation angle from 78° to 102°. Methanol induces interfacial activation in addition to significant competitive inhibition and denaturation at low water content. Our findings shed light on the importance of understanding solvent effects on enzyme function and provide practical insights for enzyme engineering and optimization in various solvent-lipase interfaces.

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

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

Preparation of a heterogeneous biocatalyst through Thermomyces lanuginosus lipase immobilization on pore-expanded SBA-15.

Heterogeneous biocatalysts were prepared by adsorbing T. lanuginosus lipase (TLL) onto uncalcined (SBAUC-TLL) and calcined (SBAC-TLL) SBA-15, using ammonium fluoride as a pore expander to facilitate TLL immobilization. At an enzyme load of 1 mg/g, high immobilization yields (>90 %) and recovered activities (>80 % for SBAUC-TLL and 70 % for SBAC-TLL) were achieved. When increasing the enzyme load to 5 mg/g, the immobilization yield of SBAUC-TLL was 80 %, and the recovered activity was 50 %, while SBAC-TLL had a yield of 100 % and a recovered activity of 36 %. Crosslinking with glutaraldehyde (GA) was conducted to improve stability (SBAUC-TLL-GA and SBAC-TLL-GA). Although SBAC-TLL-GA lost 25 % of initial activity after GA modifications, it exhibited the highest thermal (t1/2 = 5.7 h at 65 °C), when compared to SBAC-TLL (t1/2 = 12 min) and the soluble enzyme (t1/2 = 36 min), and operational stability (retained 100 % activity after 5 cycles). Both biocatalysts presented high storage stability since they retained 100 % of initial activity for 30 days. These results highlight SBA-15's potential as an enzyme support and the protocol's efficacy in enhancing stability, with implications for industrial applications in the food, chemical, and pharmaceutical sectors.

Recombinant cellobiose dehydrogenase from Thermothelomyces thermophilus: Its functional characterization and applicability in cellobionic acid production.

The fungus Thermothelomyces thermophilus is a thermotolerant microorganism that has been explored as a reservoir for enzymes (hydrolytic enzymes and oxidoreductases). The functional analysis of a recombinant cellobiose dehydrogenase (MtCDHB) from T. thermophilus demonstrated a thermophilic behavior, an optimal pH in alkaline conditions for inter-domain electron transfer, and catalytic activity on cellooligosaccharides with different degree of polymerization. Its applicability was evaluated to the sustainable production of cellobionic acid (CBA), a potential pharmaceutical and cosmetic ingredient rarely commercialized. Dissolving pulp was used as a disaccharide source for MtCDHB. Initially, recombinant exoglucanases (MtCBHI and MtCBHII) from T. thermophilus hydrolyzed the dissolving pulp, resulting in 87% cellobiose yield, which was subsequently converted into CBA by MtCDHB, achieving a 66% CBA yield after 24 h. These findings highlight the potential of MtCDHB as a novel approach to obtaining CBA through the bioconversion of a plant-based source.

The effect of carbon chain length of cross-linking agent on the functionality of carrier- free immobilized Thermomyces lanuginosa lipase particles.

The cross-linked enzyme (CLEs) of Thermomyces lanuginosa lipase (TLL) was prepared in an isocyanide-based multi-component reactions (ICMRs) platform by applying three di-acidic cross-linkers to unveil more factors contributing to the functional properties of CLEs. The linkers were 1,11-undecanedicarboxylic acid, azelaic acid, and adipic acid with 11, 7, and 4 carbon lengths, respectively, providing a proper tool to investigate the effect of linker length on the activity, stability, and selectivity of the resulting CLEs. The immobilization yields of 60-90 % and the specific activities of 168, 88.4 and 49 U/mg were obtained for the CLEs of 1,11-undecanedicarboxylic acid, azelaic acid, adipic acid, respectively. The lower activity of azelaic and adipic acid-mediated CLEs compared to the soluble TLL (110 U/mg) was explained by in silico calculations. The results revealed that as opposed to 1,11-undecanedicarboxylic acid, both linkers tended to penetrate the enzyme active site, thus resulting in a major inhibitory effect on the enzyme functionality. The thermal and co-solvent stability of the immobilized derivatives improved compared to those of free TLL. The selectivity of CLEs was also examined by catalytic release of main omega-3 fatty acids from fish oil, presenting the highest selectivity of 22 for the CLEs of azelaic acid.

Biocatalysts engineering by varying the binary CNTs-silica composition and the physicochemical characteristics of adsorbents for the immobilization of recombinant T. lanuginosus lipase.

Biocatalytic engineering was carried out by varying monotonically the binary CNTs-silica composition and, accordingly, the physicochemical characteristics of adsorbents developed for immobilization of recombinant T. lanuginosus lipase (rPichia/lip). The adsorbents based on composite carbon-silica materials (CCSMs) were produced by impregnating finely dispersed multi-walled carbon nanotubes with silica hydrosol followed by calcination in argon at 350°C; the mass ratio of the hydrophobic and the hydrophilic components varied over a wide range. Biocatalysts (BCs) for green low-temperature synthesis of various esters in a non-aqueous medium of organic solvents were prepared by adsorption of rPichia/lip with subsequent drying under ambient conditions. The characteristics of the CCSMs and BCs were characterized by thermogravimetry, nitrogen porosimetry and electron microscopy. The catalytic properties of BCs, such as enzymatic activity, substrate conversion and specificity, as well we their operational stability depending on the chemical composition of CCSMs were extensively studied in the esterification of saturated monocarboxylic acids (C4, C7, C18) and primary aliphatic alcohols (C2, C4, C16) in hexane at 20°C. It was found that the esterifying activity manyfold decreased with increasing the silica content primarily due to a decrease in adsorption ability of CCSMs toward rPichia/lip. The substrate specificity and operational stability of the lipase-active BCs did not greatly depend on the composition of CCSMs. Biocatalysts retained more than half of their initial esterifying activity after 10 reaction cycles.

Structural and molecular insights into a bifunctional glycoside hydrolase 30 xylanase specific to glucuronoxylan.

Glycoside hydrolase (GH) 30 family xylanases are enzymes of biotechnological interest due to their capacity to degrade recalcitrant hemicelluloses, such as glucuronoxylan (GX). This study focuses on a subfamily 7 GH30, TtXyn30A from Thermothelomyces thermophilus, which acts on GX in an "endo" and "exo" mode, releasing methyl-glucuronic acid branched xylooligosaccharides (XOs) and xylobiose, respectively. The crystal structure of inactive TtXyn30A in complex with 23-(4-O-methyl-α-D-glucuronosyl)-xylotriose (UXX), along with biochemical analyses, corroborate the implication of E233, previously identified as alternative catalytic residue, in the hydrolysis of decorated xylan. At the -1 subsite, the xylose adopts a distorted conformation, indicative of the Michaelis complex of TtXyn30AEE with UXX trapped in the semi-functional active site. The most significant structural rearrangements upon substrate binding are observed at residues W127 and E233. The structures with neutral XOs, representing the "exo" function, clearly show the nonspecific binding at aglycon subsites, contrary to glycon sites, where the xylose molecules are accommodated via multiple interactions. Last, an unproductive ligand binding site is found at the interface between the catalytic and the secondary ß-domain which is present in all GH30 enzymes. These findings improve current understanding of the mechanism of bifunctional GH30s, with potential applications in the field of enzyme engineering.

Application of Thermomyces lanuginosus polygalacturonase produced in Komagataella phaffii in biomass hydrolysis and textile bioscouring.

In this work, the polygalacturonase (TL-PG1) from the thermophilic fungus Thermomyces lanuginosus was heterologously produced for the first time in the yeast Komagataella phaffii. The TL-PG1 was successfully expressed under the control of the AOX1 promoter and sequentially purified by His-tag affinity. The purified recombinant pectinase exhibited an activity of 462.6 U/mL toward polygalacturonic acid under optimal conditions (pH 6 and 55 ˚C) with a 2.83 mg/mL and 0.063 µmol/minute for Km and Vmax, respectively. When used as supplementation for biomass hydrolysis, TL-PG1 demonstrated synergy with the enzymatic cocktail Ctec3 to depolymerize orange citrus pulp, releasing 1.43 mg/mL of reducing sugar. In addition, TL-PG1 exhibited efficiency in fabric bioscouring, showing potential usage in the textile industry. Applying a protein dosage of 7 mg/mL, the time for the fabric to absorb water was 19.77 seconds (ten times faster than the control). Adding the surfactant Triton to the treatment allowed the reduction of the enzyme dosage by 50% and the water absorption time to 6.38 seconds. Altogether, this work describes a new versatile polygalacturonase from T. lanuginosus with the potential to be employed in the hydrolysis of lignocellulosic biomass and bioscouring.

A thermostable and inhibitor resistant ß-glucosidase from Rasamsonia emersonii for efficient hydrolysis of lignocellulosics biomass.

The present study reports a highly thermostable ß-glucosidase (GH3) from Rasamsonia emersonii that was heterologously expressed in Pichia pastoris. Extracellular ß-glucosidase was purified to homogeneity using single step affinity chromatography with molecular weight of ~ 110 kDa. Intriguingly, the purified enzyme displayed high tolerance to inhibitors mainly acetic acid, formic acid, ferulic acid, vanillin and 5-hydroxymethyl furfural at concentrations exceeding those present in acid steam pretreated rice straw slurry used for hydrolysis and subsequent fermentation in 2G ethanol plants. Characteristics of purified ß-glucosidase revealed the optimal activity at 80 °C, pH 5.0 and displayed high thermostability over broad range of temperature 50-70 °C with maximum half-life of ~ 60 h at 50 °C, pH 5.0. The putative transglycosylation activity of ß-glucosidase was appreciably enhanced in the presence of methanol as an acceptor. Using the transglycosylation ability of ß-glucosidase, the generated low cost mixed glucose disaccharides resulted in the increased induction of R. emersonii cellulase under submerged fermentation. Scaling up the recombinant protein production at fermenter level using temporal feeding approach resulted in maximal ß-glucosidase titres of 134,660 units/L. Furthermore, a developed custom made enzyme cocktail consisting of cellulase from R. emersonii mutant M36 supplemented with recombinant ß-glucosidase resulted in significantly enhanced hydrolysis of pretreated rice straw slurry from IOCL industries (India). Our results suggest multi-faceted ß-glucosidase from R. emersonii can overcome obstacles mainly high cost associated enzyme production, inhibitors that impair the sugar yields and thermal inactivation of enzyme.

Solvent-free synthesis of hydrophobic and amphiphilic esters using a chemically modified lipase from Thermomyces lanuginosus: a comparative study with native and immobilized forms.

Using lipases to catalyze the synthesis of the most differentiated type of compounds remains one of the major challenges among scientists. Seeking more economic and advantageous catalysts is a current goal of green chemistry. In this work, we demonstrate the potential of a chemically modified form of lipase from Thermomyces lanuginosus (cmLTL) for the synthesis of both hydrophobic (heptyl heptanoate, heptyl octanoate, heptyl decanoate, decyl heptanoate, decyl octanoate and decyl decanoate) and amphiphilic (2-(2-ethoxyethoxy)ethyl oleate and 2-(2-ethoxyethoxy)ethyl linoleate) esters, in bulk. The results were compared with its native (LTL) and immobilized (imLTL) forms. The data revealed that LTL showed poor activity for all reactions performed with n-heptane (η<20 %). ImLTL was able to synthesize all hydrophobic esters (η>60 %), with exception of the short ester, heptyl heptanoate. cmLTL was the only form of LTL capable of producing hydrophobic and amphiphilic esters, without compromising the yield when the reactions were performed under solvent-free conditions (>50 %). Molecular modeling showed that the active pocket of cmLTL is able to deeply internalize transcutol, with stronger interactions, justifying the outstanding results obtained. Furthermore, owing to the possibility of cmLTL filtration, the reusability of the catalyst is ensured for at least 6 cycles, without compromising the reaction yields.

Improving the Thermostability of Thermomyces lanuginosus Lipase by Restricting the Flexibility of N-Terminus and C-Terminus Simultaneously via the 25-Loop Substitutions.

(1) Lipases are catalysts widely applied in industrial fields. To sustain the harsh treatments in industries, optimizing lipase activities and thermal stability is necessary to reduce production loss. (2) The thermostability of Thermomyces lanuginosus lipase (TLL) was evaluated via B-factor analysis and consensus-sequence substitutions. Five single-point variants (K24S, D27N, D27R, P29S, and A30P) with improved thermostability were constructed via site-directed mutagenesis. (3) The optimal reaction temperatures of all the five variants displayed 5 °C improvement compared with TLL. Four variants, except D27N, showed enhanced residual activities at 80 °C. The melting temperatures of three variants (D27R, P29S, and A30P) were significantly increased. The molecular dynamics simulations indicated that the 25-loop (residues 24-30) in the N-terminus of the five variants generated more hydrogen bonds with surrounding amino acids; hydrogen bond pair D254-I255 preserved in the C-terminus of the variants also contributes to the improved thermostability. Furthermore, the newly formed salt-bridge interaction (R27…E56) in D27R was identified as a crucial determinant for thermostability. (4) Our study discovered that substituting residues from the 25-loop will enhance the stability of the N-terminus and C-terminus simultaneously, restrict the most flexible regions of TLL, and result in improved thermostability.

Two new species of Trichocomaceae (Eurotiales), accommodated in Rasamsonia and Talaromyces section Bacillispori, from the Czech Republic.

During a previous study on microfungi associated with clematis roots, Penicillium-like fungi were isolated and identified based on morphology. In this study, we subjected those strains to a detailed examination which led to the proposal of two taxonomic novelties, named Rasamsonia chlamydospora and Talaromyces clematidis. The first taxon is characterized by rough-walled mycelium, acerose to flask shaped phialides, cylindrical conidia and by production of chlamydospore-like structures. The four-loci-based phylogeny analysis delineated the taxon as a taxonomic novelty in Rasamsonia. Talaromyces clematidis is characterized by restricted growth on Czapek yeast extract agar, dichloran 18% glycerol agar and yeast extract sucrose agar, and production of yellow ascomata on oatmeal agar. Phylogenetic analyses placed this taxon as a taxonomic novelty in Talaromyces sect. Bacillispori. Both taxa are introduced here with detailed descriptions, photoplates and information on their phylogenetic relationship with related species.

Single-Particle Tracking of Thermomyces lanuginosus Lipase Reveals How Mutations in the Lid Region Remodel Its Diffusion.

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.

Disseminated Rasamsonia argillacea infection in a dog.

CASE HISTORY: A 4-year-old, male neutered Borzoi presented for unlocalised pain and frequent episodes of vocalisation. CLINICAL FINDINGS: Pain was localised to the lumbar spine and radiographs revealed a L3-L4 lesion consistent with discospondylitis. The dog was treated for presumptive bacterial discospondylitis with surgical debridement, spinal stabilisation, and cephalexin. Samples collected from the affected intervertebral disc at the time of surgery revealed lymphoplasmacytic inflammation with no causative agent identified on histopathology or bacterial culture. After an initial period of improvement, signs recurred despite an 8-week antibiotic course, with the development of inappetence, weight loss, polydipsia, and polyuria. Repeat radiographs revealed a new cervical intervertebral lesion, and concurrent pyelonephritis was diagnosed based on blood and urine results. Fungal culture of urine resulted in growth of Rasamsonia argillacea species complex and disseminated fungal disease was clinically diagnosed. Antifungal treatment was commenced, however the dog deteriorated, and euthanasia was performed. PATHOLOGICAL FINDINGS: Multifocal white plaques were grossly visualised in the spleen, mesenteric lymph nodes, cervical vertebrae, and kidneys. Periodic acid-Schiff-positive, fine, parallel-walled, occasionally branching, septate hyphae 5-10 µm in diameter, and conidia 5-7 µm in diameter were found on sectioning all organs. R. argillacea species complex was identified by fungal culture of urine and was considered the species of fungal organism seen histologically. The isolate was subsequently confirmed as R. argillacea by DNA sequencing. DIAGNOSIS: Disseminated Rasamsonia argillacea infection. CLINICAL RELEVANCE: Rasamsonia argillacea species complex is a recognised invasive mycosis in veterinary medicine, with disseminated disease causing significant clinical complications and death. This is believed to be the first report of infection caused by R. argillacea in a dog in Australasia and highlights the importance of awareness of a potential fungal aetiology in dogs with discospondylitis.Abbreviations: CLSI: Clinical and Laboratory Standards Institute; CRI: Constant rate infusion; MEC: Minimum effective concentration; MIC: Minimum inhibitory concentration; PAS: Periodic acid-Schiff.

Studies on the morphology, phylogeny, and bioremediation potential of Penicillium citrinum and Paecilomyces variotii (Eurotiales) from oil-contaminated areas.

Crude oil pollution is one of the most arduous issues to address, as it is hazardous to both public health and the environment. The discovery of novel biosurfactants-producing fungi and bacteria is in high demand due to their excellent properties and wide range of applications. The aim of this research is to isolate a powerful biosurfactant-producing fungus from the crude oil site near Barauni oil refinery in Bihar, India. Standard protocols were used to collect samples from the site. An integrative taxonomic approach was used, which included morphological, molecular, and phylogenetic analysis. The use of plating samples on Bushnell-Hass (BH) media aided in the isolation of a fungal strain from an enrichment culture. Two fungal strains isolated from contaminated soils, Penicillium citrinum and Paecilomyces variotti, showed potent oil degrading activity in a single culture. For preliminary biosurfactants screening, drop collapse assays, oil spreading, and emulsification activity tests were used. The results showed that the cultures performed well in the screening test and were further evaluated for degradation capacity. Different treatment periods (0, 3, 6, 9, 12, and 15 days) were used to observe degradation in single cultures. A steady drop in pH, an alteration in optical density and an increase in carbon dioxide release showed the ability of fungal strain to degrade the crude oil in a single culture. Fungi mycelia provide a larger surface area for absorption and degradation of the pollutants in contaminated environment. They produce extracellular enzymes to degrade the oil, and at the same time absorb and utilise carbon, allowing them to remove toxic substances from the oil. Thus, they could be candidates for bioremediation of a hydrocarbon-contaminated site.

Anisotropic protein-protein interactions in dilute and concentrated solutions.

Interactions between biomolecules are ubiquitous in nature and crucial to many applications including vaccine development; environmentally friendly textile detergents; and food formulation. Using small angle X-ray scattering and structure-based molecular simulations, we explore protein-protein interactions in dilute to semi-concentrated protein solutions. We address the pertinent question, whether interaction models developed at infinite dilution can be extrapolated to concentrated regimes? Our analysis is based on measured and simulated osmotic second virial coefficients and solution structure factors at varying protein concentration and for different variants of the protein Thermomyces Lanuginosus Lipase (TLL). We show that in order to span the dilute and semi-concentrated regime, any model must carefully capture the balance between spatial and orientational correlations as the protein concentration is elevated. This requires consideration of the protein surface morphology, including possible patch interactions. Experimental data for TLL is most accurately described when assuming a patchy interaction, leading to dimer formation. Our analysis supports that the dimeric proteins predominantly exist in their open conformation where the active site is exposed, thereby maximising hydrophobic attractions that promote inter-protein alignment.

Immobilization of Thermomyces lanuginosus lipase on a new hydrophobic support (Streamline phenyl™): Strategies to improve stability and reusability.

This paper establishes an efficient protocol for the immobilization of Thermomyces lanuginosus lipase (TLL) on a hydrophobic resin, Streamline phenyl. The biocatalyst produced by TLL immobilization on Streamline phenyl resin was named iTLL. In addition, strategies to improve stability and reusability of iTLL were performed using polyethylenimine (PEI) or/and glutaraldehyde (GA), producing iTLL-GA, iTLL-PEI, iTLL-PEI-GA biocatalysts. The immobilization yield was about 50%, using 1 mg/g of enzyme loading, and the immobilized enzyme activity was about 77 U/g, achieving about 100% of recovered activity. Desorption assays of the enzyme from the support using 0.6% cetyltrimethylammonium bromide (CTAB) and thermal and operational stability assays were performed. Although iTLL-PEI-GA lost about 50% of its initial activity after PEI and GA modifications, it was the most thermally and operationally stable (increases its stability about 66% if comparing with soluble enzyme at 65 ºC and maintenance 90% of its initial activity after 5 cycles of pNPB hydrolysis at 25 °C and pH 7.0). Furthermore, it showed almost no desorption of enzyme molecules with 24 h of CTAB incubation. Moreover, the streamline phenyl demonstrated a high TLL loading potential, with no diffusion limitations up to 14 mg/g. These characteristics allow future application of the iTLL-PEI-GA biocatalyst in fluidized bed reactors.