Immobilization of cellulase from newly isolated strain Bacillus subtilis TD6 using calcium alginate as a support material

Bacillus subtilis TD6 was isolated from Takifugu rubripes, also known as puffer fish. Cellulase from this strain was partially purified by ammonium sulphate precipitation up to 80% saturation, entrapped in calcium alginate beads, and finally characterized using CMC as the substrate. For optimization, various parameters were observed, including pH maximum, temperature maximum, sodium alginate, and calcium chloride concentration. pH maximum of the enzyme showed no changes before and after immobilization and remained stable at 6.0. The temperature maximum showed a slight increase to 60 °C. Two percent sodium alginate and a 0.15 M calcium chloride solution were the optimum conditions for acquisition of enzyme with greater stability. K (m) and V (max) values for the immobilized enzyme were slightly increased, compared with those of free enzyme, 2.9 mg/ml and 32.1 μmol/min/mL, respectively. As the purpose of immobilization, reusability and storage stability of the enzyme were also observed. Immobilized enzyme retained its activity for a longer period of time and can be reused up to four times. The storage stability of entrapped cellulase at 4 °C was found to be up to 12 days, while at 30 °C, the enzyme lost its activity within 3 days.     

Removal of copper, lead, and zinc from contaminated water by saltbush biomass: analysis of the optimum binding, stripping, and binding mechanism

Experiments performed on the Cu(II), Pb(II), and Zn(II) binding by saltbush biomass (Atriplex canescens) showed that the metal binding increased as pH increased from 2.0 to 5.0. The highest amounts of Cu, Pb, and Zn bound by the native biomass varied from 48-89%, 89-94%, and 65-73%, respectively. The hydrolyzed biomass bound similar amount of Pb and 50% more Cu and Zn than the native. The esterified biomass had a lower binding capacity than native; however, esterified flowers bound 45% more Cu at pH 2.0 than native flowers. The optimum binding time was 10 min or less. More than 60% of the bound Cu was recovered using 0.1 mM HCl, while more than 90% of Pb was recovered with either HCl or sodium citrate at 0.1 mM. For Zn, 0.1 mM sodium citrate allowed the recovery of 75%. Results indicated that carboxyl groups participate in the Cu, Pb, and Zn binding.     

Simplified NaCl Based (68)Ga Concentration and Labeling Procedure for Rapid Synthesis of (68)Ga Radiopharmaceuticals in High Radiochemical Purity

A simple sodium chloride (NaCl) based (68)Ga eluate concentration and labeling method that enables rapid, high-efficiency labeling of DOTA conjugated peptides in high radiochemical purity is described. The method utilizes relatively few reagents and comprises minimal procedural steps. It is particularly well-suited for routine automated synthesis of clinical radiopharmaceuticals. For the (68)Ga generator eluate concentration step, commercially available cation-exchange cartridges and (68)Ga generators were used. The (68)Ga generator eluate was collected by use of a strong cation exchange cartridge. 98% of the total activity of (68)Ga was then eluted from the cation exchange cartridge with 0.5 mL of 5 M NaCl solution containing a small amount of 5.5 M HCl. After buffering with ammonium acetate, the eluate was used directly for radiolabeling of DOTATOC and DOTATATE. The (68)Ga-labeled peptides were obtained in higher radiochemical purity compared to other commonly used procedures, with radiochemical yields greater than 80%. The presence of (68)Ge could not be detected in the final product. The new method obviates the need for organic solvents, which eliminates the required quality control of the final product by gas chromatography, thereby reducing postsynthesis analytical effort significantly. The (68)Ga-labeled products were used directly, with no subsequent purification steps, such as solid-phase extraction. The NaCl method was further evaluated using an automated fluid handling system and it routinely facilitates radiochemical yields in excess of 65% in less than 15 min, with radiochemical purity consistently greater than 99% for the preparation of (68)Ga-DOTATOC.     

Investigation of factors influencing drug release using calcium alginate polymer

The effects of mixing, the sodium alginate concentration, and calcium chloride concentration on the release of sulphamethoxazole (model drug) impregnated in calcium alginate beads were investigated and evaluated. The release behaviour of the sulphamethoxazole from the calcium alginate beads was studied in a 0.1N HCl aqueous solution at 37v°C. The release rate of the sulphamethoxazole depends heavily on the type of mixers during the formation of the drug-alginate beads. The highest release rate was achieved when four-bladed rectangular agitator was used while the lowest release was achieved when magnetic stirrer was used. The amount of the released sulphamethoxazole varies slightly with the variation of the alginate concentration. The total release of sulphamethoxazole when 1% w/v solution of sodium alginate was used found to be 80% of the total drug content while 72% and 68% of the total drug content for 1.5% and 2% sodium alginate solutions. Three different calcium chloride concentrations were used (i.e., 5%, 10%, and 15% CaCl₂). The effect of the calcium chloride concentration on the release of the sulphamethoxazole is very pronounced.     

Hydrophobic modification of sodium alginate and its application in drug controlled release

Sodium alginate was hydrophobically modified by coupling of polybutyl methacrylate onto the alginate. The polybutyl methacrylate was previously prepared through polymerization of butyl methacrylate in the presence of 2-amino-ethanethiol as a chain transfer agent. The structure of the product was characterized by Fourier-transformed infrared spectrometry, nuclear magnetic resonance (¹HNMR) and thermogravimetry. The result of fluorescence analysis showed that the hydrophobicity of the modified alginate was obviously increased. The modified alginate conjugate was used for immobilization of bovine serum albumin in the presence of calcium chloride. In addition, the release behavior of the drug-loaded alginate in deionized water and Tris–HCl buffer solution (pH 7.2) was investigated. It was found that the modified sodium alginate possessed prolonged release behavior compared to unmodified sodium alginate, and it had potential application in controlled release as a drug carrier.     

Nitrogen and sulphur requirements of Colletotrichum inamdarii Lal

Summary Nitrogen and sulphur requirements ofColletotrichum inamdarii Lal isolated from the leaves ofCarissa carandas L. have been studied. DL-serine, L-asparagine and L-phenylalanine have been found to be of good nitrogen source followed by potassium nitrate, calcium nitrate, magnesium nitrate, DL-alanine, ammonium nitrate, glutamic acid, ammonium sulphate, DL-valine, aspartic acid, ammonium chloride, ammonium hydrogencarbonate, L-histidine and potassium nitrite. There was no growth in the absence of nitrogen.Sporulation was excellent on calcium nitrate and sodium nitrate, Very good on DL-serine, potassium nitrate, and magnesium nitrate. Good on L-asparagine, L-phenylalanine and ammonium oxalate. Fair on DL-alanine, DL-leucine, ammonium sulphate, DL-valine, ammonium chloride and L-histidine whereas poor on glutamic acid, aspartic acid, ammonium tartarate and ammonium nitrate. Few spores were observed on ammonium hydrogencarbonate but potassium nitrite did not show any sporulation.Amongst the sulphur compounds sodium bisulphate gave the best growth and good sporulation, followed by sodium thiosulphate, magnesium sulphate, ammonium sulphate and potassium sulphate. Thiourea gave negligible growth whereas it failed to grow on zinc sulphate and potassium persulphate.     

An improved method of microencapsulation and its evaluation to protect Lactobacillus spp. in simulated gastric conditions

An improved method of microencapsulation was developed to increase the efficacy of capsules in protecting the encapsulated bacteria under simulated gastric conditions. Lactobacillus acidophilus CSCC 2400 was encapsulated in calcium alginate and tested for its survival in simulated gastric conditions. The effects of different capsule sizes (200, 450, 1000 microm), different sodium alginate concentrations (0.75%, 1%, 1.5%, 1.8% and 2% w/v) and different concentrations of calcium chloride (0.1, 0.2, 1.0 M) on the viability of encapsulated bacteria were investigated. The viability of the cells in the microcapsules increased with an increase in alginate capsule size and gel concentration. There was no significant difference (p>0.05) in the viability of encapsulated cells when the concentration of calcium chloride was increased. Increase in cell load during encapsulation increased the number of bacterial survivors at the end of 3-h incubation in simulated gastric conditions. Hardening the capsule in calcium chloride solution for a longer time (8 h) had no impact on increasing the viability of encapsulated bacteria in a simulated gastric environment. The release of encapsulated cells at different phosphate buffer concentrations was also studied. When encapsulated L. acidophilus CSCC 2400 and L. acidophilus CSCC 2409 were subjected to low pH (pH 2) and high bile concentration (1.0% bile) under optimal encapsulation conditions (1.8% (w/v) alginate, 10(9) CFU/ml, 30 min hardening in 0.1 M CaCl(2) and capsule size 450 microm), there was a significant increase (p<0.05) in viable cell counts, compared to the free cells under similar conditions. Thus the encapsulation method described in this study may be effectively used to protect the lactobacillus from adverse gastric conditions.     

Pilot plant scale extraction of alginate from Macrocystis pyrifera. 1. Effect of pre-extraction treatments on yield and quality of alginate

In the extraction of alginate from brown seaweeds, the acid pre-extraction treatment has been considered by many authors as an essential step because it makes the alginate more readily soluble in an alkaline solution. At pilot plant level, extractions were made (i) using formalin treatment prior to the acid pre-extraction treatment (ii) using different acid treatments so the calcium ions exchanged varied from 83% to 4%. The use of formalin treatment gave a product with less color. During the acid pre-extraction treatment, it was possible to reduce the calcium exchanged from 33.4% to almost zero with a maximum reduction in alginate yield of 7%. The degree of acid treatment was positively correlated to calcium exchanged and yield but negatively correlated with alginate viscosity. Using strong acid conditions the viscosity was 168 mPa s, while mild acid conditions produced an alginate with 623 mPa s. The direct extraction from calcium alginate to sodium alginate is possible because strong alkaline conditions were used, pH 10 at 80 °C for two hours and with a low water volume. The best pre-extraction treatment to obtain an alginate with high viscoity is to hydrate the alga with 0.1% formalin overnight, then wash the alga once with hydrochloric acid at pH 4 using a batch system with continuous agitation during 15 min. This revised version was published online in August 2006 with corrections to the Cover Date.     

Influence of chitosan type on the properties of extruded pellets with low amount of microcrystalline cellulose

The purpose of this research was to study the influence of type of chitosan with different molecular weights, ie, 190 and 419 kDa, on properties of pellets prepared by extrusion/ spheronization. The formulations, consisting of acetaminophen as model drug, chitosan, microcrystalline cellulose (MCC), and dibasic calcium phosphate dihydrate with/without sodium alginate, were extruded using a twin-screw extruder and water as the granulating liquid. With 30% wt/wt MCC and no added sodium alginate, spherical pellets were produced containing low and high molecular weight chitosan at a maximum amount of 60% and 40% wt/wt, respectively. With sodium alginate (2.5% wt/wt), pellets with either type of chitosan (60% wt/wt), MCC (17.5% wt/wt), and acetaminophen (20% wt/wt) could be produced indicating an improved pelletforming ability. Type and amount of chitosan and added sodium alginate affected physical properties of pellets including size, roundness, crushing force, and drug release. Low molecular weight chitosan produced pellets with higher mean diameter, sphericity, and crushing force. Additionally, the pellets made of low molecular weight chitosan and added sodium alginate showed faster drug release in 0.1 N HCl but had slower drug release in pH 7.4 phosphate buffer. This indicated that drug release from pellets could be modified by the molecular weight of chitosan. In conclusion, the molecular weight of chitosan had a major influence on formation, physical properties, and drug release from the obtained pellets. Published: August 10, 2007     

Stabilization of alginate beads using radiation polymerized polyacrylamide.

A technique has been described for the stabilization of calcium alginate beads using radiation polymerized acrylamide. The technique involved dropping a mixture containing the cells (20%), sodium alginate (2%), acrylamide (2.5%) and N-N'-methylene-bis-acrylamide (0.1%) through a syringe needle into cold (-75 degrees C) toluene. The frozen beads obtained were exposed to 60Co gamma-rays (0.5 KGy) and were then thawed in 0.1 M CaCl2 solution. Unlike the calcium alginate beads the conjugate beads were not found to be dissolved when incubated in 3% trisodium citrate solution. Stabilized beads containing entrapped yeast cells could be reused for over 15 batches for the inversion of sucrose without loss in activity or chemical integrity of the beads.     

Nitrite as an energy-conserving electron sink for the acetogenic bacterium Moorella thermoacetica

Nitrite served as an energy-conserving electron acceptor for the acetogenic bacterium Moorella thermoacetica. Growth occurred in an undefined (0.1% yeast extract) medium containing 20 m M glyoxylate and 5 m M nitrite and was essentially equivalent to that observed in the absence of nitrite. In the presence of nitrite, acetate (the normal product of glyoxylate-derived acetogenesis) was not detected during growth. Instead, growth was coupled to nitrite dissimilation to ammonium, and acetogenesis was limited to the stationary phase. Furthermore, membranes from glyoxylate-grown cells under nitrite-dissimilating conditions were deficient in the b-type cytochrome that is typically found in the membranes of acetogenic cells. Unlike glyoxylate, other acetogenic substrates (fructose, oxalate, glycolate, vanillin, and hydrogen) were not growth supportive in the undefined medium containing nitrite, and glyoxylate-dependent growth did not occur in a nitrite-supplemented, basal (without yeast extract) medium. Glyoxylate-dependent growth by Moorella thermoautotrophica was not observed in the undefined medium containing nitrite.     

Utilization in alginate beads for Cu(II) and Ni(II) adsorption of an exopolysaccharide produced by <Emphasis Type="Italic">Chryseomonas luteola</Emphasis> TEM05

Copper and nickel adsorption onto calcium alginate, sodium alginate with an extracellular polysaccharide (EPS) produced by the activated sludge bacterium Chryseomonas luteola TEM05 and the immobilized C. luteola TEM05 from aqueous solutions were studied. After that, the multi metal ions containing these ions together were prepared and partial competitive adsorptions of these mixtures were also investigated. The metal adsorption of gel beads were carried out at pH 6.0, 25 °C. The maximum adsorption capacities in Langmuir isotherm for calcium alginate, calcium alginate + EPS, calcium alginate + C. luteola TEM05 and calcium alginate + EPS + C. luteola TEM05 were 1.505, 1.989, 1.976, 1.937 mmol/g dry weight for Cu(II) and 0.996, 1.224, 1.078, 1.219 mol/g dry weight for Ni(II), respectively.The competitive biosorption capacities of the carrier for all metal ions were lower than single conditions.     

An analysis of physiological states responsible for antero-posterior disintegration in Planaria dorotocephala

Summary Planaria were treated with equi-molal solutions of ammonium, potassium, sodium, magnesium, and calcium chlorides, made up in distilled water and the rates of cytolysis compared with cytolysis in distilled water. Potassium and ammonium accelerate cytolysis; some protection is afforded by sodium; still more by magnesium, and complete protection by calcium in the concentrations employed.In distilled water solutions of calcium chloride no cytolysis occurs in concentrations from M/500 to M/40,000; cytolysis is distinctly delayed in M/100,000. The protective action of M/1,000,000 is detectable.Potassium oxalate accelerates disintegration in hypotonic solutions.One per cent ethyl alcohol in distilled water causes cytolysis more rapidly than does distilled water alone, but in M/500 molal calcium chloride the alcohol solution is much less effective.Ringer's solution minus calcium affords no protection against death due to absence of calcium and death due to potassium oxalate but completely protects against cytolysis. Death in Ringer's solution minus calcium and in Ringer's solution with potassium oxalate occurs first in the anterior region and describes an antero-posterior gradient.Cytolysis in distilled water, in potassium oxalate solutions, in alcohol solutions, and in hypotonic calcium solutions of extreme dilution is initiated in the anterior end and describes an antero-posterior gradient within a zooid.Earlier work of the writer on the disintegrative action of lipoid solvents, heat, KNC, hyper- and hypotonic solutions is discussed. It is concluded that inPlanaria dorotocephala the antero-posterior gradient in cytolytic disintegration represents an antero-posterior differential in sensitivity to disturbance of the calcium-lipoid-water relation in the organism.     

Capillary electrophoresis of the major anions and cations in leaf extracts of woody species

Capillary electrophoresis methods are described for the analysis of the major inorganic anions (nitrite, nitrate, chloride, sulphate, phosphate), organic acids (oxalate, malate, citrate, succinate) and inorganic cations (ammonium, potassium, sodium, calcium, magnesium) in leaf extracts. Analytical performance was validated for extracts from leaves of four sclerophyllous species: Eucalyptus globulus, E. cladocalyx, E. nitens and Pinus radiata. Inorganic anions and organic acids were analysed in a single run within 5 min using a background electrolyte of 2,6-pyridinedicarboxylic acid (20 mM) and cetyltrimethylammonium bromide (0.5 mM). Cations were analysed in a separate run also within 5 min using imidazole (10 mM) and 18-crown-6 (2 mM) as background electrolyte. Replicate injections were highly repeatable when the capillary was rinsed between runs with hydrochloric acid (0.25 M) and background electrolyte, but not when the acid rinse was omitted or replaced by a rinse with sodium hydroxide (0.25 M). Standard curves for all analytes were linear over the range of 0.05-1 mm. Standard curves constructed by serial dilution of a leaf extract were also highly linear, and this, combined with the excellent recovery of added solutes in a spike and recovery test, suggests quantification was unaffected by the complex matrix that is present in un-purified, hot water extracts of leaves. There were significant differences in concentrations of the major anions and cations between the species studied.     

Some properties of alginate gels derived from algal sodium alginate

Alginic acid in soluble sodium alginate turns to insoluble gel after contact with divalent metal ions, such as calcium ions. The sodium alginate character has an effect on the alginate gel properties. In order to prepare a suitable calcium alginate gel for use in seawater, the effects of sodium alginate viscosity and M/G ratio (the ratio of D-mannuronate to L-guluronate) on the gel strength were investigated. The wet tensile strengths of gel fibers derived from high viscosity sodium alginate were higher than those from low viscosity sodium alginate. The tensile strength increased with diminishing sodium alginate M/G ratio. Among the gel fibers tested, the gel fiber obtained from a sodium alginate I-5G (1% aqueous solution viscosity = 520 mPa·s, M/G ratio = 0.6) had the highest wet tensile strength. After 13 days treatment in seawater, the wet tensile strength of the gel fiber retained 36% of the original untreated gel strength. For sodium alginates with similar viscosities, the seawater tolerance of low M/G ratio alginate was greater than that of the high M/G ratio one. This study enables us to determine a suitable calcium alginate gel for use in seawater.     

Recovery of canola meal proteins by precipitation

Recovery of rapeseed proteins from defatted canola meal by precipitation was investigated. The ability of different precipitating agents, such as sodium hexametaphosphate (HMP), carboxymethylcellulose (CMC), ammonium sulphate, and isoelectric precipitation using HCl, were evaluated based on the yield and mean size of protein aggregates. Almost 94% of dissolved protein was precipitated in the presence of 2.7M ammonium sulphate, while the largest mean protein particle size (32 mum) was obtained in the presence of HMP at pH 3.3.     

Cell immobilization technique for the enhanced production of alpha-galactosidase by Streptomyces griseoloalbus

Streptomyces griseoloalbus was immobilized in calcium alginate gel and the optimal immobilization parameters (concentrations of sodium alginate and calcium chloride, initial biomass and curing time) for the enhanced production of alpha-galactosidase were determined. The immobilization was most effective with 3% sodium alginate and 0.1M calcium chloride. The optimal initial biomass for immobilization was approximately 2.2g (wet wt.). The alginate-entrapped cells were advantageous because there was a twofold increase in the enzyme yield (55 U/ml) compared to the highest yield obtained with free cells (23.6 U/ml). Moreover, with immobilized cells the maximum yield was reached after 72 h of incubation in batch fermentation under optimal conditions, whereas in the case of free cells the maximum enzyme yield was obtained only after 96 h of incubation. The alginate beads had good stability and also retained 75% ability of enzyme production even after eight cycles of repeated batch fermentation. It is significant that this is the first report on whole-cell immobilization for alpha-galactosidase production.     

Photolytic depolymerization of alginate

A photochemical reaction has been developed for the partial de-polymerization of sodium alginate, a polysaccharide utilized in medicine, pharmacy, basic sciences and foods. An aqueous solution of sodium alginate was photochemically depolymerized to ∼40% of its average molecular weight using ultraviolet light in the presence of titanium dioxide catalyst at pH 7 over a period of 3 h. The products were separated giving four fractions all having an average molecular weight that was smaller than that of the starting material. Characterization of the guluronate (G) and mannuronate (M) contents, and determination of the M/G ratio of photochemically depolymerized alginate, were accomplished using ¹H NMR spectroscopy. The resulting M/G ratio was compared to that obtained for alginate fractions produced by acid hydrolysis. The M and G content, of each alginate fraction, was also assigned with regards to their occurrence in G-rich, M-rich or M/G heteropolymeric domains. This new depolymerization method might also be applicable in the preparation of alginate oligosaccharides for use in the food and pharmaceutical industries.     

Purification and properties of pig brain guanine deaminase.

Guanine deaminase (guanine aminohydrolase, EC 3.5.4.3) from pig brain was purified to homogeneity by column chromatography and ammonium sulphate fractionation. Homogeneity was established by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulphate (SDS). The molecular weight of 110 000 was determined by gel filtration and sucrose density gradient centrifugation. SDS polyacrylamide gel electrophoresis indicated subunits of a molecular weight of 50 000. The amino acid composition, the isoelectric point and the number of -SH groups were determined. 5.5'-Dithiobis-(2-nitrobenzoic acid) reacts with about seven -SH groups in the native enzyme, but upon denaturation with SDS, 10 -SH groups react with this former reagent. Using electrolytic reduction, 44 half-cystines were determined in accordance with the number of cysteic acid residues determined by amino acid analysis after performic acid oxidation. The Km values determined for substrates of the enzyme were 1.1 . 10(-5) M for guanine in 0.1 M Tris. HCl buffer (pH 8.0) and 3.3 . 10(-4) M for 8-azaguanine in 0.1 M phosphate buffer, pH 6.4. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 6.2 and pH 8.2. The chemical and kinetic evidence suggests that cysteine and histidine may be essential for the catalysis.     

Effects of sterilization treatments on some properties of alginate solutions and gels.

Aqueous sodium alginate solutions were subjected to various heat sterilization treatments. Sodium alginate powder was also treated by both gamma-irradiation and ethylene oxide sterilization. The effects of these treatments on the viscosities of sodium alginate solutions and both the diameter and strength of the beads formed in 0.1 M CaCl2 solutions were determined quantitatively. The viscosity of sodium alginate solutions and the gel strength of the calcium alginate beads decreased with increasing sterilization temperature while the bead diameters were found to increase. All these effects can be attributable to a reduction in the degree of polymerization of the alginate molecules as a result of the heat treatments. Ethylene oxide and gamma-irradiation treatments caused similar effects. Standard conditions for sterilization are necessary for comparative studies with alginate beads.