Biocatalyst-mediated selective acylation and deacylation chemistry on the secondary hydroxyl/amine groups of nucleosides.

Nucleosides play a pivotal role in biological systems and therefore have attracted a lot of interest as chemotherapeutic agents in drug discovery. Over the years biocatalysts have emerged as a sustainable alternative to conventional synthetic catalysts. As a nature's catalyst, they exhibit excellent selectivity, remarkable tolerance, and help in carrying out eco-friendly benign processes. The use of a biocatalyst as a regio- and enantioselective catalyst is particularly relevant in the transformations of nucleosides and their analogs because of the presence of multiple chiral centres. Herein, we discuss the recent advances in the Pseudomonas Cepacia Lipase mediated selective acylation and deacylation reactions of the secondary hydroxyl and amino groups of nucleosides and their analogs.

Continuous production of monoacylglycerol via glycerolysis of babassu oil by immobilized Burkholderia cepacia lipase in a packed bed reactor.

This study investigated the glycerolysis of babassu oil by Burkholderia cepacia lipase immobilized on SiO2-PVA particles in a continuous packed bed reactor. Experiments were conducted in a solvent-free system at 273.15 K either in an inert atmosphere or in the presence of cocoa butter to prevent lipid oxidation. The reactor (15 × 55 mm) was run at a fixed space time of 9.8 h using different molar ratios of babassu oil to glycerol (1:3, 1:6, 1:9, 1:12, and 1:15) to assess the effects of reactant molar ratio on monoacylglycerol productivity and selectivity. Nitrogen atmosphere and cocoa butter were equally effective in inhibiting lipid oxidation, indicating that addition of cocoa butter to glycerolysis reactions may be an interesting cost-reduction strategy. An oil/glycerol molar ratio of 1:9 resulted in the highest productivity (52.3 ± 2.9 mg g-1 h-1) and selectivity (31.5 ± 1.8%). Residence time distribution data were fitted to an axial dispersion model for closed-vessel boundary conditions, giving a mass transfer coefficient (kc) of 3.4229 × 10-6 m s-1. A kinetic model based on elementary steps of the studied reaction was written in Scilab and compared with experimental data, providing standard deviations in the range of 5.5-7.5%.

Computational and experimental analysis on the preferential selectivity of lipases for triglycerides in Licuri oil.

In the present study, we demonstrated the use of molecular docking as an efficient in silico screening tool for lipase-triglyceride interactions. Computational simulations using the crystal structures from Burkholderia cepacia lipase (BCL), Thermomyces lanuginosus lipase (TLL), and pancreatic porcine lipase (PPL) were performed to elucidate the catalytic behavior with the majority triglycerides present in Licuri oil, as follows: caprilyl-dilauryl-glycerol (CyLaLa), capryl-dilauryl-glycerol (CaLaLa), capryl-lauryl-myristoyl-glycerol (CaLaM), and dilauryl-myristoyl-glycerol (LaLaM). The computational simulation results showed that BCL has the potential to preferentially catalyze the major triglycerides present in Licuri oil, demonstrating that CyLaLa, (≈25.75% oil composition) interacts directly with two of the three amino acid residues in its catalytic triad (Ser87 and His286) with the lowest energy (-5.9 kcal/mol), while other triglycerides (CaLaLa, CaLaM, and LaLaM) interact with only one amino acid (His286). In one hard, TLL showed a preference for catalyzing the triglyceride CaLaLa also interacting with His286 residue, but, achieving higher binding energies (-5.3 kcal/mol) than found in BCL (-5.7 kcal/mol). On the other hand, PPL prefers to catalyze only with LaLaM triglyceride by His264 residue interaction. When comparing the computational simulations with the experimental results, it was possible to understand how BCL and TLL display more stable binding with the majority triglycerides present in the Licuri oil, achieving conversions of 50.86 and 49.01%, respectively. These results indicate the production of fatty acid concentrates from Licuri oil with high lauric acid content. Meanwhile, this study also demonstrates the application of molecular docking as an important tool for lipase screening to reach a more sustainable production of fatty acid concentrates from vegetable oils.

Bacterial Lipase Neutralized Toxicity of Lipopolysaccharide on Chicken Embryo Cardiac Tissue.

It has been shown that near all organs, especially the cardiovascular system, are affected by bacterial lipopolysaccharide via the activation of Toll-like receptor signaling pathways. Here, we tried to find the blunting effect of bacterial lipase on lipopolysaccharide (LPS)-induced cardiac tissue toxicity in chicken embryos. 7-day fertilized chicken eggs were divided randomly into different groups as follows; Control, Normal Saline, LPS (0.1, 0.5 and 1 mg/kbw), and LPS (0.1, 0.5 and 1 mg/kbw) plus 5 mg/ml Lipase. On day 17, the hearts were sampled. The expression of genes such as GATA4, NKX2.5, EGFR, TRIF, and NF-ƙB was monitored using real-time PCR analysis. Using western blotting, we measured NF-ƙB protein level. Total antioxidant capacity, glutathione peroxidase, and Catalase activity were also studied. Microvascular density and anterior wall thickness were monitored in histological samples using H&E staining. High dose of LPS (1 mg/kbw) increased the expression of TRIF but not NF-ƙB compared to the control group (p < 0.05). We found a statistically significant reduction in groups that received LPS + Lipase compared to the control and LPS groups (p < 0.05). Western blotting revealed that the injection of Lipase could reduce LPS-induced NF-ƙB compared to the control group (p < 0.05). The expression of GATA4, NKx2.5, and EGFR was not altered in the LPS group, while the simultaneous application of LPS and Lipase significantly reduced GATA4, NKx2.5, and EGFR levels below the control (p < 0.05). We found non-significant differences in glutathione peroxidase, and Catalase activity in all groups (p > 0.05), while total antioxidant capacity was increased in groups that received LPS + Lipase. Anterior wall thickness was diminished in LPS groups and the use of both lipase and LPS returned near-to-control values (p < 0.05). Despite a slight increase in microvascular density, we found statistically non-significant differences in all groups (p > 0.05). Bacterial lipase reduces detrimental effects of LPS on chicken embryo heart induced via Toll-like receptor signaling pathway.

Lipase activation by molecular bioimprinting: The role of interactions between fatty acids and enzyme active site.

Bioimprinting is an easy, sustainable and low-cost technique that promotes a printing of potential substrates on enzyme structure, inducing a more selective and stable conformation. Bioimprinting promotes conformational changes in enzymes, resulting in better catalytic performance. In this work, the effect of bioimprinting of Burkholderia cepacia lipase (BCL) and porcine pancreatic extracts (PPE) with four different fatty acids (lauric acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0), and stearic acid (C18:0)) was investigated. The results demonstrated that the better bioimprinting effect was in BCL with lauric acid in esterification reaction, promoting BCL activation in which relative enzyme activity was 70 times greater than nonimprinted BCL. Bioimprinting results were influenced by the carbon chain length of fatty acids imprinted in the BCL, in which the effects were weaker with the chain increase. Molecular docking was performed to better understand the bioimprinting method. The results of these simulations showed that indeed all fatty acids were imprinted in the active site of BCL. However, lauric acid presented the highest imprinting preference in the active site of BCL, resulting in the highest relative activity. Furthermore, Fourier transform infrared (FTIR) analysis confirmed important variations in secondary structure of bioimprinting BCL with lauric acid, in which there was a reduction in the α-helix content and an increase in the ß-sheet content that facilitated substrate access to the active site of BCL and led higher rigidity, resulting in high activity. Bioimprinted BCL with lauric acid showed excellent operational stability in esterification reaction, maintaining its original relative activity after five successive cycles. Thus, the results show that bioimprinting of BCL with lauric acid is a successful strategy due to its high catalytic activity and reusability.

Synthesis of propyl benzoate by solvent-free immobilized lipase-catalyzed transesterification: Optimization and kinetic modeling.

The present study aimed to analyze reaction kinetics and mechanism for the synthesis of propyl benzoate in solvent-free conditions. Lipase was immobilized on Hydroxypropyl methylcellulose (HPMC) and polyvinyl alcohol (PVA) polymer blend by entrapment method. Among different lipases immobilized on a support, Candida cylindracea (CCL) showed excellent activity. Systematic studies were done to optimize the reaction conditions. The activation energy was found to be 16.2 kcal/mol for immobilized CCL. Kinetic parameters were calculated, which depicted that propyl benzoate synthesized using immobilized CCL followed the ternary complex model in which propanol inhibits lipase activity at higher concentrations. Recyclability of the catalyst was checked up to four catalytic cycles and 40% retention of activity was observed up to the fourth cycle. Finally, the applicability of developed protocol to synthesize various alkyl benzoates was explored.

Design for preparation of more active cross-linked enzyme aggregates of Burkholderia cepacia lipase using palm fiber residue.

A new design of cross-linked enzyme aggregates (CLEAs) of Burkholderia cepacia lipase (BCL) based mainly on the use of lignocellulosic residue of palm fiber as an additive was proposed. Different parameters for the preparation of active CLEAs in the hydrolysis of olive oil, such as precipitation agents, crosslinking agent concentration, additives, and coating agents were investigated. The highest activity yield (121.1 ± 0.1%) and volumetric activity (1578.1 ± 2.5 U/mL) were achieved for CLEAs prepared using the combination of a coating step with Triton® X-100 and polyethyleneimine plus the use of palm fiber as an additive. The variations of the secondary structures of BCL-CLEAs were analyzed by second-derivative infrared spectra, mainly indicating a reduction of the α-helix structure, which was responsible for the lipase activation in the supramolecular structure of the CLEAs. Thus, these results provided evidence of an innovative design of BCL-CLEAs as a sustainable and biocompatible opportunity for biotechnology applications.

Production of starch-polyester bio-support for lipases immobilization: synergistic action of itaconic acid and nanoclay.

The objective of the present work was to develop biodegradable polymeric films (starch-PBAT) as support for the immobilization of lipases using sodium montmorillonite (MMT) as a reinforcing agent (2% w/w) and itaconic acid (IA - 0.5-1.5% w/w) as a compatibilizing agent. The films were produced through a two steps blow-extrusion. The addition of MMT increased the tensile strength and Tg of the films, while the presence of IA made the films more flexible, reducing their Tg. Lipases from Burkholderia cepacia LTEB11 were immobilized in the films by the adsorption method. The ester yield (% of ethyl oleate synthesis) has shown best results (96%, 6 h) for immobilized enzyme in the MMT film and six cycles of reuse were carried out until a reduction of 50% in the catalytic activity of the enzyme.

Enzymatic transesterification of coconut oil by using immobilized lipase on biochar: An experimental and molecular docking study.

Guava seed biochar appears as a new alternative of the effective support to the immobilization of Burkholderia cepacia lipase (BCL) by physical adsorption. The objective of this work was to evaluate the potential of this immobilized biocatalyst in the transesterification reaction of crude coconut oil and ethanol and to understand the mechanism of the reaction through the study of molecular docking. The best loading of BCL was determined to be 0.15 genzyme /gsupport having a hydrolytic activity of 260 U/g and 54% immobilization yield. The products of transesterification reaction produced a maximum yield at 40 °C under different reaction conditions. The monoacylglycerols (MAGs) conversion of 59% was using substrate molar ratio oil:ethanol of 1:7 with the reaction time of 24 H. In addition, the highest ethyl esters yield (48%) had the molar ratio of 1:7 with the reaction time of 96 H and maximum conversion of diacylglycerols (DAGs) was 30% with the molar ratio of 1:6 with the reaction time of 24 H. Molecular Docking was applied to clarify the mechanisms of transesterification reaction at the molecular level. MAGs and DAGs are compounds with excellent emulsifying properties used in industrial production of several bioproducts such as cosmetic, pharmaceuticals, foods, and lubricants.

Production of biodiesel by Burkholderia cepacia lipase as a function of process parameters.

Despite the already established route of chemically catalyzed transesterification reaction in biodiesel production, due to some of its shortcomings, biocatalysts such as lipases present a vital alternative. Namely, it was noticed that one of the key shortcomings for the optimization of the enzyme catalyzed biodiesel synthesis process is the information on the lipase activity in the reaction mixture. In addition to making optimization difficult, it also makes it impossible to compare the results of the independent research. This article shows how lipase intended for use in biodiesel synthesis can be easily and accurately characterized and what is the enzyme concentration that enables achievement of the desired level of fatty acid methyl esters (FAME) in the final product mixture. Therefore, this study investigated the effect of two different activity loads of Burkholderia cepacia lipase on the biodiesel synthesis varying the pH and temperature optimal for lipase activity. The optimal lipase pH and temperature were determined by two different enzyme assays: spectrophotometric and titrimetric. The B. cepacia lipase pH optimum differentiated between assays, while the lipase optimally hydrolyzed substrates at 50°C. The analysis of FAME during 24 hr of biodiesel synthesis, at two different enzyme concentrations, pH 7, 8, and 10, and using two different buffers, revealed that the transesterification reaction at optimal pH, 1 hr reaction time and lipase activity load of 250 U per gram of reaction mixture was sufficient to produce more than 99% FAME.

Preparation of Lipase-Electrospun SiO2 Nanofiber Membrane Bioreactors and Their Targeted Catalytic Ability at the Macroscopic Oil-Water Interface.

Lipase is one of the most widely used enzymes in biocatalysis. Because of the special structure of the catalytic active center, lipases show high catalytic activity at oil-water interfaces. Hence, the interface plays a key role in activating and modulating lipase biocatalysis. Compared with traditional catalytic systems that offer interfaces, such as emulsions, a lipase-membrane bioreactor exhibits many obvious advantages when at the macroscopic oil-water system. In our current research, a series of new Burkholderia cepacia lipase (BCL)-SiO2 nanofiber membrane (NFM) bioreactors prepared via combined electrospinning and immobilization strategies were reported. These SiO2 NFMs assisted BCL in reaching the oil-water interface for efficient catalysis. The enzyme loading capacity and catalytic efficiency of BCL-SiO2 NFMs varied with the surface hydrophobicity of the electrospun NFMs. As the hydrophobicity increased, the activity decreased from 2.43-fold to 0.74-fold that of free BCL. However, the lipase-loading capacity increased obviously when the hydrophobicity of the SiO2 NFMs increased from 0 to 143°, and no significant change was observed when the hydrophobicity of the SiO2 NFMs increased from 143 to 153°. The gel trapping technique proved that the hydrolytic activity of the different BCL-SiO2 NFM bioreactors depends on the contact area of the membrane at the oil-water interface. BCL-SiO2 NFM, BCL-SiO2 NFM-C12, and BCL-SiO2 NFM-C18 retained 32, 83, and 42% of activity, respectively, after five cycles of reuse. The current work was a useful exploration of the construction and modification of lipase-membrane reactors based on electrospun inorganic silicon.

Enantioselective synthesis of a chiral intermediate of himbacine analogs by Burkholderia cepacia lipase A.

The enantiomers of (4R/S)-4-hydroxy-N, N-diphenyl-2-pentynamide are key chiral synthons for the synthesis of thrombin receptor antagonists such as vorapaxar. In this paper, we report the enzymatic preparation of enantiomerically enriched (4R)-4-hydroxy-N, N-diphenyl-2-pentynamide using lipase A from Burkholderia cepacia ATCC 25416 as the catalyst. First, the lipase gene (lipA) and its chaperone gene (lipB) was cloned and expressed in Escherichia coli system. After purification, lipase A activation was performed with the assistance of foldase lipase B. Enzyme assay revealed that the activated lipase A showed the optimal catalytic activity at 60 ºC and pH 7. The effects of various metals on the activity were investigated and results demonstrated that most of the metals inhibited the activity. To further improve the catalytic outcome, two-phase reaction was studied, and n-hexane proved to be a good organic solvent for the combination system. Using the optimize conditions, (4R)-4-hydroxy-N, N-diphenyl-2-pentynamide with 94.5% ee value and 48.93% conversion ratio was achieved. Our investigation on this lipase reveals lipase A as a promising biocatalyst for producing chiral propargyl alcohol for preparation of novel himbacine analogs.

Greener approach for synthesis of N,N,N-trimethyl chitosan (TMC) using ternary deep eutectic solvents (TDESs).

N,N,N-trimethyl chitosan (TMC), quaternized hydrophilic derivative of chitosan, has been projected to have wide applications in the pharmaceutical industry owing to its improved solubility at physiological conditions. However, the conventional synthesis of TMC involves toxic organic agents, which complicates its use for biological applications. Moreover, these reactions result into unwanted O-methylation and scission of the parent polymer. In the present study we have addressed these limitations by employing a green approach to synthesize TMC, by using lipase as the biocatalyst and dimethyl carbonate (DMC) as the green methylating agent, in a reaction medium comprising of ternary deep eutectic solvents (TDESs). Synthesis of TMC was carried out by using two different lipases from Burkholderia cepacia and Candida rugosa. The resulting TMC was characterized by using FTIR, 1H NMR, DSC, XRD. Methylation was confirmed by FTIR analysis (-CH at 1666 cm-1) and 1H NMR (?? = 3.3 ppm). DSC study revealed a lower thermal stability of TMC as compared to chitosan. These results indicated the possibility of using DMC as a green methylating agent, along with TDESs as green and sustainable solvents, for lipase catalyzed reactions. TMC was successfully synthesized and exhibited a degree of quaternization of about 12.5%, 15.69%, when synthesized used lipases from Burkholderia cepacia and Candida rugosa, respectively.

Comprehensive semisyntheses of catathelasmols C, D, and E from D-glutamic acid, utilizing lipase-catalyzed site-selective reactions on intermediates.

Catathelasmols C, D, and E, which had been isolated from Catathelasma imperiale as inhibitors for 11-hydroxysteroid dehydrogenases, were comprehensively semisynthesized from commercially available D-glutamic acid. The key synthetic intermediate, (R)-pentane-1,2,5-triol, was site-selectively acetylated by treatment with vinyl acetate and Candida antarctica lipase B (Novozym 435) in tetrahydrofuran (THF) at 25°C to furnish 1,5-diacetate (catathelasmol E, quantitative). The acetylation occurred site-selectively on the primary alcohols at the C-1 and C-5 positions over the secondary alcohol at the C-2 position. Dichromic acid oxidation provided 2-oxopentane-1,5-diyl diacetate (catathelasmol C, 78%). Burkholderia cepacia lipase-catalyzed transesterification with methanol in THF at - 5°C proceeded preferentially on the acetate at C-1 located adjacent to the C-2 carbonyl group over the other terminal acetate at the C-5 position. 5-Hydroxy-4-oxopentyl acetate (catathelasmol D) was obtained in 53% yield.

Microchamber arrays made of biodegradable polymers for enzymatic release of small hydrophilic cargos.

The encapsulation of small hydrophilic molecules and response to specific biological triggers in a controlled manner have become two of the significant challenges in biomedical research, in particular in the field of localized drug delivery and biosensing. This work reports the fabrication of free-standing microchamber array films made of biodegradable polymers for the encapsulation and enzymatically triggered release of small hydrophilic molecules. Polycaprolactone (PCL) microchamber arrays were demonstrated to fully biodegrade within 5 hours of exposure to lipase from Pseudomonas cepacia (lipase PS) at a concentration of 0.5 mg ml-1, with lower concentrations producing correspondingly longer degradation times. The gradual process of deterioration was real-time monitored utilising laser Fraunhofer diffraction patterns. Additionally, a small hydrophilic molecule, 5(6)-carboxyfluorescein (CF), was loaded into the PCL microchamber arrays in a dry state; however, the substantial permeability of the PCL film led to leakage of the dye molecules. Consequently, polylactic acid (PLA) was blended with PCL to reduce its permeability, enabling blended PCL-PLA (1 : 2 ratio correspondingly) microchamber arrays to trap the small hydrophilic molecule CF. PCL-PLA (1 : 2) microchamber arrays hold potential for controlled release under the catalysis of lipase within 26 hours. Additionally, it is calculated that approximately 11 pg of CF dye crystals was loaded into individual microchambers of 10 µm size, indicating that the microchamber array films could yield a highly efficient encapsulation.

"Recent advances on support materials for lipase immobilization and applicability as biocatalysts in inhibitors screening methods"-A review.

With a substantial demand for new anti-obesity drugs for the treatment of obesity, screening lipase inhibitors from natural products has become a popular approach toward drug discovery. Due to the significant advantages of excellent reusability, stability and endurance in extreme pH and temperature conditions, lipase immobilization has been employed as a promising strategy to screen lipase inhibitors. Support is a key factor in the process of enzyme immobilization used to provide excellent biocompatibility, stable physical and chemical properties and abundant binding sites for enzymes. Thus, various supports, including nanofibers, polymeric monoliths, mesoporous materials, nanomaterials, membrane and cellulose paper, are systematically introduced and discussed in this review. Considering these supports, the application of the immobilization of lipase in screening compounds from natural products is also comprehensively reviewed, and the outlook for future research directions is described.

Continuous flow reactor based with an immobilized biocatalyst for the continuous enzymatic transesterification of crude coconut oil.

Here, we have assessed the use of one packed bed or two packed bed reactors in series in which Burkholderia cepacia lipase (BCL) was immobilized on protic ionic liquid (PIL)-modified silica and used as a biocatalyst for the transesterification of crude coconut oil. Reaction parameters including volumetric flow, temperature, and molar ratio were evaluated. The conversion of transesterification reaction products (ethyl esters) was determined using gas chromatography and the quantities of intermediate products (diglyceride and monoglyceride [MG]) were assessed using high-performance liquid chromatography. Packed bed reactors in series produced ethyl esters with the greatest efficiency, achieving 65.27% conversion after 96 H at a volumetric flow rate of 0.50 mL Min-1 at 40 °C and a 1:9 molar ratio of oil to ethanol. Further, within the first 24 H of the reaction, increased MG (54.5%) production was observed. Molecular docking analyses were performed to evaluate the catalytic step of coconut oil transesterification in the presence of BCL. Molecular docking analysis showed that triglycerides have a higher affinity energy (-5.7 kcal mol-1 ) than the smallest MG (-6.0 kcal mol-1 ), therefore, BCL catalyzes the conversion of triglycerides rather than MG, which is consistent with experimental results.

Controlled degradation of poly-ε-caprolactone for resorbable scaffolds.

A lipase from Burkholderia cepacia was successfully adsorbed on the surface of halloysite nanotubes and the coated tubes were incorporated into poly-ε-caprolactone (PCL). The efficiency of the halloysite in the adsorption of the enzyme was characterized by the total protein content determined with the Bradford method. The activity of the adsorbed enzyme was estimated by the kinetic resolution of racemic 1-phenylethanol. The immobilized enzyme was mixed with the polymer and compression molded films were prepared at 70 °C. Activity measurements proved that the enzyme remains active even after adsorption; in fact, larger activities were measured for the immobilized enzyme than for the neat enzyme preparation. The supported enzyme degraded PCL efficiently, the rate of degradation depended on the amount of enzyme adsorbed. The kinetics of degradation was described quantitatively with an appropriate model accounting for two of the three steps of the process, i.e. degradation and the denaturation of the enzyme. The determination of time constants allows the adjustment of degradation rate. This is the first time that the enzyme, which catalyzes degradation, is incorporated into the polymer, and not into the degradation medium, thus allowing the preparation of resorbable scaffolds with controlled lifetime.

Cloning, expression and characterization of catechol 1,2-dioxygenase from Burkholderia cepacia.

The present study reports on the cloning, expression and characterization of catechol 1,2-dioxygenase (CAT) of bacterial strains isolated from dioxin-contaminated soils in Vietnam. Two isolated bacterial strains DF2 and DF4 were identified as Burkholderia cepacia based on their 16S rRNA sequences. Their genes coding CAT was amplified with a specific pair of primers. Recombinant CAT (rCAT) was expressed in E. coli M15 cells and its activity was confirmed by the detection of cis,cis-muconic acid, a product from catechol, by high-performance liquid chromatography (HPLC) analysis. The rCAT of DF4 had an optimal pH and temperature of 7 and 30°C, respectively. Metal ions, such as Zn2+ and Mn2+, and surfactants, such as SDS, Tween 20 and Triton X100, strongly inhibited enzyme activity, while K+ slightly increased the activity.

The shift in urea orientation at protein surfaces at low pH is compatible with a direct mechanism of protein denaturation.

Surface-specific spectroscopic data has shown that urea undergoes a shift in orientation at protein surfaces in acidic media. Since urea denatures proteins at a wide range of pHs, the variable chemical nature of protein-urea interactions has been used to support an indirect mechanism of urea-induced denaturation. Here, we use molecular dynamics simulations, minimum-distance distribution functions (MDDFs), and hydrogen-bond analysis, to characterize the interactions of urea with proteins at neutral and low pH, as defined by the protonation state of acidic residues. We obtain the expected preferential solvation by urea and dehydration, consistently with urea-induced denaturation, while the MDDFs allow for a solvent-shell perspective of protein-urea interactions. The distribution functions are decomposed into atomic contributions to show that there is indeed a shift in the orientation of urea molecules in the vicinity of acidic side-chains, as shown by the experimental spectroscopic data. However, this effect is local, and the interactions of urea with the other side chains and with the protein backbone are essentially unaffected at low pH. Therefore, hydrophobic solvation and urea-backbone hydrogen bonds can play a role in a direct mechanism of urea-induced protein denaturation without contradicting the observed variations in the chemical nature of protein-urea interactions as a function of the acidity of the solution.