Papain kinetics in the presence of a water-miscible organic solvent

The effects of various concentrations of a water-miscible organic solvent [a 7:3 (v/v) mixture of N, N dimethylformamide and dimethylsulfoxide] on the kinetics of papain have been investigated. The parameters k(cat) and K(m) for the amidase and esterase activity of papain using N-alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and N-alpha-benzoyl-L-arginine ethyl ester (BAEE) as substrates were determined. For both types of activity, k(cat) initially increased (up to about 15% solvent), and then decreased with increasing concentrations of organic solvent. In contrast, K(m) increased sharply with the organic solvent concentration. Active site titration at 0 and 50% solvent indicated no change in the amount of active enzyme. Fluorometric measurements of the emission spectrum of papain did not indicate any major conformational changes with increasing concentrations of organic solvent.     

Microbial conversion of beta-ionone by immobilized Aspergillus niger in the presence of an organic solvent

Aspergillus niger JTS191 was capable of the conversion of beta-ionone to a mixture of its derivatives that is utilized to an essential oil of tobacco. The authors attempted this microbial conversion in the presence of an organic solvent to improve its reaction rate. The addition of isooctane accelerated the microbial conversion of beta-ionone. It took three days to complete the reaction whereas without isooctane it took more than six days. The addition of isooctane also improved the resistance of A. niger to the antifungal property of beta-ionone. A. niger pellets were immobilized in hydrophobic polymer, PU-3, and applied for the microbial conversion of beta-ionone. Further improvement of the resistance to the antifungal property of beta-ionone was achieved by immobilization. PU-3 immobilized A. niger was repeatedly used for microbial conversion of beta-ionone in the presence of isooctane for more than 480 hours.     

Immobilization of lipase in organic solvent in the presence of fatty acid additives

In this study porcine pancreatic lipase (PPL) was covalently immobilized on cross-linked polyvinyl alcohol (PVA) in organic media in the presence of fatty acid additives in order to improve its immobilized activity. The effects of fatty acid additions to the immobilization media were investigated choosing tributyrin hydrolysis in water and ester synthesis by immobilized PPL in n-hexane. Various fatty acids which are also the substrates of lipases in esterification reactions were used as active site protecting agents during the immobilization process in an organic solvent. The obtained results showed that covalent immobilization carried out in the presence of fatty acids as protective ligands improved the hydrolytic and esterification activity of immobilized enzyme. A remarkable increase in activity of the immobilized PPL was obtained when octanoic acid was used as an additive and the hydrolytic activity was increased from 5.2 to 19.2 μmol min⁻¹ mg⁻¹ as compared to the non-additive immobilization method. With the increase of hydrolytic activity of immobilized lipase in the presence of octanoic acid, in an analogous manner, the rate of esterification for the synthesis of butyl octanoate was also increased from 7.3 to 26.3 μmol min⁻¹ g⁻¹ immobilized protein using controlled thermodynamic water activities with saturated salt solutions. In addition, the immobilized PPL activity was maintained at levels representing 63% of its original activity value after 5 repeated uses. The proposed method could be adopted for a wide variety of other enzymes which have highly soluble substrates in organic solvent such as other lipases and esterases.     

Transacetylations to carbohydrates catalyzed by acetylxylan esterase in the presence of organic solvent

Various conditions were applied to test the ability of acetylxylan esterase (AcXE) from Schizophyllum commune to catalyze acetyl group transfer to methyl beta-D-xylopyranoside (Me-beta-Xylp) and other carbohydrates. The best performance of the enzyme was observed in an n-hexane-vinyl acetate-sodium dioctylsulfosuccinate (DOSS)-water microemulsion at a molar water-detergent ratio (w(0)) of about 4-5. Although the enzyme was found to have a half-life of about 1 h in the system, more than 60% conversion of Me-beta-Xylp to acetylated derivatives was achieved. Under identical reaction conditions, the enzyme acetylated other carbohydrates such as methyl beta-D-cellobioside (Me-beta-Cel), cellotetraose, methyl beta-D-glucopyranoside (Me-beta-Glcp), 2-deoxy-D-glucose, D-mannose, beta-1,4-mannobiose, -mannopentaose, -mannohexaose, beta-1,4-xylobiose and -xylopentaose. This work is the first example of reverse reactions by an acetylxylan esterase and a carbohydrate esterase belonging to family 1.     

Lipase catalyzed polytransesterification in an organic solvent

Summary Lipase-catalyzed polytransesterification ofbis(2,2,2-trifluoroethyl) dodecanedioate with aliphatic diols (from 1,2-ethanediol to 1,6-hexanediol) was studied with 4 enzymes and a number of solvents. The effects of experimental parameters were investigated with the purpose of obtaining a polyester of as high as possible average molar mass. The highest mass average molar mass (M _w) of 34,750 g mol^-1 (DP = 122) was obtained under vacuum with 1,4-butanediol,Mucor miehei lipase, and diphenyl ether as solvent.     

Yeast-mediated preparation of l-PAC in an organic solvent

The yeast-mediated acyloin condensation of benzaldehyde and pyruvic acid to form l-PAC occurs in a petroleum spirit solvent system at room temperature with moderate conversion (30%) and high enantioselectivity (86%ee) after 24 h. The addition of a small amount of ethanol (0.5% mL) to the reaction mixture inhibits the formation of the side product benzyl alcohol and increases the conversion to l-PAC. Conducting the reaction using 13C labeled pyruvate indicated that the pyruvate was incorporated into the l-PAC and that the excess pyruvate was converted into ethanol. Conducting the reaction at 5 degrees C results in similar conversion but higher enantioselectivity.     

Baker''s yeast activity in an organic solvent system

The baker's yeast mediated reduction of ethyl 3-oxobutanoate-3-¹³C in hexane was conducted in an NMR tube at 20°C and a ¹³C NMR spectrum recorded each hour. A plot of relative peak intensity against time allowed the progress of the reaction to be monitored. A series of reactions was carried out in which the yeast was pretreated with the organic solvent system for 3, 6, 12 and 24 h prior to the addition of the substrate. From the initial rate of these reactions it was determined that in hexane the enzyme activity remained constant for about 12 h and then rapidly decreased until after 24 h very little activity remained. The reaction was also carried out at 10°C and 30°C. At the lower temperature, the reaction was slower but enzyme activity was maintained for more than 60 h, while at 30°C the enzyme activity had ceased after 8 h.     

Lipid-induced conformational transitions of beta-lactoglobulin

Bovine beta-lactoglobulin (betaLG) provides an excellent model protein system for beta-to-alpha conformational change, but its behavior varies when the change is induced by alcohols, surfactants, or lipid vesicles. Here the interaction and orientation of betaLG in association with various artificial lipid vesicles at neutral and acidic pH have been studied by use of several complementary spectroscopic techniques. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectra demonstrated that betaLG acquires a non-native alpha-helical structure upon binding with anionic lipids, while zwitterionic lipids do not have a significant effect on its conformation. The degree of induced alpha-helix depends on the lipid concentration and is strongly affected by the charge of the protein and lipids as well as the ionic strength of the solution. Near-UV CD and Trp emission spectra revealed that the tertiary structure of lipid-bound betaLG is highly expanded but not completely disrupted. Fluorescence quenching together with a Trp emission blue shift showed that the Trp residues remain largely shielded from the solvent when interacting with DMPG, which would be consistent with at least some portions of betaLG having been inserted into the lipid membrane. The orientations of the alpha-helix and beta-sheet axes in membrane-bound betaLG were found to be parallel and perpendicular, respectively, to the membrane film normal, as determined by use of polarized attenuated total reflection (ATR) FTIR spectra. Our findings reveal that the lipid-induced beta-to-alpha transition in betaLG, accompanied by a substantial disruption in tertiary structure, is mainly driven by strong electrostatic interactions. Once the tightly packed betaLG is disrupted, hydrophobic residues become exposed and available for insertion into the lipid bilayer, where hydrophobic interaction with the lipids may play a role in stabilizing the helical components.     

Predicting order of conformational changes during protein conformational transitions using an interpolated elastic network model

The decryption of sequence of structural events during protein conformational transitions is essential to a detailed understanding of molecular functions ofvarious biological nanomachines. Coarse‐grained models have proven useful by allowing highly efficient simulations of protein conformational dynamics. By combining two coarse‐grained elastic network models constructed based on the beginning and end conformations of a transition, we have developed an interpolated elastic network model to generate a transition pathway between the two protein conformations. For validation, we have predicted the order of local and global conformational changes during key ATP‐driven transitions in three important biological nanomachines (myosin, F₁ ATPase and chaperonin GroEL). We have found that the local conformational change associated with the closing of active site precedes the global conformational change leading to mechanical motions. Our finding is in good agreement with the distribution of intermediate experimental structures, and it supports the importance of local motions at active site to drive or gate various conformational transitions underlying the workings of a diverse range of biological nanomachines. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Lipase-catalyzed methanolysis of palm oil in presence and absence of organic solvent for production of biodiesel

Enzymatic production of methyl esters (biodiesel) by methanolysis of palm oil in presence and absence of organic solvent was investigated using Candida antarctica lipase immobilized on acrylic resin as a biocatalyst. Although, at least molar equivalent of methanol (methanol-palm oil ratio 3:1) is required for the complete conversion of palm oil to methyl esters, lipase catalyzed methanolysis of palm oil in absence of organic solvent was poisoned by adding more than 1/3 molar equivalent of methanol. The use of polar organic solvents prevented the lipase to be poisoned in methanolysis with a molar equivalent of methanol, and tetrahydrofuran (THF) was found to be the most effective. The presence of water in methanolysis of palm oil both in presence and absence of THF inhibited the reaction rate but this inhibition was considerably low in THF containing system. The palm oil-lipase (w/w) ratio significantly influenced the activity of lipase and the optimal ratio in presence and absence of THF was 100 and 50, respectively.     

Multinuclear NMR study of enzyme hydration in an organic solvent

Multinuclear NMR spectroscopy has been used to study water bound to subtilisin Carlsberg suspended in tetrahydrofuran (THF), with the water itself employed as a probe of the hydration layer's physicochemical and dynamic characteristics. The presence of the enzyme did not affect the intensity, chemical shift or linewidth of water (up to 8% v/v) added to THF, as measured by 17O- and 2H-NMR. This finding suggests that hydration of subtilisin can be described by a three-state model that includes tightly bound, loosely bound, and free water. Solid-state 2H-NMR spectra of enzyme-bound D2O support the existence of a non-exchanging population of tightly bound water. An important implication is that the loosely-bound water is the same as free water from an NMR viewpoint. This loosely bound water must also be the water responsible for the large increase in catalytic activity observed in previous hydration studies.     

The use of nanosecond fluorometry in detecting conformational transitions of an allosteric enzyme

Analysis of the time decay of fluorescence of 1-anilino-naphthalene-8-sulfonate bound to phosphorylase b reveals the presence of two components with different decay times. In glycylglycine buffer, pH 7.0, the addition of the activators AMP or IMP, or the conversion to phosphorylase a results in a preferential decrease in the amplitude of the component of longer decay time, arising probably from a preferential dissociation of 1-anilino-naphthalene-8-sulfonate from the corresponding site as a consequence of a change in molecular state.     

Geometry-based sampling of conformational transitions in proteins

The fast and accurate prediction of protein flexibility is one of the major challenges in protein science. Enzyme activity, signal transduction, and ligand binding are dynamic processes involving essential conformational changes ranging from small side chain fluctuations to reorientations of entire domains. In the present work, we describe a reimplementation of the CONCOORD approach, termed tCONCOORD, which allows a computationally efficient sampling of conformational transitions of a protein based on geometrical considerations. Moreover, it allows for the extraction of the essential degrees of freedom, which, in general, are the biologically relevant ones. The method rests on a reliable estimate of the stability of interactions observed in a starting structure, in particular those interactions that change during a conformational transition. Applications to adenylate kinase, calmodulin, aldose reductase, T4-lysozyme, staphylococcal nuclease, and ubiquitin show that experimentally known conformational transitions are faithfully predicted.     

Effective artificial proteases synthesized by covering silica gel with aldehyde and various other organic groups

Organic artificial proteases with broad substrate specificity were synthesized by covering the surface of silica gel with aldehyde and the functional groups present in amino acids. The artificial proteases hydrolyzed ovalbumin, albumin, hemoglobin, gamma-globulin with half-lives as short as 50 min at 25 degrees C or 7 min at 50 degrees C.     

Energetics of solvent and ligand-induced conformational changes in alpha-lactalbumin

The energetics of structural changes in the holo and apo forms of a-lactalbumin and the transition between their native and denatured states induced by binding Ca2+ and Na+ have been studied by differential scanning and isothermal titration microcalorimetry and circular dichroism spectroscopy under various solvent conditions. Removal of Ca2+ from the protein enhances its sensitivity to pH and ionic conditions due to noncompensated negative charge-charge interactions at the cation binding site, which significantly reduces its overall stability. At neutral pH and low ionic strength, the native structure of apo-alpha-lactalbumin is stable below 14 C and undergoes a conformational change to a native-like molten globule intermediate at temperatures above 25 degrees C. The denaturation of either holo- or apo-alpha-lactalbumin is a highly cooperative process that is characterized by an enthalpy of similar magnitude when calculated at the same temperature. Measured by direct calorimetric titration, the enthalpy of Ca2+-binding to apo-LA at pH 7.5 is -7.1 kJ mol(-1) at 5.0 degrees C. which is essentially invariant to protonation effects. This small enthalpy effect infers that stabilization of alpha-lactalbumin by Ca2+ is primarily an entropy driven process, presumably arising from electrostatic interactions and the hydration effect. In contrast to the binding of calcium, the interaction of sodium with apo-LA does not produce a noticeable heat effect and is characterized by its ionic nature rather than specific binding to the metal-binding site. Characterization of the conformational stability and ligand binding energetics of alpha-lactalbumin as a function of solvent conditions furnishes significant insight regarding the molecular flexibility and regulatory mechanism mediated by this protein.     

Unfolding of ervatamin C in the presence of organic solvents: sequential transitions of the protein in the O-state

The folding of ervatamin C was investigated in the presence of various fluorinated and non-fluorinated organic solvents. The differences in the unfolding of the protein in the presence of various organic solvents and the stabilities of O-states were interpreted. At pH 2.0, non-fluorinated alkyl alcohols induced a switch from the native alpha-helix to a beta-sheet, contrary to the beta-sheet to alpha-helix conversion observed for many proteins. The magnitude of ellipticity at 215 nm, used as a measure of beta-content, was found to be dependent on the concentration of the alcohol. Under similar conditions of pH, fluorinated alcohol enhanced the intrinsic a-helicity of the protein molecule, whereas the addition of acetonitrile reduced the helical content. Ervatamin C exhibited high stability towards GuHCl induced unfolding in different O-states. Whereas the thermal unfolding of O-states was non-cooperative, contrary to the cooperativity seen in the absence of the organic solvents under similar conditions. Moreover, the differential scanning calorimetry endotherms of the protein acquired at pH 2.0 were deconvoluted into two distinct peaks, suggesting two cooperative transitions. With increase in pH, the shape of the thermogram changed markedly to exhibit a major and a minor transition. The appearance of two distinct peaks in the DSC together with the non-cooperative thermal transition of the protein in O-states indicates that the molecular structure of ervatamin C consists of two domains with different stabilities.