Arginine ethylester prevents thermal inactivation and aggregation of lysozyme.

Arginine is a versatile additive to prevent protein aggregation. This paper shows that arginine ethylester (ArgEE) prevents heat-induced inactivation and aggregation of hen egg lysozyme more effectively than arginine or guanidine. The addition of ArgEE decreased the melting temperature of lysozyme. This data could be interpreted in terms of ArgEE binding to unfolded lysozyme, possibly through the ethylated carboxyl group, which leads to effective prevention of intermolecular interaction among aggregation-prone molecules. The data suggest that ArgEE could be used as an additive to prevent inactivation and aggregation of heat-labile proteins.     

Prevention of thermal inactivation and aggregation of lysozyme by polyamines.

Proteins tend to form inactive aggregates at high temperatures. We show that polyamines, which have a relatively simple structure as oligoamids, effectively prevent thermal inactivation and aggregation of hen egg lysozyme. In the presence of additives, including arginine and guanidine (100 microM), more than 30% of 0.2 mg x mL(-1) lysozyme in sodium phosphate buffer (pH 6.5) formed insoluble aggregates by heat treatment (98 degrees C for 30 min). However, in the presence of 50 mm spermine or spermidine, no aggregates were observed after the same heat treatment. The residual activity of lysozyme after this heat treatment was very low (< 5%), even in the presence of 100 microM arginine and guanidine, while it was maintained at approximately 50% in the presence of 100 microM spermine and spermidine. These results imply that polyamines are new candidates as molecular additives for preventing the thermal aggregation and inactivation of heat-labile proteins.     

Characteristics of thermal inactivation of lysozyme in solution

In the buffer solution (pH 6,2) at 20-80 degrees, the lysozyme thermoinactivation was studied by monitoring of its activity decrease in the lysis of M. lysodeicticus cells. Protein inactivation was characterized by effective pseudofirst order rate constants which depend on enzyme concentration and are described by equation k = k0 . exp [-alpha 0 (1-gamma/T) [E]0], where k0 is inactivation rate constant at "infinite" enzyme dilution, [E0] is an initial lysozyme concentration, alpha 0 and gamma are the coefficients independent on [E0]. By extrapolation of the "k" dependencies on [E]0 the constants k0 were determined. In the range 40-70 degrees C, the rate constant k0 is equal 4,0 X 10(11) . exp (-24 200/RT) sec-1.     

Curcumin and kaempferol prevent lysozyme fibril formation by modulating aggregation kinetic parameters

Interaction of small molecule inhibitors with protein aggregates has been studied extensively, but how these inhibitors modulate aggregation kinetic parameters is little understood. In this work, we investigated the ability of two potential aggregation inhibiting drugs, curcumin and kaempferol, to control the kinetic parameters of aggregation reaction. Using thioflavin T fluorescence and static light scattering, the kinetic parameters such as amplitude, elongation rate constant and lag time of guanidine hydrochloride-induced aggregation reactions of hen egg white lysozyme were studied. We observed a contrasting effect of inhibitors on the kinetic parameters when aggregation reactions were measured by these two probes. The interactions of these inhibitors with hen egg white lysozyme were investigated using fluorescence quench titration method and molecular dynamics simulations coupled with binding free energy calculations. We conclude that both the inhibitors prolong nucleation of amyloid aggregation through binding to region of the protein which is known to form the core of the protein fibril, but once the nucleus is formed the rate of elongation is not affected by the inhibitors. This work would provide insight into the mechanism of aggregation inhibition by these potential drug molecules.     

Irreversible lysozyme inactivation and aggregation induced by stirring: kinetic study and aggregates characterisation

Stirring strongly enhanced irreversible inactivation and aggregation of lysozyme being studied as a model enzyme. From 0 to 740 rpm (equivalent to impeller tip speeds from 0 to 0.77 m s^–1), the inactivation kinetic constant was proportional to the power imparted by the impeller. Collisions between inactive and native molecules induced inactivation of the latter and led to lysozyme aggregation. These fractal aggregates of lysozyme were made of monomers, dimers and trimers.     

The role of cysteine oxidation in the thermal inactivation of T4 lysozyme

Wild-type T4 lysozyme contains unpaired cysteine residues at positions 54 and 97. To investigate the role these residues play in the thermal inactivation of the wild-type, we constructed a double mutant with these cysteines replaced with valine and serine. This molecule, T4 lysozyme (C54V/C97S), is more stable than the wild-type to inactivation at 70 degrees C at pH 6.5 and 8.0. Guanidine hydrochloride reactivation experiments and SDS-PAGE on the inactivated products show that the wild-type is susceptible to varying degrees of oxidative damage, depending on buffer conditions, while the cysteine-minus mutant inactivates only by other pathways. The products of thermal, oxidative inactivation of the wild-type are disulfide-linked oligomers. The dependence of inactivation rate on temperature suggests that the formation of these aggregates depends on prior thermal unfolding of the T4 lysozyme molecule.     

Lysozyme inactivation and aggregation in stirred-reactor

Mechanisms of enzyme inactivation and aggregation are still poorly understood. In this work, we are considering the characterisation of both inactivation and aggregation in stirred tank reactor, with lysozyme as the model enzyme.

The inactivation kinetics are first order. For stirring speeds in the range of 0–700 rpm, the kinetic constant is found to be proportional to the power brought by the impeller. It suggests that inactivation depends on collisions between enzyme molecules. Efficient collisions between native and inactive molecules induce native molecules to turn into inactive molecules and lead to lysozyme aggregation.

During inactivation, enzymes are found to aggregate as shown by light scattering measurements. The structure of aggregates was studied on samples treated for chemical denaturation and reduction. The aggregates are supramolecular edifices, mainly made up of inactivated enzymes linked by weak forces. But aggregates are also made up of dimers and trimers of lysozyme, linked by disulfide bridges. Dimers and trimers are 18% and 5%, respectively, of the total amount of lysozyme aggregates.

Whatever the stage of aggregate formation and the initial enzyme concentration are, these aggregates are irreversibly inactivated. Enzyme activity is definitely lost even if stirring is stopped and/or temperature decreased.

This study points out the importance of hydrodynamics in bioreactors and highlights the nature of the aggregates resulting from the interactions between native and inactive enzymes.     

Denaturation and aggregation of lysozyme in water-ethanol solution

We have applied rheological methods for the analysis of ethanol-lysozyme interaction during the process of denaturation and aggregation of the protein. At low concentration of ethanol a destruction of the hydration shell of lysozyme is observed. With the increase in the ethanol concentration a structural transformation takes place. It leads to the formation of a protein aggregate with an elongated structure. The rheological characteristics of lysozyme-water-ethanol solution changes from Newtonian to pseudoplastic.     

Small molecule inhibitors of lysozyme amyloid aggregation

Protein amyloid aggregation is associated with a number of important human pathologies, but the precise mechanisms underlying the toxicity of amyloid aggregates are still incompletely understood. In this context, drugs capable of blocking or interfering with the aggregation of amyloidogenic proteins should be considered in strategies aimed at the development of novel therapeutic agents. Human lysozyme variants have been shown to form massive amyloid deposits in the livers and kidneys of individuals affected by hereditary systemic amyloidosis. Currently, there are no clinical treatments available to prevent or reverse formation of such amyloid deposits. We have recently described a number of di- and trisubstituted aromatic compounds that block the formation of soluble oligomers and amyloid fibrils of the β-amyloid peptide (Aβ) and protect hippocampal neurons in culture from Aβ-induced toxicity. Here, we show that some of those compounds inhibit the formation and disrupt preformed amyloid fibrils from both human and hen egg white lysozyme. These results suggest that these small molecule compounds may serve as prototypes for the development of drugs for the prevention or treatment of different types of amyloidoses.     

Mechanism of lysozyme inactivation and degradation by iron.

The site-specific lysozyme damage by iron and by iron-catalysed oxygen radicals was investigated. A solution of purified lysozyme was inactivated by Fe(II) at pH 7.4 in phosphate buffer, as tested on cleavage of Micrococcus lysodeikticus cells; this inactivation was time- and iron concentration-dependent and was associated with a loss of tryptophan fluorescence. In addition, it was reversible at pH 4, as demonstrated by lysozyme reactivation and by the intensity of the 14.4-kD-band on SDS-PAGE. Desferal (1 mM) and Detapac (1 mM) added before iron, prevented lysozyme inactivation, while catalase (100 micrograms/ml), superoxide dismutase (100 micrograms/ml) and bovine serum albumin (100 micrograms/ml) gave about 30 to 40% protection by competing with lysozyme for iron binding. The denaturing effect of iron on lysozyme was studied in the presence of H2O2 (1 mM) and ascorbate (1 mM); under these conditions the enzyme underwent partly irreversible inactivation and degradation different to that produced by gamma radiolysis-generated .OH. Catalase almost fully protected lysozyme; in contrast, mannitol (10 mM), benzoate (10 mM), and formate (10 mM) provided no protection because of their inability to access the site at which damaging species are generated. In this system, radical species were formed in a site-specific manner, and they reacted essentially with lysozyme at the site of their formation, causing inactivation and degradation differently than the hydroxyl radical.     

Amino acid sequence of pigeon egg-white lysozyme

The amino acid sequence of pigeon egg-white lysozyme has been determined. The protein molecule contains a single polypeptide chain of 127 amino acid residues and exhibits only about 60% homology when compared to hen egg-white lysozyme.     

The osmoprotectants glycine and its methyl derivatives prevent the thermal inactivation of protective antigen of Bacillus anthracis

Protective antigen (PA) is the main immunogenic constituent of all vaccines against anthrax. It is known to lose its biological activity even at 37 degrees C. Its thermolabile nature has, thus, remained a cause of concern as even transient exposure of the vaccine to higher temperature could compromise its efficacy. Various types of cosolvent excipients have been used to stabilize a number of proteins with variable success. However, no comprehensive and systematic study to stabilize anthrax PA molecule using this approach has ever been undertaken. We have carried out a systematic study on the effect of osmoprotectants like glycine and its methyl derivatives, sarcosine, dimethylglycine, and betaine, on the thermostability of PA. The thermal stability of PA was found to be highly sensitive to pH with maxima at pH 7.9. All the cosolvent additives used were able to enhance the thermal stability of PA as inferred from an increase in T(1/2) values, the temperature at which 50% activity was retained during short-term incubation. Glycine was found to be the best stabilizer, while the ability of its methyl derivatives to stabilize PA decreased with an increase in the number of substituted methyl groups suggesting perturbation of hydrophobic interactions. On extended incubation at 40 degrees C the half-life of PA thermal inactivation increased more than four times in the presence of glycine. Thus, glycine could be used as an effective stabilizer to enhance the shelf life of recombinant vaccine against anthrax.     

Different mechanisms of action of poly(ethylene glycol) and arginine on thermal inactivation of lysozyme and ribonuclease A

Proteins tend to undergo irreversible inactivation through several chemical modifications, which is a serious problem in various fields. We have recently found that arginine (Arg) suppresses heat‐induced deamidation and β‐elimination, resulting in the suppression of thermal inactivation of hen egg white lysozyme and bovine pancreas ribonuclease A. Here, we report that poly(ethylene glycol) (PEG) with molecular weight 1,000 acts as a thermoinactivation suppressor for both proteins, especially at higher protein concentrations, while Arg was not effective at higher protein concentrations. This difference suggests that PEG, but not Arg, effectively inhibited intermolecular disulfide exchange among thermally denatured proteins. Investigation of the effects of various polymers including PEG with different molecular weight, poly(vinylpyrolidone) (PVP), and poly(vinyl alchol) on thermoinactivation of proteins, circular dichroism, solution viscosity, and the solubility of reduced and S‐carboxy‐methylated lysozyme indicated that amphiphilic PEG and PVP inhibit intermolecular collision of thermally denatured proteins by preferential interaction with thermally denatured proteins, resulting in the inhibition of intermolecular disulfide exchange. These findings regarding the different mechanisms of the effects of amphiphilic polymers––PEG and PVP––and Arg would expand the capabilities of methods to improve the chemical stability of proteins in solution. Biotechnol. Bioeng. 2012; 109: 2543–2552. © 2012 Wiley Periodicals, Inc.     

Isolation and characterization of rhodanese intermediates during thermal inactivation and their implications for the mechanism of protein aggregation

The initial steps of heat-induced inactivation and aggregation of the enzyme rhodanese have been studied and found to involve the early formation of modified but catalytically active conformations. These intermediates readily form active dimers or small oligomers, as evident from there being only a small increase in light scattering and an increase in fluorescence energy homotransfer from rhodanese labeled with fluorescein. These species are probably not the domain-unfolded form, as they show activity and increased protection of hydrophobic surfaces. Cross-linking with glutaraldehyde and fractionation by gel filtration show the predominant formation of dimer during heat incubation. Comparison between the rates of aggregate formation at 50 degrees C after preincubation at 25 or 40 degrees C gives evidence of product-precursor relationships, and it shows that these dimeric or small oligomeric species are the basis of the irreversible aggregation. The thermally induced species is recognized by and binds to the chaperonin GroEL. The unfoldase activity of GroEL subsequently unfolds rhodanese to produce an inactive conformation and forms a stable, reactivable complex. The release of 80% active rhodanese upon addition of GroES and ATP indicates that the thermal incubation induces an alteration in conformation, rather than any covalent modification, which would lead to formation of irreversibly inactive species. Once oligomeric species are formed from the intermediates, GroEL cannot recognize them. Based on these observations, a model is proposed for rhodanese aggregation that can explain the paradoxical effect in which rhodanese aggregation is reduced at higher protein concentration.     

氨基酸生产和海洋生物的氨基酸资源开发

氨基酸在医药、食品、饲料等领域有着极为重要和广泛的用途,世界上氨基酸总需求量以５～１０％递增,市场竞争十分激烈。生物资源提取、化学合成、生物合成和综合法是生产氨基酸的４种技术,目前的发展趋势为生物合成和综合法,特别是将现代生物工程技术应用于氨基酸生产。另外,氨基酸生产领域另一个新的倾向是海洋生物氨基酸资源的开发和应用,尤其是海洋生物所产生的特殊氨基酸、肽及其衍生物的开发,同时,综合利用海产品加工后的废弃物来生产氨基酸也受到重视。