The hydrodynamics of a Graesser ("raining bucket") contactor with a reverse micellar phase

A variety of contactor types have been assessed for the liquid-liquid extraction of proteins using reversed micelles; however, many of these contactors suffer from drawbacks such as emulsion formation and poor mass transfer performance. In this study, a small (1.25 L) Graesser "raining bucket" contactor was assessed for use with this system since it has the potential to ameliorate many of these problems. The aim of the work was to evaluate the hydrodynamics of the contactor in order to use this information for future work on mass transfer performance. Hydrodynamic characteristics such as the axial mixing coefficient were determined by residence time distribution studies using a tracer injection method. The effect of rotor speed and flow rate of each phase on axial mixing was investigated, and as a result of its unusual structure, i.e., falling/rising sheet, the interfacial mass transfer area in the Graesser was determined by image analysis. It was found that rotor speed had more influence on the axial mixing coefficient in the aqueous phase than in the reverse micellar phase. The axial mixing coefficient in each phase increased by increasing the flow rate of the same phase. The images obtained in a dropping cell showed that under the conditions of this study (3 rpm, 22 degrees C), the bucket pours one phase through the other in the form of a curtain or sheet. A new image technique was developed to determine the interfacial area of both phases, and it was found that the specific area was 8.6 m(2)/m(3), which was higher than in a spray column but considerably lower than in a RDC or a Graesser run at high rotational speed (50 rpm) without the addition of a surfactant.     

The effect of demulsifiers on lysozyme extraction from hen egg white using reverse micelles

The liquid–liquid extraction of protein from buffered aqueous phases using reverse micelles (RM) has been extensively researched from a fundamental point of view. However, very little effort has been expended at scaling up this process for the extraction of real fermentation broth. When real broths are used with reverse micellar phases there are major problems with emulsion formation. In this study the effect of a variety of demulsifiers on lysozyme extraction was evaluated in terms of their influence on the separating properties of the emulsion, water content (W _o ), and, extraction yield and kinetics from both buffer and hen egg white. In addition, the use of a low shear contactor (a Graesser or `raining bucket') was assessed in terms of its suitability as a RM contactor. It was found that most of the demulsifiers reduced the settling time of the emulsion, and enhanced the yield and kinetics of lysozyme extraction from hen egg white. It was hypothesised that this was due to the demulsifier displacing the lysozyme from the interface and preventing the protein unfolding and precipitating. This effect was found to depend on both the generic type of demulsifier, and its concentration.     

高速逆流双水相色谱法纯化卵白蛋白

生物大分子的液_固色谱纯化过程中固相载体会产生产物吸附、变性等不良影响。高速逆流色谱无需固相载体 ,且具有高分便率和高回收率的优点 ,其中有机相 水相体系在分离天然产物中应用广泛 ,而应用双水相体系分离生物大分子尚处于研究阶段。双水相高速逆流色谱体系的建立与仪器设备及操作工艺条件密切相关 ,因此利用多分离柱高速逆流色谱仪 ,研究了PEG1000-无机盐双水相体系对标准蛋白质混合物以及卵白蛋白的分离。pH值和PEG浓度对不同种类蛋白质的分配系数影响不同 ,实验发现在pH9.2的150% (W/W)PEG1000 170% (W/W)磷酸钾盐体系中 ,细胞色素C、溶菌酶和肌红蛋白的分配系数差异较大 ,且分布合理 ,因而采用该体系在 0 8mL min流速 ,85 0r min转速的条件下 ,成功分离了细胞色素C、溶菌酶和肌红蛋白的混合物。实验也发现在pH9 2的 16 0 % (W/W)PEG10 0 0 17 0 % (W/W)磷酸钾盐体系中 ,鸡蛋清样品中的主要蛋白质成分:卵转铁蛋白、卵白蛋白和溶菌酶的分配系数差异最大 ,因而采用该体系在 1 8mL min流速、85 0r mi转速的条件下,200min内从鸡蛋清样品中成功分离卵白蛋白,其电泳纯度为100%,收率为95%.     

Refolding of denatured lysozyme by water-in-oil microemulsions of sucrose fatty acid esters

Water-in-oil (w/o) microemulsion of sucrose fatty acid ester was used to renature denatured hen egg white lysozyme without aggregation. After lysozyme was denatured in 5 M guanidine hydrochloride for 24 h, the resultant denatured lysozyme was held in the microemulsion, overnight at 25°C. Renatured lysozyme was transferred from the microemulsion phase to the recovery aqueous phase by conventional liquid-liquid extraction. The enzymatic activity of the recovered lysozyme was 93%.     

Effects of Lysozyme and Inorganic Anions on the Morphology of Streptococcus mutans BHT: Electron Microscopic Examination

The effects of hen egg white lysozyme and the inorganic salt sodium thiocyanate on the integrity of Streptococcus mutans BHT were studied by transmission electron microscopy. Both control cells and cells exposed to NaSCN possessed thick outer cell walls and densely staining inner cell walls juxtaposed to the plasma membranes. In the presence of NaSCN, however, the S. mutans BHT nucleoid was coagulated into thick electron-dense filaments. Exposure of S. mutans BHT to 150 μg of hen egg white lysozyme per ml resulted in the progressive destruction of both the cell walls and the plasma membranes. The enzyme appeared to affect the region of the cell wall septum, and exposure to 150 μg of hen egg white lysozyme per ml for as short a time as 10 min resulted in visible morphological cell wall alterations. At 30 min, ultrastructural observations revealed that the majority of the cells were in the process of expelling a portion of their cytoplasmic contents from the septal and other regions of the cells at the time of fixation. After 3 h of incubation in the presence of this high lysozyme concentration, gelled protoplasmic masses, which were free from the cells, were evident. In addition, extensive damage to the outer and inner cell walls and to the plasma membranes was apparent, although the cells maintained their shape. On some areas of the cell surface, the outer cell wall and plasma membrane were completely absent, whereas at other locations the outer cell wall was either split away from the inner cell wall and plasma membrane or distended from an area free of inner cell wall and plasma membrane. Upon addition of NaSCN to the hen egg white lysozyme-treated cells, both the gelled protoplasmic masses and the damaged cells exhibited an exploded appearance and existed as membrane ghosts, cell wall fragments, or dense aggregates of cytoplasmic components. The effects of a low lysozyme concentration (22.5 μg/ml) on S. mutans morphology were less pronounced at short incubation times (i.e., 10 and 30 min) than those that were observed with a high enzyme concentration; however, breaks in the cell walls and dissolution of the plasma membranes with resulting cell lysis were visible after a prolonged (3-h) incubation and after subsequent addition of NaSCN.     

Pilot-scale separation of lysozyme from hen egg white by integrating aqueous two-phase partitioning and membrane separation processes

A novel, cost-effective method of lysozyme separation from hen egg white was studied. This method integrates aqueous two-phase partitioning in the system EO₅₀PO₅₀/phosphates with membrane separation processes. The experiments were carried out in a pilot-scale on crude hen egg white.Initially, by forming an aqueous two-phase system, lysozyme was selectively extracted to the upper, polymer-rich phase while the other egg white proteins partitioned to the lower, phosphate-rich phase. Then, in order to recover lysozyme, thermoseparation of polymer-rich phase was applied. A novel approach for the simultaneous thermoseparation of the polymer-rich phase as well as for the recovery of the lysozyme was proposed, using a cross-flow microfiltration. Additionally, recovery of proteins by ultrafiltration from lower, phosphate-rich phase was also investigated.Lysozyme could be obtained after the thermal phase separation by means of microfiltration at a total recovery over the extraction steps of 47.5 and the purification factor of 10.5. The specific activity of lysozyme preparations was 34 188 U/mg of protein. Using cross-flow membrane techniques, it was found that the recovery of the polymer by microfiltration from the top phase was 83.9.     

Enhancement in the lysozyme activity of the hen egg white foam matrix by cross-linking in the presence of N-acetyl glucosamine.

Lysozyme naturally present in raw hen egg white was immobilized by cross-linking the egg white foam with glutaraldehyde. Inclusion of N-acetyl glucosamine, a competitive inhibitor of lysozyme, was found to enhance the yield of lysozyme activity by fivefold.     

Linear correlation between thermal stability and folding kinetics of lysozyme

We have studied the refolding and thermal denaturation of hen egg white lysozyme in a wide range of pH values (from 1.5 to 9.4) using stopped-flow circular dichroism (CD) and differential scanning calorimetry (DSC). A linear correlation was found between the thermal denaturation temperature (T(m)) and the logarithm of the refolding rate of the slow folding phase of hen egg white lysozyme (lnk(2)).     

Activity of crystalline turkey egg white lysozyme.

Hexagonal crystals of turkey egg white lysozyme have been examined for activity in order to evaluate their potential for use in time-resolved X-ray crystallographic experiments. Substrates used in this study were hexa-N-acetylglucosamine (hexa-GlcNAc) and a modified analogue of hexa-GlcNAc where the terminal sugar ring was opened by reduction with tritiated sodium borohydride. This gave a labeled beta-N-acetylglucosaminitol unit at the sixth position of the sugar chain and allowed easy quantitation of enzymatic cleavage on TLC plates. Using these substrates, it has been shown that turkey egg white lysozyme is enzymatically active in the crystal. Enzyme dispersed in the buffer surrounding the crystal does not show detectable activity under conditions relevant to an X-ray experiment. Unmodified hexa-GlcNAc is hydrolyzed into di-, tri-, and tetrasaccharides in the crystal. This cleavage pattern is different from that obtained with hen egg white lysozyme in solution and likely causes of the differences are discussed. The reduced radiolabeled oligosaccharide has a unique cleavage pattern with trisaccharides as the products. The specific activity of the enzyme with the radiolabelled analogue was 9.8 (+/- 1.0) x 10(-7) mmol/min/mg protein at 22 degrees C in the crystal.     

Cellular basis of genetic nonresponsiveness to egg white lysozyme

Utilizing radiation chimeras, we have investigated the cellular level at which the low immunological responsiveness to egg white lysozyme C57Bl/10 mice is expressed. Both NR----F1 (responder) and F1----NR combinations were assessed. The results demonstrate that C57Bl/10 bone marrow can give rise to hen egg white lysozyme responsive cells, but this response requires that the antigen be presented by cells derived from high responder animals.     

Initial protein concentration and residual denaturant concentration strongly affect the batch refolding of hen egg white lysozyme

The effects of several variables on the refolding of hen egg white lysozyme have been studied. Lysozyme was denatured in both urea, and guanidine hydrochloride (GuHCl), and batch refolded by dilution (100 to 1000 fold) into 0.1M Tris-HCl, pH 8.2, 1 mM EDTA, 3 mM reduced glutathione and 0.3 mM oxidised glutathione. Refolding was found to be sensitive to temperature, with the highest refolding yield obtained at 50°C. The apparent activation energy for lysozyme refolding was found to be 56 kJ/mol. Refolding by dilution results in low concentrations of both denaturant and reducing agent species. It was found that the residual concentrations obtained during dilution (100-fold dilution: [GuHCl]=0.06 mM, [DTT]=0.15 mM) were significant and could inhibit lysozyme refolding. This study has also shown that the initial protein concentration (1–10 mg/mL) that is refolded is an important parameter. In the presence of residual GuHCl and DTT, higher refolding yields were obtained when starting from higher initial lysozyme concentrations. This trend was reversed when residual denaturant components were removed from the refolding buffer.     

General Properties of Endo-N-acetylmuramidase of Bacillus subtilis YT-25

The enzymatic behaviour, amino acid composition and some physical properties of a new endo-N-acetylmuramidase (B-enzyme) of Bacillus subtilis YT–25 were determined and compared with hen’s egg white lysozyme. The molecular weight was estimated to be about 13000 by the sedimentation equilibrium method. The isoelectric point was pH 9.8. The amino acid composition indicates that the enzyme is rich in basic amino acids, especially lysin. Maximal activity on the lysis of cell walls of M. lysodeikticus occurred at pH 6.2. The enzyme was stable at pH 3.5 ~ 6.0. The specific activity for the lysis of cell walls of M. lysodeikticus was less than fourth part of that of hen’s egg white lysozyme. Digest of cell walls of M. lysodeikticus with B-enzyme consisted greater numbers of high molecular products than digest with egg white lysozyme. Substrate specificity of B-enzyme seemed to be different from that of egg white lysozyme.     

Amyloid protofilament formation of hen egg lysozyme in highly concentrated ethanol solution

Mutant human lysozymes (Ile56Thr & Asp67His) have been reported to form amyloid deposits in the viscera. From the standpoint of understanding the mechanism of amyloid formation, we searched for conditions of amyloid formation in vitro using hen egg lysozyme, which has been extensively studied from a physicochemical standpoint. It was found that the circular dichroism spectra in the far-ultraviolet region of the hen egg lysozyme changed to those characteristic of a beta-structure from the native alpha-helix rich spectrum in 90% ethanol solution. When the concentration of protein was increased to 10 mg/mL, the protein solution formed a gel in the presence of 90% ethanol, and precipitated on further addition of 10 mM NaCl. The precipitates were examined by electron microscopy, their ability to bind Congo red, and X-ray diffraction to determine whether amyloid fibrils were formed in the precipitates. Electron micrographs displayed unbranched protofilament with a diameter of approximately 70 A. The peak point of the difference spectrum for the Congo red binding assay was 541 nm, which is characteristic of amyloid fibrils. The X-ray diffraction pattern showed a sharp and intense diffraction ring at 4.7 A, a reflection that arises from the interstrand spacing in beta-sheets. These results indicate that the precipitates of hen egg lysozyme are amyloid protofilament, and that the amyloid protofilament formation of hen egg lysozyme closely follows upon the destruction of the helical and tertiary structures.     

Production of bioactive lysophosphatidic acid by lysophospholipase D in hen egg white

Lysophosphatidic acid (LPA), a lysophospholipid mediator, is produced extracellularly by lysophospholipase D (lysoPLD) secreted in several animal body fluids including blood plasma. Previously, we reported that hen egg white contains polyunsaturated fatty acid-rich LPA. In this study, we examined whether lysoPLD is involved in the production of LPA in hen egg white. LysoPLD activity was measured by determining LPA and choline by mass spectrometric and enzyme-linked fluorometric analyses, respectively. LysoPLD increased with increased dilution of egg white, indicating that one or more components of egg white strongly inhibit its lysoPLD activity. This dilution-dependent increase in the lysoPLD activity was masked by co-incubation of the egg white with lysozyme, a major protein in hen egg white. Furthermore, addition of Zn(2+), Mn(2+), Ni(2+), or Co(2+) to diluted egg white altered preference patterns of lysoPLD toward choline-containing substrates. In particular, the egg white lysoPLD activity was greatly increased when Co(2+) was added. The cation-requirement of lysoPLD activity in hen egg white resembled that of plasma autotaxin (ATX)/lysoPLD. Western blot analysis revealed that egg white contained a protein that was immunostained with anti-ATX antibody. These results suggested that LPA in hen egg white is produced from lysophospholipids, especially LPC, by the action of ATX/lysoPLD, possibly originating from hen oviduct fluid.     

Detection of chitinase activity after polyacrylamide gel electrophoresis

Commercial Streptomyces griseus and Serratia marcescens chitinases and purified wheat germ W1A and hen egg white lysozymes were subjected to polyacrylamide gel electrophoresis under native conditions at pH 4.3. After electrophoresis, an overlay gel containing 0.01% (W/V) glycol chitin as substrate was incubated in contact with the separation gel. Lytic zones were revealed by uv illumination with a transilluminator after staining for 5 min with 0.01% (W/V) Calcofluor white M2R. As low as 500 ng of purified hen egg lysozyme could be detected after 1 h incubation at 37 degrees C. One band was observed with W1A lysozyme and several bands with the commercial microbial chitinases. The same system was also used with native polyacrylamide gel electrophoresis at pH 8.9. Several bands were detected with the microbial chitinases. The same enzymes were also subjected to denaturing polyacrylamide gel electrophoresis in gradient gels containing 0.01% (W/V) glycol chitin. After electrophoresis, enzymes were renatured in buffered 1% (V/V) purified Triton X-100. Lytic zones were revealed by uv after staining with Calcofluor white M2R as for native gels. The molecular weights of chitinolytic enzymes could thus be directly estimated. In denaturing gels, as low as 10 ng of purified hen egg white lysozyme could be detected after 2 h incubation at 37 degrees C. Estimated molecular weights of St. griseus and Se. marcescens were between 24,000 and 72,000 and between 40,500 and 73,000, respectively. Some microbial chitinases were only resistant to denaturation with sodium dodecyl sulfate while others were resistant to sodium dodecyl sulfate and beta-mercaptoethanol.     

Evaluation of the performance of protein separation in figure-8 centrifugal counter-current chromatography

The performance of protein separation using the figure-8 column configuration in centrifugal counter-current chromatography was investigated under various flow rates and revolution speeds. The separation was performed with a two-phase solvent system composed of polyethylene glycol 1000/potassium phosphate each at 12.5% (w/w) in water and with lysozyme and myoglobin as test samples. In order to improve tracing of the elution curve, a hollow fiber membrane dialyzer was inserted at the inlet of the UV detector. The results showed that the retention of stationary phase (Sf) and resolution (Rs) increased with decreased flow rate and increased revolution speed. The highest Rs of approximately 1 was obtained at a flow rate of 0.01 mL/min under a revolution speed of 1200 rpm with a 3.4 mL capacity column.     

Three-dimensional structure of goose-type lysozyme from the egg white of the Australian black swan, Cygnus atratus

The egg white of C. atratus contains two forms of lysozyme, a 'chick-type' which is similar to that found in the egg white of the domestic hen, and a 'goose-type' similar to that found in the egg white of the Embden goose. The molecular structure of the goose-type lysozyme has been determined at a resolution of a 2.8 A by X-ray crystallographic analysis. The structure consists of two domains linked by a long stretch of alpha-helix. In all, there are seven helical segments in the structure. While there is no amino acid sequence homology with either hen egg-white or bacteriophage T4 lysozymes, there are portions of the structure where the folding of the main chain is similar to that found in portions of either hen egg-white lysozyme or T4 lysozyme or both. In particular, there is a consistency of structure in the arrangement of acid groups in the catalytic site. G-o plots calculated for this structure and for the bacteriophage T4 lysozyme structure show that both have similar 'modules' of structure with boundaries occurring at structurally equivalent positions. Three of the common boundaries are equivalent structurally to three of the four module boundaries observed in G-o plots of hen egg-white lysozyme. The variation in the position of the remaining boundary may be related to differences in substrate binding.     

The calcium-binding property of equine lysozyme

It was found that equine lysozyme binds one Ca2+. It was eluted with equimolar Ca2+ from a Bio-Gel P-4 column. Human lysozyme did not behave similarly. Equine lysozyme is concluded to be a calcium metallo-protein like alpha-lactalbumin, which is a homologue of hen egg white lysozyme.     

Redesign of the substrate-binding site of hen egg white lysozyme based on the molecular evolution of C-type lysozymes.

On the basis of the molecular evolution of hen egg white, human, and turkey lysozymes, three replacements (Trp62 with Tyr, Asn37 with Gly, and Asp101 with Gly) were introduced into the active-site cleft of hen egg white lysozyme by site-directed mutagenesis. The replacement of Trp62 with Tyr led to enhanced bacteriolytic activity at pH 6.2 and a lower binding constant for chitotriose. The fluorescence spectral properties of this mutant hen egg white lysozyme were found to be similar to those of human lysozyme, which contains Tyr at position 62. The replacement of Asn37 with Gly had little effect on the enzymatic activity and binding constant for chitotriose. However, the combination of Asn37----Gly (N37G) replacement with Asp101----Gly (D101G) and Trp62----Tyr (W62Y) conversions enhanced bacteriolytic activity much more than each single mutation and restored hydrolytic activity toward glycol chitin. Consequently, the mutant lysozyme containing triple replacements (N37G, W62Y, and D101G) showed about 3-fold higher bacteriolytic activity than the wild-type hen lysozyme at pH 6.2, which is close to the optimum pH of the wild-type enzyme.     

A kinetic study of the competition between renaturation and aggregation during the refolding of denatured-reduced egg white lysozyme

The recovery of proteins following denaturation is optimal at low protein concentrations. The decrease in yield at high concentrations has been explained by the kinetic competition of folding and "wrong aggregation". In the present study, the renaturation-reoxidation of hen and turkey egg white lysozyme was used as a model system to analyze the committed step in aggregate formation. The yield of renatured protein for both enzymes decreased with increasing concentration in the folding process. In addition, the yield decreased with increasing concentrations of the enzyme in the denatured state (i.e., prior to its dilution in the renaturation buffer). The kinetics of renaturation of turkey lysozyme were shown to be very similar to those of hen lysozyme, with a half-time of about 4.5 min at 20 degrees C. The rate of formation of molecular species that lead to formation of aggregates (and therefore fail to renature) was shown to be rapid. Most of the reaction occurred in less than 5 s after the transfer to renaturation buffer, and after 1 min, the reaction was essentially completed. Yet, by observing the effects of the delayed addition of denatured hen lysozyme to refolding turkey lysozyme, it was shown that folding intermediates become resistant to aggregation only much more slowly, with kinetics indistinguishable from those observed for the appearance of native molecules. The interactions leading to the formation of aggregates were nonspecific and do not involve disulfide bonds. These observations are discussed in terms of possible kinetic and structural aspects of the folding pathway.