Marked effect of cations on the separation of spinach thylakoid chlorophyll-proteins on polyacrylamide gel electrophoresis at 0 degree C in the presence of alkanolammonium dodecyl sulfates

The separation of spinach thylakoid chlorophyll-proteins on polyacrylamide gel electrophoresis at 0 degrees C in the presence of dodecyl sulfate is markedly influenced by the kind of surfactant cation in the media used for solubilization and electrophoresis. The mode of separation can thus be modulated through the cation selection. Three kinds of alkanolammonium dodecyl sulfates were tested and their abilities as to the dissociation of the molecular assemblies of chlorophyll-proteins were found to decrease in the following order: tris(hydroxymethyl)methylammonium, triethanolammonium and triisopropanolammonium. Comparison of the electrophoretic patterns obtained with different kinds of cation may help clarify the hierarchy of the molecular assemblies of chlorophyll-proteins.     

Multiple forms of chlorophyll-protein complexes from a thermophilic cyanobacterium Synechococcus sp

Eight chlorophyll-proteins were resolved from the thylakoid membranes, or digitonin particles, of a thermophilic cyanobacterium Synechococcus sp. by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six chlorophyll-proteins with slower electrophoretic mobilities were shown to be P700-chlorophyll a-protein complexes (CP1), whereas faster-moving proteins (CP2) were related to photosystem 2. Extraction of CP1 complexes from the membranes with different detergent/chlorophyll ratios and reelectrophoresis of extracted CP1 complexes indicated that the chlorophyll-proteins are closely interrelated with each other; any CP1 complex could be transformed to other CP1 complexes with faster electrophoretic mobilities. This, together with the Ferguson plot and the polypeptide composition, showed that six CP1 complexes are different in terms of polypeptide composition, oligomerization, SDS-binding, or conformation of the proteins but represent, in the order of increasing electrophoretic mobility, increasing degree of modification of the native P700-chlorophyll a-protein.     

Effects of medium components on L-ornithine production byBrevibacterium ketoglutamicum

Effects of yeast extract, and ammonium sulfate were investigated on the production of L-ornithine by an arginine auxotroph.Brevibacterium ketoglutamicum in flask and batch cultures. Yeast extract as an arginine source and ammonium sulfate as an inorganic nitrogen source had significant effects on L-ornithine, production and cell growth. L-ornithine production was repressed by the excessive addition of arginine. Reversion of auxotrophic cells to the wild type was observed when the initial yeast extract concentration was too low. There existed optimum concentrations of yeast extract and ammonium sulfate for L-ornithine production. The effects of yeast extract and ammonium sulfate concentrations on the Leudeking-Piret model parameters were examined to analyze, the relationship between cell growth and L-ornithine production.     

Influence of ammonium ions on calcite and struvite formation byazotobacter in chemically defined media

The formation of calcite and struvite crystals byAzotobacter vinelandii andA.chroococcum was studied on chemically defined media. The crystals were identified microscopically and by X-ray differentiation analysis. Calcite crystal formation was inhibited specifically with 0.05 % ammonium sulfate. However, 0.2 % ammonium sulfate was required for struvite crystal formation.     

Solubilization and partial purification of n,n'-dicyclohexylcarbodiimide-sensitive ATPase from pea cotyledon mitochondria

The N,N′-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase of pea (Pisum sativum L.) cotyledon mitochondria was solubilized from submitochondrial particle membranes with sodium cholate and ammonium sulfate. Ammonium sulfate precipitation of the enzyme resulted in an increase in specific activity. At between 38% and 45% saturated ammonium sulfate, 20% of the ATPase activity was precipitated, with a specific activity 4 to 5 times higher than that of the crude enzyme. The precipitate was highly sensitive to DCCD.

The properties of the ammonium sulfate preparation were investigated. It contained levels of cytochrome and NADH dehydrogenase contamination comparable to those of the highly purified F₀F₁ preparations from animal tissue. The high degree of purification was corroborated by sodium dodecyl sulfate electrophoresis.

     

Production of Nitrous Oxide by Ammonia-Oxidizing Chemoautotrophic Microorganisms in Soil

Gas chromatographic studies showed that nitrous oxide was produced in each instance when sterilized (autoclaved) soil was incubated after treatment with ammonium sulfate and inoculation with pure cultures of ammonia-oxidizing chemoautotrophic microorganisms (strains of Nitrosomonas, Nitrosospira, and Nitrosolobus). Production of N₂O in ammonium-treated sterilized soil inoculated with Nitrosomonas europaea increased with the concentration of ammonium and the moisture content of the soil and was completely inhibited by both nitrapyrin and acetylene. Similar effects of nitrapyrin, acetylene, ammonium concentration, and soil moisture content were observed in studies of factors affecting N₂O production in nonsterile soil treated with ammonium sulfate. These observations support the conclusion that, at least under some conditions, most of the N₂O evolved from soils treated with ammonium or ammonium-producing fertilizers is generated by chemoautotrophic nitrifying microorganisms during oxidation of ammonium to nitrite.     

Medium optimization of carbon and nitrogen sources for the production of spores from Bacillus amyloliquefaciens B128 using response surface methodology

The production of spores from Bacillus amyloliquefaciens B128 was investigated in shaker-flask cultivation. Optimization of the culture medium was carried out by using a two-step approach. A quick identification of a suitable source of carbon and nitrogen was obtained by a simple screening experiment, which was followed by an application of response surface methodology (RSM) for further optimization design. A five-level four-factor central composite design was employed to determine the maximum spore yield at optimum levels for lactose, tapioca, ammonium sulfate and peptone. Tapioca and peptone showed a significant linear main effect, while lactose and ammonium sulfate had no significant linear effect. The spore production was also significantly affected by lactose–ammonium sulfate and lactose–peptone. Optimum cultivation parameters were (g/L): 12.7 of lactose, 16.7 of tapioca, 1.8 of ammonium sulfate and 8.0 of peptone. The prediction spore yield was 5.93 × 10⁸ (no/mL). The actual experimental results were in agreement with the prediction.     

Purification of (S)-3-cyano-5-methylhexanoic acid from bioconversion broth using an acetone/ammonium sulfate aqueous two-phase system

(S)-3-Cyano-5-methylhexanoic acid ((S)-CMHA) is the key chiral intermediate of pregabalin. In this paper, an aqueous two-phase system (ATPS) was developed to extract (S)-CMHA from nitrilase-catalyzed bioconversion broth. Inorganic salts and hydrophilic solvents were screened to form ATPS, among which an acetone/ammonium sulfate ATPS was investigated in detail, including phase diagram, effect of phase composition and stability of (S)-CMHA. The maximum product recovery of 99.15% was obtained by an optimized ATPS system composed of 15% (w/w) ammonium sulfate and 35% (w/w) acetone with the removal of 99% cells and 86.27% proteins. The total (S)-CMHA yield reached 92.11% after back-extraction. The recycling use of ammonium sulfate was investigated, and 93.10% of salt in the salt-rich phase was recovered with the addition of methanol. The results demonstrated the efficiency of the two-step extraction process for separation of (S)-CMHA.     

Milk-clotting Enzyme from Microorganisms: V. Purification and Crystallization of Mucor Rennin from Mucor pusillus var. Lindt.1

A rennin crystal was obtained from the crude milk-clotting enzyme of Mucor pusillus var. Lindt. The crude enzyme was purified by using columns of Amberlite CG-50, diethylaminoethyl Sephadex A-50, and Sephadex G-100. This purified enzyme was dissolved in 0.1 M sodium acetate (pH 5.0) buffer to a final concentration of 2 to 3%; ammonium sulfate (to 40% saturation) was added, and the resulting solution was placed in cellophane tubes. The enzyme solution was dialyzed against 0.1 M sodium acetate buffer (pH 5) containing ammonium sulfate was added dropwise to the outside solution of the cellophane tube, and the concentration of ammonium sulfate in the cellophane tube increased gradually. The crystals of enzyme were formed in the cellophane tube when the concentration reached approximately 50% saturation. After the enzyme solution was concentrated in the freezer, the crystals were obtained. The activity of the crystalline enzyme was inhibited by Hg²⁺, Ag⁺, Zn²⁺, and KMnO₄.     

Milk-clotting Enzyme from Microorganisms: V. Purification and Crystallization of Mucor Rennin from Mucor pusillus var. Lindt

A rennin crystal was obtained from the crude milk-clotting enzyme of Mucor pusillus var. Lindt. The crude enzyme was purified by using columns of Amberlite CG-50, diethylaminoethyl Sephadex A-50, and Sephadex G-100. This purified enzyme was dissolved in 0.1 M sodium acetate (pH 5.0) buffer to a final concentration of 2 to 3%; ammonium sulfate (to 40% saturation) was added, and the resulting solution was placed in cellophane tubes. The enzyme solution was dialyzed against 0.1 M sodium acetate buffer (pH 5) containing ammonium sulfate was added dropwise to the outside solution of the cellophane tube, and the concentration of ammonium sulfate in the cellophane tube increased gradually. The crystals of enzyme were formed in the cellophane tube when the concentration reached approximately 50% saturation. After the enzyme solution was concentrated in the freezer, the crystals were obtained. The activity of the crystalline enzyme was inhibited by Hg²⁺, Ag⁺, Zn²⁺, and KMnO₄.     

Hydrophobic chromatography of galactosyltransferase.

O-Glycosidic analogs of N-acetylglucosamine are good substrates for galactosyltransferase, and as the O-substituted group becomes more hydrophobic, the apparent K_m decreases as much as 2000-fold. l-leucine, leucine-amide, norleucine, valine, ?-amino-n-caproic acid and tyrosine-agaroses all retain galactosyltransferase in the presence of 1.25 m ammonium sulfate. The enzyme is eluted quantitatively with a five- to tenfold purification by a decreasing linear gradient of ammonium sulfate. Galactosyltransferase was not specifically bound on any of a series of ω-aminoalkylagaroses tested. A simple and highly efficient procedure for the isolation of galactosyltransferase from bovine skim milk was developed and consisted of a 40–60% ammonium sulfate precipitation of the enzyme from skim milk followed by chromatography on (1) norleucine-Sepharose, (2) UDP-hexanolamine-Sepharose, and (3) α-lactalbumin-Sepharose.     

The structure and function of CPa-1 and CPa-2 in Photosystem II

This review presents a summary of recent investigations examining the structure and function of the chlorophyll-proteins CPa-1 (CP47) and CPa-2 (CP43). Comparisons of the derived amino acid sequences of these proteins suggest sites for chlorophyll binding and for interactions between these chlorophyll-proteins and other Photosystem II components. Hydropathy plot analysis of these proteins allows the formulation fo testable hypotheses concerning their topology and orientation within the photosynthetic membrane. The role of these chlorophyll-proteins as interior light-harvesting chlorophyll-a antennae for Photosystem II is examined and other possible additional roles for these important Photosystem II components are discussed.This work was supported by NSF Grant DMB-8740292.     

Lucrative pectinase production by novel strain Pseudozyma sp. SPJ with statistical optimization techniques using agro-industrial residues

Production of high titers of an alkaline, extracellular and thermo-tolerant pectinase by a newly isolated yeast Pseudozyma sp. SPJ was carried out under solid state fermentation. Citrus peel, the inexpensive agro-industrial residue used as substrate, was experienced to be unsurpassed. Response surface methodology was conducted to optimize the culture conditions for Pseudozyma sp. SPJ for hyper production of pectinase. Plackett Burman design was applied to identify the most effective culture variables. Out of nine variables studied, incubation time, moisture content and ammonium sulfate were detected as most important. A full factorial Central Composite Design was used to optimize the levels of these variables, which resulted in 17-fold increase （71.19 IU/g to 1215.66 IU/g dry substrate） in the enzyme yield. The results of analysis of variance and multiple regression analysis implies that the effect of incubation time （p 〈 0.000） and moisture content （p 〈 0.018） is more than ammonium sulfate. And also the interaction of moisture content with ammonium sulfate （p 〈 0.002） is more significant.     

Effect of salts and Triton X-100 on ultrafiltration purification and properties of extracellular glucose oxidase from <Emphasis Type="Italic">Penicillium adametzii</Emphasis> LF F-2044.1

We compared the effectiveness of glucose oxidase isolation from the culture fluid of Penicillium adametzii LF F-2044.1 in the presence of ammonium sulfate, ammonium chloride, and Triton X-100. Ammonium chloride inhibited glucose oxidase in the culture fluid. This compound increased K _M (by 1.2–1.3 times), but decreased V _max for D-glucose oxidation (by 1.7–1.8 times). Ammonium sulfate had little effect on kinetic parameters. Combined treatment with salts and Triton X-100 was followed by a significant increase in the effectiveness of ultrafiltration purification of the culture fluid. The samples of glucose oxidase were electrophoretically characterized. The dependence of kinetic parameters on glucose oxidase concentration during oxidation of D-glucose was evaluated. The catalytic constant and k _cat/K _M ratio for glucose oxidase samples from the culture fluid isolated in the presence of additives significantly surpassed those for enzyme samples, which were obtained by ultrafiltration of the culture fluid with no additives and chromatography on aluminum oxide. The activity of glucose oxidase isolated from the culture fluid in the presence of ammonium chloride was lower compared to that of the enzyme obtained in the presence of ammonium sulfate. This agent is preferable for ultrafiltration of the culture fluid.     

Inactivation of Kanamycin,Neomycin, and Streptomycin by Enzymes Obtained in Cells of Pseudomonas aeruginosa

Ten strains of Pseudomonas aeruginosa were disrupted and centrifuged. The supernatant fluids from centrifugation at 105,000 × g contained enzymes inactivating kanamycin, neomycin, and streptomycin in the presence of adenosine triphosphate. Kanamycin-inactivating enzyme was precipitated with ammonium sulfate at 66% of saturated concentration, and the inactivated kanamycin was shown to be kanamycin-3′-phosphate in which the C-3 hydroxyl group of 6-amino-6-deoxy-d-glucose moiety was phosphorylated. This is identical with kanamycin inactivated by Escherichia coli carrying R factor. Streptomycin-inactivating enzyme was precipitated with ammonium sulfate at 33% of saturated concentration.     

Purification of Escherichia coli amplifiable plasmids by high-salt Sepharose chromatography

This new method allows an easy and rapid purification of amplifiable Escherichia coli plasmids such as pBR 322 without the use of cesium chloride centrifugation. After gentle lysis, centrifugation, and phenol extraction, the material is reextracted with acid phenol to remove the bacterial DNA. The high-molecular-weight ribosomal RNA is removed by precipitation with 2 m ammonium sulfate and the tRNA by passage through a small column of Sepharose CL 4B in the presence of 2 m ammonium sulfate.     

Effects of iron sources on the growth and lipid/carbohydrate production of marine microalga <Emphasis Type="Italic">Dunaliella tertiolecta</Emphasis>

The effects of iron sources with different speciation and anionic moieties (ferric chloride, ferrous chloride, ferric EDTA, ferrous EDTA, ferric ammonium sulfate, and ferrous ammonium sulfate) on the cell growth and the production of energy storage (lipid and carbohydrate) from Dunaliella tertiolecta were investigated. The influence of iron dosage was also compared in the range from 0.65 mg/L (1X) to 6.5 mg/L (10X) as Fe concentration. Best cell growth rate was achieved when ferrous ammonium sulfate was used. Ferric EDTA resulted in higher lipid content than other iron sources, while ferrous ammonium sulfate favored the accumulation of carbohydrate among six iron sources. The accumulations of lipid and carbohydrate as energy storage competed each other and thus both contents did not increase together. In the presence of ferric EDTA, lipid content is increasing, while carbohydrate content is decreasing. On the contrary, lipid content is decreasing while carbohydrate is increasing in the presence of ferric ammonium sulfate. Because the overall carbohydrate content was larger than that of lipid, bioethanol production would be more advantageous than biodiesel production with the present D. tertiolecta strain if the carbohydrate in D. tertiolecta contains a high fraction of glucose with a good saccharification yield.     

The modulation of cell surface cAMP receptors from Dictyostelium disscoideum by ammonium sulfate

Dictyostelium discoideum cells contain a heterogeneous population of cell surface cAMP receptors with components possessing different affinities (K_d between 15 and 450 nM) and different off-rates of the cAMP-receptor complex ($\text{t}\text{1}\text{2}$ between 0.7 and 150 s). The association of cAMP to the receptor and the dissociation of the cAMP-receptor complex still occur in the presence of 3.4 M ammonium sulfate. However, these processes are strongly altered. (1) Low concentrations of ammonium sulfate (≈ 50 mM) induce an approx. 2-fold increase of the number of cAMP binding sites. The same effect is induced by millimolar concentrations of CaCl₂. Ammonium sulfate and CaCl₂ are not additive, which suggests that these salts may act via the same mechanism. (2) High concentrations of ammonium sulfate (3.4 M) induce an alteration in the proportioning of the various cAMP binding sites to the components with the highest affinity. (3) High concentrations of ammonium sulfate (3.4 M) retard the dissociation of all binding sites about 3–6-fold, thus giving rise to an increase in the affinity of all cAMP-binding components.     

Three-phase partitioning as a rapid and efficient method for purification of invertase from tomato

Three-phase partitioning (TPP), a technique used in protein purification, was used to purify invertase from tomato (Lycopersicon esculentum). The method consists of simultaneous addition of ammonium sulfate and t-butanol to the crude enzyme extract in order to obtain the three phases. Different parameters (ammonium sulfate saturation, crude extract to t-butanol ratio and pH) essential for the extraction and purification of invertase were optimized to get highest purity fold and yield. It was seen that, 50% (w/v) ammonium sulfate saturation with 1:1 (v/v) ratio of crude extract to t-butanol at pH 4.5 gave 8.6-fold purification with 190% activity recovery of invertase in a single step. Finally, the purified enzyme was also characterized and the general biochemical properties were determined. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of enzyme showed considerable purification and its molecular weight was nearly found to be as 20 kDa. This work shows that, TPP is a simple, quick and economical technique for purification of invertases.     

Differences in chlorophyll-protein complexes and composition of polypeptides between thylakoids from bundle sheaths and mesophyll cells in maize

Thylakoids from enzymatically separated bundle sheath and mesophyll tissue chloroplasts were examined for their chlorophyll-proteins by tube sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). Differences were found in distribution of chlorophyll among peaks. The chlorophyll-protein a peak (CPa), considered to be the photosystem II (PSII) reaction centre by many authors, was seen to be absent in bundle sheath thylakoid samples. The slab SDS-PAGE revealed the absence of the polypeptides present in PSII preparations of chloroplast subfractions having only PSII activity. This finding confirms Anderson's hypothesis of the structure of grana and stroma thylakoids.