Cloning,over expression and functional attributes of serine proteases from Oceanobacillus iheyensis O.M.A18 and Haloalkaliphilic bacterium O.M.E12

Cloning, over-expression, characterization and structural and functional analysis of two alkaline proteases from the newly isolated haloalkaliphilic bacteria: Oceanobacillus iheyensis O.M.A₁8 and Haloalkaliphilic bacterium O.M.E₁2 were carried out. The cloned protease genes were over-expressed in Escherichia coli within 6 h of the IPTG induction. The protease genes were sequenced and the sequence submitted to the GenBank with the accession numbers, HM219179 and HM219182. The recombinant proteases were active in the range of pH 8–11 and temperature 30–50 °C. The amino acid sequences of the alkaline proteases displayed hydrophobic character and stable configurations. The amino acids Asp 141, His 171 and Ser 324 formed the catalytic triad, while Ile, Leu and Ser were other amino acid moieties present in the active site. The characteristics of the recombinant proteases were compared and found to be similar to their native counterparts. On the basis of the in-silico analysis and inhibitor studies, the enzymes were confirmed as serine proteases. The study hold significance as only limited enzymes from the haloalkaliphilic bacteria have been cloned, sequenced and analyzed for the structure and function analysis.     

Removal of Cr6+ from groundwater using zero-valence iron in the laboratory

Abstract

In this paper, we describe a series of laboratory experiments which quantify the rate of Cr⁶⁺ reduction by Fe⁰. The main goal of these experiments was to determine the removal efficiency of Cr⁶⁺ by iron. The results indicate that Fe⁰ reduces Cr⁶⁺ to Cr³⁺ under alkaline and slightly acidic conditions. The removal efficiency rises with an increase of the initial concentration of Cr⁶⁺ (1 mg/L to 10 mg/L) when the quantity of Fe⁰ is stable. The removal efficiency increases as the quantity of Fe⁰ is raised when other conditions are constant. The removal efficiency would not be affected by other inorganic ions unless they were present at very high concentrations. When the initial concentration Cr⁶⁺ is 10mg/L and pH is 6.5–7.7, the final concentration of Cr⁶⁺ in effluent is less than 0.05 mg/L and the total Fe is less than 0.3 mg/L in effluent.     

Ciliate communities in shallow groundwater: seasonal and spatial characteristics

1. Ciliated protozoans (Phylum Ciliophora) were collected from five sites in a shallow groundwater system in southern Ontario, Canada over a 13‐month period: one at the spring source, two along the channel banks, and two in the stream channel. Ciliates and environmental data were collected from surface water and at five depths into the sediment, located at 20, 40, 60, 80 and 100 cm. 2. Species richness was high (170 ciliate species belonging to 89 genera were identified) and variable, both spatially and temporally. Highest species richness (86) occurred between 20 and 60 cm, and typically decreased below 60 cm. 3. Ciliate densities were also seasonally and spatially variable. Densities peaked in March between 40 cm (as high as 69 900 cells L⁻¹) and 60 cm, and again in May and June at 80 and 100 cm. Densities were lowest in winter. The surface‐water ciliate community had a different species composition and lower population densities. 4. At all depths, small (<50 μm) bacterivorous ciliates typically dominated, but omnivorous and predatory species were also present (combined, up to 30% of the average density). 5. Several ciliate genera, traditionally considered planktonic, occurred at low densities from 40 cm down to 100 cm. 6. Ordination analysis indicated that the main factors influencing the shallow groundwater ciliate communities were depth and temperature. 7. Dissolved oxygen also appeared to influence these communities in that they typically comprised genera that preferred either low‐oxygen or anaerobic conditions.     

Differentiation of smooth muscle cells in the fetal rat testis and ovary: localization of alkaline phosphatase,smooth muscle myosin,F-actin,and desmin

Summary The histochemical demonstration of alkaline phosphatase (AP) activity and localization of smooth muscle myosin (SMM), F-actin, and desmin were carried out on frozen sections of testes and ovaries from 15-day-old fetal to newborn rats. The presence of immunocytochemically localized SMM and desmin was confirmed by Western blot analysis of proteins from isolated gonads. The development of smooth muscle cells was predominant in the testis. The first SMM-positive cells with an increasing intensity for F-actin and desmin appeared in the testicular tunica albuginea and around the testicular cords by the age of 16 days. A continuous layer of SMM- and F-actin-positive (but not uniformly desmin-positive) myoid cells was detected in the newborn testis. In the early gonads and in the newborn ovary, a majority of the interstitial cells expressed desmin, indicating that, in undifferentiated tissues, non-myogenic cells may also express desmin. During fetal development, male and female gonocytes showed a decrease in F-actin content but retained their high AP activity. In the cortex of the newborn rat ovary, the observed high AP activity and the presence of desmin may be associated with the postnatal histogenesis of the follicles. The presence of SMM-containing cells in the hilus of the ovary may be required for the demarcation of the ovary from the mesonephros by the constriction of the mesovarium. The occurrence of SMM-positive cells predominantly in male fetuses suggests that the development of the contractile cells in the fetal testis may be induced by testicular androgens.     

Regulation of polyphosphate metabolism in Acinetobacter strain 210A grown in carbon- and phosphate-limited continuous cultures

The response of Acinetobacter strain 210A to low phosphate concentrations was investigated in P- or C-limited chemostat cultures. The organism accumulated poly--hydroxybutyric acid under P-deprivation, at phosphate concentrations ranging from 0.1 to 0.7 mM. The amount of biomass was proportional to the phosphate concentration in the medium and no polyphosphate was formed. When shifting a culture from P- to C-limitation phosphate was accumulated as polyphosphate. No poly--hydroxybutyrate could be detected in these cells. The amount of polyphosphate in the cell showed a hysteresis. When cultures were shifted from low to high phosphate concentrations, polyphosphate reached a maximum of about 60 mg P per gram of dry weight at about 3 times excess phosphate (ca. 2.5 mM P_i). It decreased to 45 mg P per gram dry weight at approximately 5 times the phosphate needed for growth (ca. 3.5 mM P_i). In the reverse case (high to low) polyphosphate did never exceed 45 mg P per gram dry weight. The specific activities of alkaline phosphatase and the phosphate uptake system were induced at residual P_i concentrations below the detection limit (<10 M). The specific uptake rate followed also a hysteresis. The specific activities of polyphosphatase and polyphosphate: AMP phosphotransferase increased when polyphosphate formation was possible.Abbreviations HPP High polymeric polyphosphates - PHB Poly--hydroxybutyric acid - PP_n Polyphosphate - PQQ Pyrrolo-quinoline quinone - U 1 mol product formed · min^-1     

Alkaline phosphatase activities of anAnabaena sp. from deep-water rice

Anabaena sp. grew with mono- and di-ester phosphate compounds as sources of phosphate, indicating the presence of phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) activities. Cell-bound PMEase and PDEase activities were detected during growth in 0.5 and 10 mg PO₄l^–1 only when the cellular phosphate concentration fell to 0.46% of cell protein and the activities increased as cellular phosphate content decreased. The K_m values for these enzymes were 0.3mm forp-nitrophenyl phosphate and 0.2mm for bis-p-nitrophenyl phosphate, respectively. Only PMEase activity was found extracellularly. The pH optima for PMEase and PDEase were 10.2 and 10.4, respectively, and the temperature optima at pH 10.2 were 37°C and 40°C, respectively. Ca²⁺ increased the enzyme activities while Zn²⁺ caused marked inhibition. The inorganic phosphate repressed the cellular PMEase activity after a lag of 4 h.     

Effect of metabolic acidosis on phosphate transport by the renal brush-border membrane

Metabolic acidosis produces a phosphaturia which is independent of parathyroid hormone or dietary phosphorus intake. To study the underlying mechanism, inorganic phosphate (P_i) and glucose transport were studied in brush-border membrane vesicles prepared from the renal cortex of parathyroidectomized rats gavaged for three days with either 7.5 ml of 1.6% NaCl (control) or 1.5% NH₄Cl (acidosis). At killing, blood pH and plasma bicarbonate were $\text{7.36 ± 0.01}$ and $\text{21.8 ± 0.8}\text{mequiv./l}$, respectively, in control and $\text{7.12 ± 0.03}$ ($\text{P < 0.01}$) and $\text{11.1 ± 1.2}$ ($\text{P < 0.01}$) in acidotic rats. Serum P_i was similar in both groups, while 24 h urine P_i excretion was higher in the acidotic group ($\text{P < 0.01}$). Peak sodium-dependent uptake of P_i, measured after 1.5 min of incubation, was higher in controls than acidotic rats ($\text{4442 ± 464}$ vs. $\text{2412 ± 259}\text{pmol/mg}$ protein, $\text{P < 0.01}$), whereas peak glucose uptake at 1.5 min was not significantly different between the groups. Equilibrium values for P_i and glucose uptake were similar in the two groups. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for P_i uptake in the control and acidotic animals were not different, 0.036 and 0.040 mM, respectively. By contrast, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ was higher in controls than in the acidotic group, 3.13 vs. 1.15 nmol/mg protein per 15 s. These results suggest that metabolic acidosis directly inhibits P_i uptake by the brush border of the proximal tubule by decreasing the availability of P_i carriers of the renal brush-border membrane.     

耐碱性芽孢杆菌碱性淀粉酶的研究：Ⅱ.菌株的诱变与产酶条件

耐碱性巨大芽孢杆菌（Ｂａｃｉｌｌｕｓ ｍｅｇａｔｅｒｉｕｍ）９－Ａ２经紫外线、甲基磺酸乙酯和硫酸二乙酯等多次诱变处理,获得一株产碱性α－淀粉酶能力较高的变异株Ｌ－４９。Ｌ－４９菌株比出发株的产酶能力提高近２．５倍,当以酵母膏为氮源,可溶性淀粉为碳源,初始ｐＨ９￣１０,种龄１８ｈ,接种量５％（Ｖ／Ｖ）,３０℃培养４８ｈ,酶活力可达７３０μ／ｍｌ。     

Optimization and maintenance of soluble methane monooxygenase activity inMethylosinus trichosporium OB3b

Soluble methane monooxygenase (sMMO) maximization studies were carried out as part of a larger effort directed towards the development and optimization of an aqueous phase, multistage, membrane bioreactor system for treatment of polluted groundwater. A modified version of the naphthalene oxidation assay was utilized to determine the effects of methane:oxygen ratio, nutrient supply, and supplementary carbon sources on maximizing and maintaining sMMO activity inMethylosinus trichosporium OB3b.Methylosinus trichosporium OB3b attained peak sMMO activity (275–300 nmol of naphthol formed h^–1 mg of protein^–1 at 25°C) in early stationary growth phase when grown in nitrate mineral salts (NMS) medium. With the onset of methane limitation however, sMMO activity rapidly declined. It was possible to define a simplified nitrate mineral salts (NMS) medium, containing nitrate, phosphate and a source of iron and magnesium, which allowed reasonably high growth rates (_max 0.08 h^–1) and growth yields (0.4–0.5 g cells/g CH₄) and near maximal activities of sMMO. In long term batch culture incubations sMMO activity reached a stable plateau at approximately 45–50% of the initial peak level and this was maintained over several weeks. The addition of d-biotin, pyridoxine, and vitamin B₁₂ (cyanocobalamin) increased the activity level of sMMO in actively growing methanotrophs by 25–75%. The addition of these growth factors to the simplified NMS medium was found to increase the plateau sMMO level in long term batch cultures up to 70% of the original peak activity.Abbreviations sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - NMS nitrate mineral salts - TCE trichloroethene - NADH reduced nicotinamide adenine dinucleotide     

Evaluation of health risks from a buried mass of diesel fuel before and after bioremediation

Plans are being formulated for in situ bioremediation of a subsurface plume of diesel fuel No. 2 that resulted from an accidental fuel release. Raoult's law and the aqueous solubilities of the toxic components were used to estimate organic contaminant concentrations in leachate from the untreated fuel mass. Carcinogenic risks and noncarcinogenic hazard indices were calculated for undiluted leachate. An 80% decrease in hydrocarbon mass and increases in the average molecular weights of the component fractions were assumed to result from the treatment. Sample calculations are provided to show how to evaluate results of analyses for petroleum hydrocarbons after bioremediation.