Initial Protein Concentration Research Articles

Facile synthesis of copper(II)-decorated magnetic particles for selective removal of hemoglobin from blood samples

In this report, the Cu2+-immobilized magnetic particles were prepared by a facile route and they were used as adsorbents for removal of high abundance of hemoglobin in blood based on immobilized metal affinity chromatography. Ethylenediaminetetraacetic acid modified magnetic particles (EDTA-Fe3O4) were first synthesized through a one-pot solvothermal method and then charged with copper ions. The as-prepared Cu2+-EDTA-Fe3O4 particles were characterized by Fourier transform infrared spectrometry, scanning electron microscopy, transmission electron microscopy, energy dispersive X-ray spectroscopy, vibrating sample magnetometry and zeta potential. Factors affecting the adsorption of bovine hemoglobin on Cu2+-EDTA-Fe3O4 particles (including contact time, solution pH, ionic strength and initial concentration of protein) were investigated. The adsorption process followed a pseudo-second-order kinetic model and the adsorption equilibrium could be achieved in 60min. The adsorption isotherm data could be well described by a Langmuir model and the maximum adsorption capacity was 1250mgg−1. The as-prepared particles showed high efficiency and excellent selectivity for removal of hemoglobin from bovine and human blood. The removal process integrated the selectivity of immobilized metal affinity chromatography and the convenience of magnetic separation. The results demonstrated that Cu2+-EDTA-Fe3O4 particles had potential application in removal of abundant histidine-rich proteins in biomedical diagnosis analysis.

On the Relationship of Protein and mRNA Dynamics in Vertebrate Embryonic Development

A biochemical explanation of development from the fertilized egg to the adult requires an understanding of the proteins and RNAs expressed over time during embryogenesis. We present a comprehensive characterization of protein and mRNA dynamics across early development in Xenopus. Surprisingly, we find that most protein levels change little and duplicated genes are expressed similarly. While the correlation between protein and mRNA levels is poor, a mass action kinetics model parameterized using protein synthesis and degradation rates regresses protein dynamics to RNA dynamics, corrected for initial protein concentration. This study provides detailed data for absolute levels of ∼10,000 proteins and ∼28,000 transcripts via a convenient web portal, a rich resource for developmental biologists. It underscores the lasting impact of maternal dowry, finds surprisingly few cases where degradation alone drives a change in protein level, and highlights the importance of transcription in shaping the dynamics of the embryonic proteome.

Artificial neuronal network modeling of the enzymatic hydrolysis of horse mackerel protein using protease mixtures

In enzymatic reactions, the modeling of the degree of hydrolysis (DH) is desirable given its influence on many functional and biological activities. Empirical approaches are preferred to phenomenological ones to model the complexity of enzymatic reactions. Artificial Neuronal Networks (ANN) are able to process large sets of data where no linear relationships between them are expected. In this work, a feedforward ANN, comprising 10 neurons in the hidden layer, was successfully employed to model the DH as a function of the initial concentration of horse mackerel protein (tested at 2.5g/L, 5g/L and 7.5g/L), the reaction temperature (40°C, 47.5°C and 55°C), the time of reaction (up to 4h) and the percentage of subtilisin in the enzyme mixture (0%, 25%, 50%, 75% and 100%). The resulting ANN model was optimized by an evolutionary algorithm, obtaining a maximum (DH 17.1%) at 2.54g/L, 40°C, 4h and an enzyme mixture comprising 38.3% of subtilisin and the rest of trypsin. The combination of trypsin and subtilisin led to higher DH than the sole use of subtilisin (DH 15.5% at 52°C). Furthermore, the former optimum was attained at lower reaction temperature, which reduces both the operational costs and the nutritional losses.

Effects of water management practices on residue decomposition and degradation of Cry1Ac protein from crop-wild Bt rice hybrids and parental lines during winter fallow season

Rice is the staple diet of over half of the world's population and Bacillus thuringiensis (Bt) rice expressing insecticidal Cry proteins is ready for deployment. An assessment of the potential impact of Bt rice on the soil ecosystem under varied field management practices is urgently required. We used litter bags to assess the residue (leaves, stems and roots) decomposition dynamics of two transgenic rice lines (Kefeng6 and Kefeng8) containing stacked genes from Bt and sck (a modified CpTI gene encoding a cowpea trypsin inhibitor) (Bt/CpTI), a non-transgenic rice near-isoline (Minghui86), wild rice (Oryza rufipogon) and crop-wild Bt rice hybrid under contrasting conditions (drainage or continuous flooding) in the field. No significant difference was detected in the remaining mass, total C and total N among cultivars under aerobic conditions, whereas significant differences in the remaining mass and total C were detected between Kefeng6 and Kefeng8 and Minghui86 under the flooded condition. A higher decomposition rate constant (km) was measured under the flooded condition compared with the aerobic condition for leaf residues, whereas the reverse was observed for root residues. The enzyme-linked immunosorbent assay (ELISA), which was used to monitor the changes in the Cry1Ac protein in Bt rice residues, indicated that (1) the degradation of the Cry1Ac protein under both conditions best fit first-order kinetics, and the predicted DT50 (50% degradation time) of the Cry1Ac protein ranged from 3.6 to 32.5 days; (2) the Cry1Ac protein in the residue degraded relatively faster under aerobic conditions; and (3) by the end of the study (~154 days), the protein was present at a low concentration in the remaining residues under both conditions. The degradation rate constant was negatively correlated with the initial carbon content and positively correlated with the initial Cry1Ac protein concentration, but it was only correlated with the mass decomposition rate constants under the flooded condition. No Cry1Ac protein was detected in the soils surrounding the buried residue. Our results did not reveal any evidence that the stacked genes (Bt/CpTI) or the presence of the Cry1Ac protein influenced the decomposition dynamics of the rice residues. Furthermore, our results suggested that field drainage after residue incorporation would promote Cry1Ac protein degradation.

Valorization of tomato waste proteins through production of antioxidant and antibacterial hydrolysates by proteolytic Bacillus subtilis: optimization of fermentation conditions.

In this study, protein-rich waste of tomato processing industries was fermented by Bacillus subtilis A14h to produce hydrolysates with antioxidant and antibacterial activities. The effects of different levels of initial pH, incubation temperature, fermentation time, protein concentration and inoculum size on proteolytic activity, release of amino acids and peptides, antioxidant and antibacterial activities of hydrolysates were evaluated and optimized by using response surface methodology (RSM). Results showed that all the evaluated variables significantly influenced the hydrolysis and bioactivities of hydrolysates in polynomial models. Hydrolysates showed remarkable 2, 2'-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity (up to 70%), ferric ion reducing power, and inhibitory activity against B. cereus (up to 69.8%) and E. coli (up to 29.8%). Overall, good correlation between the concentration of amino acids and peptides, and antioxidant as well as antibacterial activities (in particular for B. cereus inhibition activity) was observed. Finally, optimum conditions for fermentative conversion of tomato waste proteins to antioxidant and antibacterial hydrolysates were established. Results of this study showed that tomato waste protein can be valorized to produce antioxidant and antibacterial hydrolysates in a fermentative system using B. subtilis A14h.

Immobilization, Regiospecificity Characterization and Application of Aspergillus oryzae Lipase in the Enzymatic Synthesis of the Structured Lipid 1,3-Dioleoyl-2-Palmitoylglycerol.

The enzymatic synthesis of 1,3-dioleoyl-2-palmitoylglycerol (OPO), one of the main components of human milk fats, has been hindered by the relatively high cost of sn-1,3-specific lipases and the deficiency in biocatalyst stability. The sn-1,3-specific lipase from Aspergillus oryzae (AOL) is highly and efficiently immobilized with the polystyrene-based hydrophobic resin D3520, with a significant 49.54-fold increase in specific lipase activity compared with the AOL powder in catalyzing the synthesis of OPO through the acidolysis between palm stearin and oleic acid (OA). The optimal immobilization conditions were investigated, including time course, initial protein concentration and solution pH. The sn-1,3 specificity of lipases under different immobilization conditions was evaluated and identified as positively associated with the lipase activity, and the pH of the immobilization solution influenced the regiospecificity and synthetic activity of these lipases. Immobilized AOL D3520, as the biocatalyst, was used for the enzymatic synthesis of the structured lipid OPO through the acidolysis between palm stearin and OA. The following conditions were optimized for the synthesis of structured lipid OPO: 65 °C temperature; 1:8 substrate molar ratio between palm stearin and OA; 8% (w/w) enzyme load; 3.5% water content of the immobilized lipase; and 1 h reaction time. Under these conditions, highly efficient C52 production (45.65%) was achieved, with a tripalmitin content of 2.75% and a sn-2 palmitic acid (PA) proportion of 55.08% in the system.

Aggregate structure, morphology and the effect of aggregation mechanisms on viscosity at elevated protein concentrations

Non-native aggregation is a common issue in a number of degenerative diseases and during manufacturing of protein-based therapeutics. There is a growing interest to monitor protein stability at intermediate to high protein concentrations, which are required for therapeutic dosing of subcutaneous injections. An understanding of the impact of protein structural changes and interactions on the protein aggregation mechanisms and resulting aggregate size and morphology may lead to improved strategies to reduce aggregation and solution viscosity. This report investigates non-native aggregation of a model protein, α-chymotrypsinogen, under accelerated conditions at elevated protein concentrations. Far-UV circular dichroism and Raman scattering show structural changes during aggregation. Size exclusion chromatography and laser light scattering are used to monitor the progression of aggregate growth and monomer loss. Monomer loss is concomitant with increased β-sheet structures as monomers are added to aggregates, which illustrate a transition from a native monomeric state to an aggregate state. Aggregates grow predominantly through monomer-addition, resulting in a semi-flexible polymer morphology. Analysis of aggregation growth kinetics shows that pH strongly affects the characteristic timescales for nucleation (τn) and growth (τg), while the initial protein concentration has only minor effects on τn or τg. Low-shear viscosity measurements follow a common scaling relationship between average aggregate molecular weight (Mwagg) and concentration (σ), which is consistent with semi-dilute polymer-solution theory. The results establish a link between aggregate growth mechanisms, which couple Mwagg and σ, to increases in solution viscosity even at these intermediate protein concentrations (less than 3w/v %).

Effective incorporation of insulin in mucus permeating self-nanoemulsifying drug delivery systems

The development of a novel, mucus permeating SNEDDS formulation for oral insulin delivery containing a hydrophobic ion pair of insulin/dimyristoyl phosphatidylglycerol (INS/DMPG) is presented. Three oil/surfactant/cosurfactant combinations and 27 weight ratios of oil, surfactant and cosurfactant for each combination were evaluated with the aid of ternary phase diagrams, for the incorporation of the protein/phospholipid complex. The developed formulation was characterized by an average droplet diameter of 30–45nm. Depending on the initial protein concentration, the loading of insulin in SNEDDS varied between 0.27 and 1.13wt%. The therapeutic protein was found to be efficiently protected from enzymatic degradation by intestinal enzymes (i.e., trypsin, α-chymotrypsin). The SNEDDS formulation exhibited increased mucus permeability and did not appear to be affected by ionic strength. The incorporation of INS/DMPG in SNEDDS prevented an initial burst release of insulin. INS/DMPG loaded SNEDDS were found to be non-cytotoxic up to a concentration of 2mg/ml. According to the reported results, the incorporation of the hydrophobic ion pair of INS/DMPG in SNEDDS could be regarded as a promising strategy for the oral delivery of insulin.

Circulating Mitochondrial DAMPs Are Not Effective Inducers of Proteinuria and Kidney Injury in Rodents.

Mitochondria in eukaryotic cells are derived from bacteria in evolution. Like bacteria, mitochondria contain DNA with unmethylated CpG motifs and formyl peptides, both of which have recently been shown to be damage associated molecular patterns (DAMPs) and induce immune response and cell injury. Based on the facts that circulating mitochondrial DAMPs (mtDAMPs) are increased in the patients of trauma or burn injury who also have proteinuria, that mtDAMPs can activate immune cells which in turn secrete glomerular permeability factors, that renal intrinsic cells express a variety of DAMP receptors, and that mtDAMPs can directly increase endothelial cell permeability in vitro, we hypothesized that mtDAMPs may be novel circulating factors inducing proteinuria and kidney injury. We tested this hypothesis by directly injecting mtDAMPs into rodents and examining urinary protein and kidney histology. We prepared mtDAMP samples, including mitochondrial DNA (mtDNA) and mitochondrial debris (MTD), from rodent liver. In mice, injection of mtDNA for 20 μg/ml initial concentration in circulation (much higher than the clinical range), did not cause any renal manifestations. However, an increased dose leading to 45 μg/ml initial concentration in circulation resulted in a transient, slight increase in urinary albumin. In rats, MTD injection resulting in 450 μg/ml initial concentration of MTD protein in circulation, which was much higher than the clinical range, caused mild, transient proteinuria and lung lesions. Multiple injections of such large amount of either mtDNA or MTD into rodents on 3 consecutive days also failed in inducing proteinuria and kidney injury. In summary, clinical levels of circulating mtDAMPs do not induce proteinuria and clinically irrelevant high levels of mtDAMPs cause only a transient and slight increase in urinary protein in rodents, suggesting that circulating mtDAMPs may not be responsible for the proteinuria and kidney injury in patients with trauma, burn injury, and other diseases.

Selective adsorption of bovine hemoglobin on functional TiO2 nano-adsorbents: surface physic-chemical properties determined adsorption activity

Surface functionalized nanoparticles are efficient adsorbents which have shown good potential for protein separation. In this work, we chose two different types of organic molecules, oleic acid (OA) and 3-glycidoxypropyltrimethoxy silane (GPTMS), to functionalize the surface of TiO2 nanoparticles, and we studied the effects of this modification on their surface physicochemical properties in correlation with their selective adsorption of proteins. The results showed that the surface zeta potential and the surface water wettability of the modified TiO2 were significantly changed in comparison with the original TiO2 nanoparticles. The adsorption activities of bovine hemoglobin (BHb) and bovine serum albumin (BSA) on these functionalized TiO2 samples were investigated under different conditions, including pH values, contact time, ion strength, and initial protein concentration. In comparison with the non-specific adsorption of original TiO2, however, both the OA–TiO2 and GPTMS–TiO2 exhibited increased BHb adsorption and decreased BSA adsorption at the same time. Using a binary protein mixture as the adsorption object, a higher separation factor (SF) was obtained for OA–TiO2 under optimum conditions. The different adsorption activities of BHb and BSA on the modified TiO2 were correlated with different interactions at the protein/solid interface, and the chemical force as well as the electrostatic force played an important role in the selective adsorption process.

Protein freeze concentration and micro-segregation analysed in a temperature-controlled freeze container

To examine effects of varied freezing conditions on the development of spatial heterogeneity in the frozen protein solution, macroscopic freeze concentration and micro-segregation of bovine serum albumin (BSA) were investigated in a temperature-controlled 200-ml freeze container. Freezing to −40°C promoted formation of protein concentration gradients (69–114μgml−1) in frozen samples taken from 12 different freezer positions, whereby slow freezing in 4h or longer facilitated the evolution of strong spatial heterogeneities and caused local concentration increases by 1.15-fold relative to the initial protein concentration (100μgml−1). To visualize protein micro-segregation during phase separation, BSA was conjugated with fluorescein isothiocyanate and confocal laser scanning fluorescence microscopy was used to localize and size the freeze-concentrated protein regions. Slow freezing resulted in distinctly fewer and larger protein domains in the frozen bulk than fast freezing. Surface stress on the protein during freezing would therefore be minimized at low cooling rates; microscopic freeze concentration would however be highest under these conditions, potentially favoring protein aggregation.

Preparation, structure and stability of sodium caseinate and gelatin micro-particles

Protein particles are promising candidates for texturing food products and can be produced in several ways. Here, we produced protein particles using a two-step emulsification method. This method is suitable to change the size of the particles and to control the protein concentration inside the particles. In this study, we prepared protein particles from two different protein sources, sodium caseinate (NaCas) and gelatin, that are gelled by acidification and cooling, respectively. The size and the internal protein concentration of the particles, their stability against heating and pH changes were studied. Although similar emulsification conditions were used to prepare the particles, NaCas particles were found to be 10 times smaller (average diameter 400 nm) than the gelatin particles (average diameter 4 μm). The internal protein concentration of the NaCas particles (16.8% w/w) is approximately twice as high compared to that of gelatin particles (7.6% w/w) (using an initial protein concentration of the solution of 10% (w/w)). The NaCas particle dispersions were found to be stable between pH 3 and pH 4. The particles disintegrated at pH values further away from the iso-electric point. Upon heating the dispersions at 90 °C, the NaCas particles were shown to be heat stable. Dispersions of gelatin particles were stable against aggregation at all pH values studied, except at pH 6, while the particles melted above 40 °C. Swelling of both particles was observed for both acidic and alkaline pH values. We conclude that emulsification method is robust for different protein sources used.

Determination of multivalent protein-ligand binding kinetics by second-harmonic correlation spectroscopy.

Binding kinetics of the multivalent proteins peanut agglutinin (PnA) and cholera toxin B subunit (CTB) to a GM1-doped 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer were investigated by both second-harmonic correlation spectroscopy (SHCS) and a traditional equilibrium binding isotherm. Adsorption and desorption rates, as well as binding affinity and binding free energy, for three bulk protein concentrations were determined by SHCS. For PnA binding to GM1, the measured adsorption rate decreased with increasing bulk PnA concentration from (3.7 ± 0.3) × 106 M–1·s–1 at 0.43 μM PnA to (1.1 ± 0.1) × 105 M–1·s–1 at 12 μM PnA. CTB–GM1 exhibited a similar trend, decreasing from (1.0 ± 0.1) × 109 M–1·s–1 at 0.5 nM CTB to (3.5 ± 0.2) × 106 M–1·s–1 at 240 nM CTB. The measured desorption rates in both studies did not exhibit any dependence on initial protein concentration. As such, 0.43 μM PnA and 0.5 nM CTB had the strongest measured binding affinities, (3.7 ± 0.8) × 109 M–1 and (2.8 ± 0.5) × 1013 M–1, respectively. Analysis of the binding isotherm data suggests there is electrostatic repulsion between protein molecules when PnA binds GM1, while CTB–GM1 demonstrates positive ligand–ligand cooperativity. This study provides additional insight into the complex interactions between multivalent proteins and their ligands and showcases SHCS for examining these complex yet technologically important protein–ligand complexes used in biosensors, immunoassays, and other biomedical diagnostics.

A novel affinity disks for bovine serum albumin purification.

The adsorption characteristics of bovine serum albumin (BSA) onto the supermacroporous poly(hydroxyethylmethacrylate)-Reactive Green 19 [p(HEMA)-RG] cryogel disks have been investigated in this paper. p(HEMA) cryogel disks were prepared by radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. Reactive Green (RG) 19 was covalently attached to the p(HEMA) cryogel disks. These disks were used in BSA adsorption studies to interrogate the effects of pH, initial protein concentration, ionic strength, and temperature. BSA adsorption capacity of the p(HEMA)-RG cryogel disk was significantly improved after the incorporation of RG. Adsorption capacity reached a plateau value at about 0.8 mg/mL at pH 4.0. The amount of adsorbed BSA decreased from 37.7 to 13.9 mg/g with increasing NaCl concentration. The enthalpy of BSA adsorption onto the p(HEMA)-RG cryogel disk was calculated as -58.4 kJ/mol. The adsorption equilibrium isotherm was fitted well by the Freundlich model. BSA was desorbed from cryogel disks (over 90 %) using 0.5 M NaSCN, and the purity of desorbed BSA was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The experimental results showed that the p(HEMA)-RG cryogel disks have potential for the quick protein separation and purification process.

Screening carbohydrate libraries for protein interactions using the direct ESI-MS assay. Applications to libraries of unknown concentration.

A semiquantitative electrospray ionization mass spectrometry (ESI-MS) binding assay suitable for analyzing mixtures of oligosaccharides, at unknown concentrations, for interactions with target proteins is described. The assay relies on the differences in the ratio of the relative abundances of the ligand-bound and free protein ions measured by ESI-MS at two or more initial protein concentrations to distinguish low affinity (≤10(3) M(-1)) ligands from moderate and high affinity (>10(5) M(-1)) ligands present in the library and to rank their affinities. Control experiments were performed on solutions of a single chain antibody and a mixture of synthetic oligosaccharides, with known affinities, in the absence and presence of a 40-component carbohydrate library to demonstrate the implementation and reliability of the assay. The application of the assay for screening natural libraries of carbohydrates against proteins is also demonstrated using mixtures of human milk oligosaccharides, isolated from breast milk, and fragments of a bacterial toxin and human galectin 3.

Analysis of the fractional modification of the monocyclic enzyme cascades, defined in an alternative way involving the two forms of the modified protein

This paper presents the discussion of a more complete definition of the fractional modification of an monocyclic enzyme cascade suggested, but not discussed, by Varon et al. (Bull Math Biol 68(7):1461-1493, 2006) as the quotient of the sum of all forms of the modified protein, i.e. the free one and the intermediate complex converter enzyme of the original protein-modified protein, between the initial concentration of the target protein. From this general equation, obtained under three assumptions necessary to linearize the set of differential equations describing the kinetic of the system, we derive, as particular cases, other simpler expressions, by applying additional simplifying assumptions, which are, therefore, of a smaller range of validity. We discuss the relationships between the kinetic parameters and concentrations needed for the fulfillment of both the necessary and unnecessary assumptions. The goodness of the analysis was tested by using the shape in the steady state of the simulated time progress curves obtained by numerical integration. Seven arbitrarily chosen examples differing in one or more of the values of the concentrations and/or kinetic parameters have been used to support the results.

Optimization of citric acid production from a carrot juice-based medium by Yarrowia lipolytica using response surface methodology

In this study, diluted and fortified carrot juice was used for modelling and optimization of citric acid production by a new mutant strain, Yarrowia lipolytica K-168. Protein concentrate obtained from fine flour -a byproduct of semolina production- was used as a nitrogen source in the fermentation medium. Interactive effects of selected independent variables, initial total sugar concentration, initial pH, initial concentration of protein concentrate obtained from fine flour of semolina and temperature, on the growth and citric acid production of the yeast were investigated. An experimental design including 30 experiments was conducted by using the method of central composite design. Modelling the effects of these independent variables on maximum citric acid concentration, maximum citric acid production rate, citric acid yield, the ratio of maximum citric acid concentration to maximum isocitric acid concentration and specific growth rate were performed by response surface methodology. The variations of all of the responses with the independent variables were defined by a quadratic model. Numeric optimization was performed by using the desireability function. The conditions with 190.83 g/L initial sugar concentration, 5.90 initial pH, 0.07 g/L initial concentration of fine flour protein concentrate and 27.86 °C were determined as optimal conditions for citric acid production. The maximum citric acid concentration reached to 80.53 g/L in optimal conditions.

Immobilized metal ion affinity nanospheres for $\alpha $-amylase immobilization

Immobilized metal chelate affinity chromatography (IMAC) support was practiced for a-amylase immobilization. Poly(hydroxyethylmethacrylate-methacryloylamidotryptophan)-Ni2+ [p(HEMA-MAT)-Ni2+] nanospheres, average diameter 100 nm, were produced by surfactant free emulsion polymerization. Characterizations of p(HEMA-MAT)-Ni2+ nanospheres were carried out by Fourier transform infrared (FTIR) spectroscopy and scanning electron microscope (SEM). In addition, average particle size, size distribution, and surface charge were specified. The amount of N-methacryloylamidotryptophan (MAT) incorporated to polymer was determined as 1.95 mmol/g polymers by using nitrogen stoichiometry. The specific surface areas of poly(hydroxyethylmethacrylate) [p(HEMA)] and p(HEMA-MAT) nanospheres were calculated as 1856 m2/g and 1914 m2/g, respectively. Protein adsorption increased with increasing initial protein concentration and maximum a-amylase adsorption on p(HEMA-MAT)-Ni2+ nanospheres was observed at pH 4.0. Both free and immobilized a-amylase showed pH optimum at pH 7.0. It was determined that the immobilized a-amylase had better thermostability than the free one. Immobilization of the enzyme did not significantly change the kinetic parameters. The storage stability of a-amylase increased upon immobilization. It was also observed that p(HEMA-MAT)-Ni2+ nanospheres can be repeatedly used for a-amylase immobilization.

Immobilization of β-glucosidase onto mesoporous silica support: Physical adsorption and covalent binding of enzyme

This paper investigates ?-glucosidase immobilization onto mesoporous silica support by physical adsorption and covalent binding. The immobilization was carried out onto micro-size silica aggregates with the average pore size of 29 nm. During physical adsorption the highest yield of immobilized ?-glucosidase was obtained at initial protein concentration of 0.9 mg ml-1. Addition of NaCl increased 1.7-fold, while Triton X-100 addition decreased 6-fold yield of adsorption in comparison to the one obtained without any addition. Covalently bonded ?-glucosidase, via glutaraldehyde previously bonded to silanized silica, had higher yield of immobilized enzyme as well as higher activity and substrate affinity in comparison to the one physically adsorbed. Covalent binding did not considerably changed pH and temperature stability of obtained biocatalyst in range of values that are commonly used in reactions in comparison to unbounded enzyme. Furthermore, covalent binding provided biocatalyst which retained over 70% of its activity after 10 cycles of reuse.

Shape, Shell, and Vacuole Formation during the Drying of a Single Concentrated Whey Protein Droplet

The drying of milk concentrate droplets usually leads to specific particle morphology influencing their properties and their functionality. Understanding how the final shape of the particle is formed therefore represents a key issue for industrial applications. In this study, a new approach to the investigation of droplet-particle conversion is proposed. A single droplet of concentrated globular proteins extracted from milk was deposited onto a hydrophobic substrate and placed in a dry environment. Complementary methods (high-speed camera, confocal microscopy, and microbalance) were used to record the drying behavior of the concentrated protein droplets. Our results showed that whatever the initial concentration, particle formation included three dynamic stages clearly defined by the loss of mass and the evolution of the internal and external shapes of the droplet. A new and reproducible particle shape was related in this study. It was observed after drying a smooth, hemispherical cap-shaped particle, including a uniform protein shell and the nucleation of an internal vacuole. The particle morphology was strongly influenced by the drying environment, the contact angle, and the initial protein concentration, all of which governed the duration of the droplet shrinkage, the degree of buckling, and the shell thickness. These results are discussed in terms of specific protein behaviors in forming a predictable and a characteristic particle shape. The way the shell is formed may be the starting point in shaping particle distortion and thus represents a potential means of tuning the particle morphology.