Studies on the Dissociation of the Soluble Ovomucin by Sonication

The soluble ovomucin obtained from the liquid part of thick white by gel filtration on a Sepharose 4B was an aggregated and polymerized molecule (intrinsic viscosity was 365 ml/g and molecular weight was 8.3 × 10⁶) and it was unable to dissociate the soluble ovomucin into two components without modifications.

Molecular weight and reduced viscosity of the soluble ovomucin decreased markedly with time of sonication. By the sonication for 10 min, it was successful to fractionate it into carbohydrate rich and poor component by density gradient electrophoresis, cellulose acetate electrophoresis and DEAE-cellulose column chromatography.

Concerning carbohydrate and amino acid compositions of two components obtained from the sonicated soluble ovomucin, it was found that the carbohydrate poor component corresponded to the reduced S-component or the reduced α-ovomucin, and the carbohydrate rich component to the reduced F-component or the reduced β-ovomucin.

It was considered that the sonicated soluble ovomucin was an intermediate of the aggregated, polymerized ovomucin (the soluble ovomucin) and the monomeric ovomucin (the sonicated and reduced soluble ovomucin).     

Characterization of Aqueous Dispersions and Gels Made of Sodium Caseinate and Basil Seed Gum: Phase Behavior,Rheology, and Microstructure

The interactions between sodium caseinate (NaCas) and basil seed gum (BSG) in the presence of calcium chloride (CaCl₂) were investigated. The phase behavior of the mixed aqueous dispersions and their gels revealed a homogeneous mixture, obtained at the higher concentrations of both CaCl₂ and BSG. The Herschel-Bulkley model sufficiently fitted the flow behavior of the mixture solution data. Apparent viscosity increased significantly (p < 0.05) by increasing the concentration of BSG, where the addition of CaCl₂ had no significant effect on the viscosity of the samples (p > 0.05). Furthermore, there was an increase in thixotropy due to the higher concentrations of BSG and CaCl₂. Based on the frequency sweep test, at the low frequencies, a more gel-like behavior was observed in the case of the higher concentrations of either BSG or CaCl₂. The rheological and SEM data suggested that the stronger structure of NaCas-BSG gel in the presence of the higher concentrations of CaCl₂ was related to the induction of complex formation between the two biopolymers.

     

The Release of Carbohydrate Rich Component from Ovomucin Gel during Storage

The fractionation pattern of OMG₀, ovomucin gel(B) in fresh egg white, by density gradient column electrophoresis showed two peaks. Each peak was shown to migrate as a single component, with a mobility of either the fast or slow moving component of ovomucin gel(B). Each peak was named as F-component and S-component.

Carbohydrate and sulfate contents of F-component were much higher than these of S-component. The carbohydrate content of F-component and S-component was found to be about 50 and 15 percents of dry matter, respectively. Serine and threonine contents in F-component were much higher than those in S-component.

The fractionation pattern of OMG₂₀, ovomucin gel(B) in egg white stored for 20 days at 30°C, by density gradient electrophoresis showed only one peak which corresponded to S-component, and that of OMS₂₀, ovomucin sol (B) in egg white stored for 20 days at 30°C, showed two peaks which corresponded to F- and S-component.

Ability of F-component to interact with lysozyme was greater than that of S-component.     

Improvement of the transfucosylation activity of α-l-fucosidase from Thermotoga maritima for the synthesis of fucosylated oligosaccharides in the presence of calcium and sodium

The influence of CaCl₂ and NaCl in the hydrolytic activity and the influence of CaCl₂ in the synthesis of fucosylated oligosaccharides using α-l-fucosidase from Thermotoga maritima were evaluated. The hydrolytic activity of α-l-fucosidase from Thermotoga maritima displayed a maximum increase of 67% in the presence of 0.8 M NaCl with water activity (a_w) of 0.9672 and of 138% in the presence of 1.1 M CaCl₂ (a_w 0.9581). In addition, the hydrolytic activity was higher when using CaCl₂ compared to NaCl at a_w of 0.8956, 0.9581 and 0.9672. On the other hand, the effect of CaCl₂ in the synthesis of fucosylated oligosaccharides using 4-nitrophenyl-fucose as donor substrate and lactose as acceptor was studied. In these reactions, the presence of 1.1 M CaCl₂ favored the rate of transfucosylation, and improved the yield of synthesis duplicating and triplicating it with lactose concentrations of 58 and 146 mM, respectively. CaCl₂ did not significatively affect hydrolysis rate in these reactions. The combination of the activating effect of CaCl₂, the decrement in a_w and lactose concentration had a synergistic effect favoring the synthesis of fucosylated oligosaccharides.

     

Studies on Changes in Stored Shell Eggs

Being solubilized by treatment with 0.01 m mercaptoethanol, the ovomucin gel(B) was found in free boundary electrophoresis to contain subunits which were consisted of two components. Changes in the physicochemical properties of all the insoluble ovomucin gel(B) and sol(B) obtained from stored egg white were studied after this treatment.

The fast moving component of the ovomucin gel(B) in free boundary electrophoresis decreased during storage and disappeared completely after 30 days. On the other hand, the fast moving component of the ovomucin sol(B) increased during storage.

The acid mucoprotein concentration of the ovomucin gel(B) in acrylamide gel electrophoresis decreased and that of the ovomucin sol(B) increased during storage, although the protein pattern did not show significant changes.

The interaction of the ovomucin gel(B) with lysozyme decreased whereas that of the ovomucin sol(B) increased during storage.

By summarizing these results, a model of ovomucin gel structure and a mechanism of egg white thinning were proposed.     

A novel immobilised design for the production of the heterologous protein lysozyme by a genetically engineered Aspergillus niger strain

Abstract A novel immobilisation design for increasing the final concentration of the heterologous protein lysozyme by a genetically engineered fungus, Aspergillus niger B1, was developed. A central composition design was used to investigate different immobilised polymer types (alginate and pectate), polymer concentration [24% and 4% (w/v)], inoculum support ratios (1:2 and 1:4) and gel-inducing agent concentration [CaCl₂, 2% and 3.5% (w/v)]. Studies of the kinetics of production showed that optimum lysozyme productivity occurred after 10 days. Lysozyme production was significantly affected by polymer type, polymer concentration, and inoculum support ratio. Overall, immobilisation in Ca-pectate resulted in higher lysozyme production compared to that in Ca-alginate. Similar effects were observed when the polymer concentration was reduced. Regardless of polymer type and concentration, increasing the fungal inoculum level increased lysozyme production. A significantly higher lysozyme yield was achieved with Ca-pectate in comparison to Ca-alginate (approximately 20–23 mg l^–1 and 0.5–2 mg l^–1, respectively). The maximum lysozyme yield achieved was about 23 mg l^–1 by immobilisation in Ca-pectate 2% (w/v) with 33% (v/v) mycelium and 3.5% (w/v) gel-inducing agent (CaCl₂). Response surface methodology was used to investigate the effect of pH and water activity (a_w). The best medium pH was 4.5–5.0, and bead a_w for optimum lysozyme yield was 0.94, regardless of polymer type.     

Inactivation of ntcA gene revealed differential proteome expression and induction of some hypothetical proteins in the cyanobacterium Anabaena sp. PCC 7120 and its derivative ntcA mutant under different levels of calcium

Abstract

Calcium is an important macronutrient for both prokaryotes and eukaryotes. It acts as an important second messenger mediating rapid response to environmental conditions. The present investigation deals with proteome profiling of Anabaena 7120 and its derivative ntcA mutant in response to varied calcium doses (0, 1 and 10?mM CaCl₂). Concentration of 1?mM CaCl₂ salt was the optimum concentration whereas 10?mM CaCl₂ was the inhibitory concentration for both the wild type and mutant strains. The results showed highly significant alteration in terms of protein abundance and differential response related to key processes of photosynthesis, energy and metabolism, nitrogen metabolism, oxidative and antioxidative defence, transport and signalling and fatty acid metabolism. In the wild type proteins related to photosynthesis and nitrogen metabolism showed upregulation at 1?mM CaCl₂ concentration while antioxidative defence related proteins were down-regulated. In the mutant however, proteins related to photosynthesis and nitrogen metabolism exhibited severe down-regulation. Some hypothetical proteins were also realized during proteome analysis. Overall, our results suggested that NtcA have a potential role in regulation of calcium ion dependent key processes underlying in various metabolic activities of the cyanobacterium Anabaena 7120.     

Solubilization and Characterization of Ovomucin without Chemical Modification

1. The egg white, thick and thin fractions, was solubilized in 1.0% SDS solution by vigorous mixing and subjected to gel filtration on a Sepharose 4B column, eluted with 1.0% SDS. The isolated thick and thin ovomucins were found by analytical disc electrophoresis to be free from contamination with lysozyme.

2. In the velocity sedimentation the two ovomucin fractions behave similarly, both comprising at least two components with sedimentation coefficients 35 S and 30 S.

3. The chemical compositions of the two ovomucin fractions showed only notable difference in that the carbohydrate content of the thick white ovomucin was somewhat higher than that of the thin white ovomucin. The amino acid profiles of the two fractions were similar.

     

Activation of Macrophages by Sulfated Glycopeptides in Ovomucin,Yolk Membrane,and Chalazae in Chicken Eggs

Sulfated glycopeptides in ovomucin, chalazae and yolk membrane were found to activate cultured macrophage-like cells, J774.1, and TGC-induced macrophages from the peritoneal cavity of male mice. The macrophage-stimulating activity was estimated by the growth and morphology of the cells, H₂O₂ generation, and interleukin-1 (IL-1) production from the cells. The in vitro culture assay with macrophages showed that the protease digests of ovomucin, yolk membrane, and chalazae induced morphologic alteration and increased H₂O₂ generation and IL-1 production in lower concentration (100 μg/ml). The isolation of the components having macrophage-stimulating activity was attempted to elucidate the molecular mechanism. The O-linked carbohydrate chains, consisting of N-acetylgalactosamine, galactose, N-acetylneuraminic acid and sulfate, in the sulfated glycopeptide were identified as a component having macrophage-stimulating activity.     

Effect of potassium chloride and calcium chloride induced stress on in vitro cultures of Bacopa monnieri (L.) Pennell and accumulation of medicinally important bacoside A

Growth, osmotic adjustment, antioxidant enzyme defense and principle medicinal component bacoside A was studied in in vitro raised shoots of Bacopa monnieri under different concentrations of KCl and CaCl₂ (0, 50, 100, 150 or 200 mM). Significant reduction was observed in shoot number per culture; shoot length, fresh weight, dry weight and tissue water content (TWC) when shoots were exposed to increasing KCl and CaCl₂ concentrations (50–200 mM) as compared to control. Minimum damage to the membrane as assessed by malondialdehyde (MDA) content was noticed in control in contrast to sharp increase in KCl and CaCl₂ stressed shoots. Higher amounts of free proline, glycine betaine and total soluble sugars (TSS) accumulated in KCl and CaCl₂ exposed shoots compared to the controls. Among different concentrations of KCl and CaCl₂, increasing concentration of CaCl₂ showed more increase in osmolyte accumulation. Na⁺ content decreased with increasing concentrations of KCl and CaCl₂. Accumulation of K⁺ increased significantly in KCl (50–100 mM) stressed shoots as compared to control, while it decreased in CaCl₂ treated shoots indicating that it prevents the uptake of K⁺ ions. Ca²⁺ accumulation significantly increased with increasing concentrations of CaCl₂ up to 150 mM but decreased at higher concentrations. Shoots treated with KCl and CaCl₂ (0–100 mM) showed higher antioxidant enzyme (SOD, CAT, APX and GPX) activities but KCl suppressed the activities at higher concentrations. Accumulation of bacoside A was enhanced with an increase in KCl and CaCl₂ concentration up to 100 mM. It appears from the data that accumulation of osmolytes, and elevated activities of antioxidant enzymes play an important role in osmotic adjustment in shoot cultures of Bacopa and the two salts tested have a positive effect on bacoside accumulation.     

Effect of exogenous calcium on post-thaw growth recovery and subsequent plant regeneration of cryopreserved embryogenic calli of <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> (Müll. Arg.)

A reliable cryopreservation technique was developed for friable embryogenic callus lines of Hevea brasiliensis. The study showed that reducing the CaCl₂ concentration of the pre-culture medium from 9 mM to 1 or 0 mM CaCl₂ before cryopreservation promoted post-thaw callus growth, 1 mM being the optimum CaCl₂ concentration for embryo regeneration. Post-thaw callus proliferation decreased in line with the increase of plated callus weight. The effect of cryopreservation was assessed on 39 independent lines showing that cryopreservation did not affect embryogenic and plant regeneration for a majority of lines. The decrease in CaCl₂ concentration of the pre-culture medium led to a drop in callus calcium content indicating a direct link between the CaCl₂ concentration of the pre-culture medium and the endogenous calcium content of the calli. It also highlighted the implication of tissue calcium content in cryotolerance. Callus water status and the different ways by which calcium could prevent cryoinjury is also discussed.     

Effect of 1-Anilino-8-naphthalenesulfonate on Properties and Associations of αs-, β- and κ-Caseins

It was indicated from fluorescence spectra and fluorescence titration that a hydrophobic probe, 1-anilino-8-naphthalenesulfonate (ANS), binds to casein components (α_s-, β- and κ-caseins). Fluorescence intensity and affinity of ANS-κ-casein complex were larger than that of ANS-α_s- and ANS-β-casein complexes. Enhancements of fluorescence intensity of complexes of casein components were observed by the addition of KCI or CaCl₂. Reason for the enhancement was postulated to be the increase of the quantum yield of the ANS fluorescence caused by the environmental change of ANS binding region of the casein components.

Marked increase of sedimentation coefficient of β-casein in the presence of KCl or CaCl₂ at 10°C was caused by the addition of ANS. This may be responsible for the stimulation of the Ca-dependent precipitation of β-casein by the addition of ANS.

It was found that α_s · κ-association was prevented by ANS and that hydrophobic interaction have an important role for α_s · κ-association.     

Optimization of α‐Amylase Immobilization in Calcium Alginate Beads

Abstract

α‐Amylase enzyme was produced by Aspergillus sclerotiorum under SSF conditions, and immobilized in calcium alginate beads. Effects of immobilization conditions, such as alginate concentration, CaCl₂ concentration, amount of loading enzyme, bead size, and amount of beads, on enzymatic activity were investigated. Optimum alginate and CaCl₂ concentration were found to be 3% (w/v). Using a loading enzyme concentration of 140 U mL^?1, and bead (diameter 3 mm) amount of 0.5 g, maximum enzyme activity was observed. Beads prepared at optimum immobilization conditions were suitable for up to 7 repeated uses, losing only 35% of their initial activity. Among the various starches tested, the highest enzyme activity (96.2%) was determined in soluble potato starch hydrolysis for 120 min at 40°C.     

Effects of the Storage in an Atomosphere of Carbon Dioxide on Ovomucin

The yolk index of newly laid egg was 0.50, while that of the egg stored at 30°C for 15 days in an atmosphere of carbon dioxide at a pressure of 2 kg/cm² and in air was 0.43 and 0.25, respectively.

The carbohydrate content of ovomucin gel (B) obtained from the eggs stored in an atmosphere of carbon dioxide was higher than that of ovomucin gel (B) obtained from the eggs stored in air.

The free boundary electrophoretic pattern of ovomucin gel (B) obtained from the eggs stored in an atmosphere of carbon dioxide consisted of two peaks, and the relative mobility of each peak showed the same value as that of the corresponding each peak of ovomucin gel (B) obtained from newly laid egg white.

The fractionation pattern obtained by density gradient column electrophoresis of ovomucin gel (B) obtained from the eggs stored in an atmosphere of carbon dioxide showed two peaks, peak-F and peak-S, while that of the eggs stored in air showed a considerable diminution in peak-F.

From these results, discussion was made about the occurrence and mechanism of egg white thinning when eggs were stored in an atmosphere of carbon dioxide.     

Calcium alginate entrapment of the yeast Rhodosporidium toruloides for the kinetic resolution of 1,2-epoxyoctane

Resting cells of the yeast Rhodosporidium toruloides (UOFS Y-0471) were immobilised in calcium alginate beads for the enantioselective kinetic resolution of racemic-1,2-epoxyoctane. The initial activity exhibited by immobilised cells was almost 50% lower than that of the free counterpart but was extremely stable when compared to the free cells. The concentration of the immobilised biomass had no effect on apparent enzyme activity but did lead to a decrease in single cell activity. An increase in both the alginate and CaCl₂ concentrations used for bead preparation led to a decrease in enzyme stability. An increase in the alginate concentration led to an increase in bead diameter. The stoichiometric equation for cross-linking of alginate was only obeyed when CaCl₂ concentrations higher than 0.4 M were utilised for bead preparation.     

Studies on Changes in Stored Shell Eggs

The intrinsic viscosity of ovomucin(B) from the thick white was higher than that of the thin white. The relative area of the fast moving component in the electrophoretic pattern of ovomucin(B) from the thick white was about twice that of the thin white. The hexose, hexosamine and sialic acid contents of ovomucin(B) from the thick white were higher than those of the thin white. The intrinsic viscosity and carbohydrate content of ovomucin from the egg white previously freed from lysozyme were low in comparison with those of ovomucin(B). The carbohydrate contents of crude lysozyme from the thick white were higher than those of the thin white, and crude lysozyme obtained from the thick white contained larger amounts of ovomucin-iike material than the thin white. From these results, discussion were made about the relation between the properties of ovomucin(B) and ovomucin-lysozyme interaction.     

The Isolation and Fractionation of Chicken Egg White Ovomucin

Three fractions of chicken egg white ovomucin were obtained from ovomucin precipitated by the classical method of dilution of egg white with water. The first fraction was obtained by dilute salt extraction of the precipitated ovomucin and consisted of smaller components of ovomucin. The second fraction was obtained by extraction with 1 M KCl of the precipitate remaining after dilute salt extraction and consisted of a heterogeneous mixture of components of larger mass. The third fraction, the ovomucin remaining after concentrated salt extraction, was insoluble.

The two soluble fractions were found to be unstable with time (pH 8, μ 0.2). The rate of breakdown was slower in concentrated salt solution. The final breakdown product(s) for both fractions appeared to be a 6 S component (s) of molecular mass 1.5 × 10⁵ daltons.     

Annuario della Società Botanica Italiana 1932

A sandy culture experiment was conducted to investigate the effects of exogenous CaCl₂ on the indole alkaloid accumulation in Catharanthus roseus under salt stress. One-month seedlings of C. roseus were treated with the different concentrations of NaCl (0, 50, and 100 mmol l^? 1) and 7.5 mmol l^? 1 CaCl₂. The plant samples were analyzed after 7 days of the treatments. The NaCl-stressed plants showed decrease of fresh and dry weight and increase of malondialdehyde (MDA) content compared to control. Tryptophan decarboxylase (TDC) activity increased significantly under 50 mmol l^? 1 NaCl without CaCl₂ addition, 50 mmol l^? 1 NaCl with 7.5 mmol l^? 1 CaCl₂, and CaCl₂ treatment without NaCl addition. There was a significant increase in peroxidase activity under NaCl stress compared to control. The vindoline, catharanthine, vincristine, and vinblastine contents increased under salt stress (especially with 50 mmol l^? 1 NaCl treatment with or without CaCl₂). Addition of CaCl₂ to NaCl-stressed plants increased biomass, TDC activity, vindoline, and catharanthine contents and lowered MDA and vincirstine contents compared to the plants without CaCl₂. The plants treated with CaCl₂ alone showed higher TDC activity, vindoline, catharanthine, and vinblastine content when compared to control. The results showed that exogenous CaCl₂ could promote the indole alkaloid metabolism under salt stress.     

Promoting larval settlement of coral Pocillopora damicornis by calcium

Larval settlement is a critical bottleneck in the process of coral sexual propagation. Promoting coral larval settlement by inducers is a promising strategy in coral reef restoration engineering. In this study, the settlement-promoting effect of Ca²⁺ on larvae of the brooding coral Pocillopora damicornis was investigated for the first time. Treatment with 40 mM CaCl₂ for 24 h effectively promoted coral larval settlement (~ 80%). Moreover, CaCl₂ is comparable with the natural inducer, crustose coralline algae (CCA), in both promoting coral larval settlement and post-settlement growth. CaCl₂ showed toxic effects on larval survival and growth at high concentrations, and this could be minimized by optimizing CaCl₂ concentration and shortening the exposure period. Our study suggests that applying Ca²⁺ to effectively and efficiently induce coral larval settlement is viable for laboratory research and small-scale aquaculture systems, and it might become a useful tool in future coral reef restoration engineering.

     

Phosphate potential and phosphate capacity of soils

Summary The relationship between the phosphate potential (I) and the amount of phosphate (Q), added to the soil has been examined by equilibrating soil samples with 0.001M or 0.01M CaCl₂ solutions containing various amounts of phosphate. For one neutral and two alkaline soils the Q/I relationship depends on the CaCl₂ concentration and the pH in such a way that the apparent values of I decrease when the CaCl₂ concentration increases from 0.001 M to 0.01M. The difference between the two values increases when the pH increases. When correction is made for the formation of the soluble calcium phosphate complex, CaHPO₄, the Q/I relationship becomes independent of the CaCl₂ concentration. The initial phosphate potential (I₀) determined by interpolation, is also found to be independent of the CaCl₂ concentration. The necessary amount of phosphate to be added or removed per gram of soil in order to obtain a certain alteration of the phosphate potential is designated the differential phosphate potential buffering capacity, DPBC. For ten soils DPBC-values are determined on the basis of the Q/I relationships, (ΔQ/ΔI)_Io, and found to be independent of the CaCl₂ concentration. The content of colloids and of inorganic phosphate accounts for a significant part of the variation in the DPBC for different soils. The importance of the DPBC for characterization of the phosphate status of soils in respect to phosphate supply to plants is briefly discussed. The author is indebted to professor, Dr. H. C. Aslyng, head of the department for his suggestions and helpful criticism during the progress of this work.