Characterization of Pb<Superscript>2+</Superscript> biosorption from aqueous solution by<Emphasis Type="Italic"> Rhodotorula glutinis</Emphasis>

The yeast Rhodotorula glutinis was examined for its ability to remove Pb(2+) from aqueous solution. Within 10 min of contact, Pb(2+) sorption reached nearly 80% of the total Pb(2+) sorption. The optimum initial pH value for removal of Pb(2+ )was 4.5-5.0. The percentage sorption increased steeply with the biomass concentration up to 2 g/l and thereafter remained more or less constant. Temperature in the range 15-45 degrees C did not show any significant difference in Pb(2+ )sorption by R. glutinis. The light metal ions such as Na(+), K(+), Ca(2+), and Mg(2+) did not significantly interfere with the binding. The Langmuir sorption model provided a good fit throughout the concentration range. The maximum Pb(2+ )sorption capacity q(max) and Langmuir constant b were 73.5 mg/g of biomass and 0.02 l/mg, respectively. The mechanism of Pb(2+) removal by R. glutinis involved biosorption by direct biosorptive interaction with the biomass through ion exchange and precipitation by phosphate released from the biomass.     

Endo-polygalacturonase production from untreated lemon peel byAspergillus sp. CH-Y-1043

Summary Endo-polygalacturonase (endo-PG) production byAspergillus sp. CH-Y-1043 using untreated lemon peel as the sole carbon source was investigated. This strain was observed to produce more activity of endo-PG at 37°C than at 29°C. Untreated lemon peel proved to be a beeter substrate than citrus pectin for endo-PG production. Modification of the culture medium and lowering of the initial pH to 2.8 caused a 10-fold increase in the production of endo-PG activity using lemon peel.     

Development of an Optimized Method to Obtain a Limonene-Rich Concentrate from the Discarded Lemon Peels

In this study, lemon peels were used as volatile component source. Automatic solvent extraction has been used for the recovery of limonene rich citrus volatile extract for the first time. The process parameters (amount of raw material, immersion time and washing time) were analyzed to optimize the process by means of Box-Behnken design via response surface methodology. The optimum conditions were achieved by ~10 g fresh lemon peel, and ~15 min immersion time and ~13 min washing time. The difference between the actual (89.37 mg/g limonene) and predicted (90.85 mg/g limonene) results was satisfactory (<2 %). α-Terpinene, β-pinene, citral, ɣ-terpinene and linalool were determined as other major volatiles in the peel extract. FT-IR and ¹H- and ¹³C-NMR spectroscopies were applied to verify the identified volatile compounds.     

Impact of Cell Wall Structure on the Behavior of Bacterial Cells as Sorbents of Cadmium and Lead

Batch metal sorption studies were conducted to compare the behavior of Gram-positive Bacillus subtilis and Gram-negative Escherichia coli as sorbents of Cd 2+ and Pb 2+ . A pH range from 3.0 to 6.5 was investigated at total metal concentrations of 1 2 10 -4.0 and 3.2 2 10 -5 M. Concentration apparent equilibrium sorption constants (K s n M ) and sorption capacity (S max n ) values were determined for the bacteria by fitting experimental data to one- ( n = 1) and two-site ( n = 2) Langmuir sorption isotherms. The sorption data for each of the bacteria were described well by a one-site model (r 2 > 0.9), Cd 2+ exhibited somewhat lower sorption affinities (log K s M =- 1.5 for B. subtilis , and -0.7 for E. coli ) than Pb 2+ (log K s M =-0.6 for B. subtilis and -0.8 for E. coli ). Corresponding S max values for Cd 2+ and Pb 2+ on B. subtilis were 0.36 mmole/g and 0.27 mmole/g, respectively. For E. coli Cd 2+ and Pb 2+ S max values were lower at 0.10 mmole/g and 0.21 mmole/g. A two-site sorption model yielded an improved fit for only the E. coli data with several orders of magnitude difference evident between high (Cd 2+ log K s1 M = 0.9; Pb 2+ log K s1 M = 1.5) and low (Cd 2+ log K s2 M =- 1.1; Pb 2+ log K s2 M = -1.6) affinity sorption sites. In addition, allowing for the presence of low affinity sorption (i.e., S max2 ) sites further increased the total E. coli metal sorption capacity closer to that of B. subtilis . As expected, the sorption of Cd 2+ and Pb 2+ by the bacteria exhibited a strong dependence on pH with sorption edges in the range of pH 4.2 to 5.6. The results of this study show that, despite differences in cell wall structure and composition, B. subtilis and E. coli exhibit remarkably similar sorption behavior toward Cd 2+ and Pb 2+ , respectively. These similarities can be attributed to the specific chemical reactivity of acidic functional groups (e.g., carboxyl, phosphoryl) that occur in the cell walls of both bacteria.     

Production of pectinases byAspergillus sp using differently pretreated lemon peel as the carbon source

Summary Aspergillus sp strains from decaying lemons were tested for extracellular pectinase production, testing differently pretreated lemon peel as the carbon source instead of pectin. It was found that the production of extracellular polygalacturonase was about the same and that of pectinesterase substantially higher when unwashed fresh lemon peel was used instead of pectin. The culture filtrate obtained showed a clarifying capacity similar to that of a commercial pectinase preparation, but the vitamin C of the juice was less affected by the treatment.     

Purification and characteristics of alkaline proteinase from alkalophylic Bacillus sp.]

Alkaline protease was purified from Bacillus sp. isolated from soil. The pH optimum was 11.5 at 37 degrees C. Calcium divalent cation was effective to stabilize the enzyme especially at higher temperatures. The proteolytic activity was inhibited by active site inhibitors of PMSF (Phenylmethylsulfonyl fluoride), and ions of Mg, Mn, Pb, Li, Zn, Ag, Hg. The enzyme was stable in the presence of some detergents, such as Triton-X-100, Tween-80, SDS (sodium dodecyl sulfate) and EDTA (ethylendiaminetetraacetic acid), pH 11.5 and 37 degrees C for 30 min. The optimum pH was 11.5 at 37 degrees C and the optimum temperature was 62 degrees C at pH 11.5.     

Batch and column studies of biosorption of heavy metals by Caulerpa lentillifera

The biosorption of Cu(II), Cd(II), and Pb(II) by a dried green macroalga Caulerpa lentillifera was investigated. The sorption kinetic data could be fitted to the pseudo second order kinetic model. The governing transport mechanisms in the sorption process were both external mass transfer and intra-particle diffusion. Isotherm data followed the Sips isotherm model with the exponent of approximately unity suggesting that these biosorption could be described reasonably well with the Langmuir isotherm. The maximum sorption capacities of the various metal components on C. lentillifera biomass could be prioritized in order from high to low as: Pb(II)>Cu(II)>Cd(II). The sorption energies obtained from the Dubinin-Radushkevich model for all sorption systems were in the range of 4-6 kJ mol(-1) indicating that a physical electrostatic force was potentially involved in the sorption process. Thomas model could well describe the breakthrough data from column experiments. Ca(II), Mg(II), and Mn(II) were the major ions released from the algal biomass during the sorption which revealed that ion exchange was one of the main sorption mechanisms.     

Inhibition and activation of mannan synthesis in Saccharomyces cerevisiae spheroplast lysates.

Mannan synthetase activity in spheroplast lysates prepared from Saccharomyces cerevisiae was measured by following the incorporation of [14C]mannose from guanosine 5'-diphosphate-[14C]mannose into material precipitable with cold 0.3 M perchloric acid. When enzyme activity was assayed at high concentrations of spheroplast lysate protein (10 mg/ml) in the presence of 7.5 mM MnCl2, a severe inhibition was observed. This inhibition could be relieved by preincubation of the spheroplast lysate at 4 degrees C for 16 to 32 h before assay, by repeated freezing and thawing of the spheroplast lysate, or by the omission of MnCl2 from assay mixtures. The addition of ethylenediaminetetraacetic acid or monovalent cations removed inhibition in the presence of Mn2+. No similar inhibition was observed when a washed membrane fraction was substituted for spheroplast lysate as the source of mannan synthetase. The supernatant fluid obtained by centrifuging spheroplast lysate at 100,000 x g, when added to assay mixtures containing either spheroplast lysate preincubated at 4 degrees C or washed membrane fraction, also caused inhibition of enzyme activity. This inhibition required 7.5 mM MnCl2 and was destroyed by heating the supernatant fluid at 60 degrees C for 10 min, or by trypsin treatment at 30 degrees C. These results indicate the existence of a protein inhibitor of mannan synthesis whose inhibitory activity in spheroplast lysates may be modulated by preincubation at low temperature or by varying the available Mn2+ concentration.     

Production of biomass and beta-D-galactosidase by Candida pseudotropicalis grown in continuous culture on whey

The production of biomass and beta-D-galactosidase by the lactose-utilizing yeast Candida pseudotropicalis NCYC 744 in whey medium was studied. Apparent optimization of growth conditions and medium was done in continuous culture. Optimaql pH and temperature were 2.6 and 36-38 degrees C, respectively, Limitations in Cu, Zn, and possbily Mn were detected in deproteinized whey medium. Additions of tryptophan estimulated growth of the yeast. Under optimal conditions in medium supplemented with excess tryptophan, Cu, Zn, and Mn the maximum values obtained: yeast concentration, 4.6 g/L; yeast productivity, 1.4 g/L h (at D = 0.35 h(-1)); enzyme volumetric productivity, 2100 U/L h (at D = 0.25 h(-1)); maintenance coefficient, 5-10 mg lactose/g cell h; saturation constant (K(s)) for lactose, 4.76mM; maximum specific growth rate, (mu(max)), 0.47 h(-1). No significant increase in specific enzyme activity (U/mg cell) was observed after medium optimiztion evidencing the importance of regulatory controls in enzyme synthesis.     

Purification and properties of rat kidney catechol-O-methyltransferase]

The S-adenosyl-methionine: catechol-O-methyltransferase (EC 2.1.1.6) from rat kidney was purified about 650 fold as compared with the homogenate and the result of disc electrophoresis presented. The purification involved extraction, precipitation at pH 5, ammonium sulfate fractionation, Chromatographies on Biogel 0.5 m, Ultrogel AcA 44 and DE Sephadex A 50. Affinity chromatography was tried but unsuccessful. The enzyme exhibited two pH optima at 7.9 and 9.6 with a minimum at about 8.9. The COMT had a temperature optimum of 50 degrees C, with activation energy of 23.1 Kcal/Mole between 25-35 degrees C, 18.9 Kcal/mole between 35-45 degrees C and the Q10 within the range of 25-35 degrees amounted to 3.5. The molecular weight was estimated to be 21500+/-1000 daltons from its behavior on Ultrogel AcA 44 and the pH1 determined by electrofocalisation was near 5.50. The time of half life of the best purified enzymatic extract was found to be 2 h 10 min. at -20 degrees C. At basic pH the instability of the enzyme was increased. Since O-methylation required the presence of divalent cations, our results show that apparent Michaelis constants for Mg++ and Mn++ were respectively 0.50 X 10(-3) M and 0.33 X 10(-5) M. The study of their Hill's number indicated that there was only one point of fixation on the enzyme. The Km value determined by Florini and Vestling's method were 2.5 X 10(-4) M and 11.9 X 10(-5) M for epinephrine and S-adenosyl-methionine respectively. All results were discussed with respect to other investigations.     

An ESR study of the Mn(II)-heavy meromyosin system.

The Mn(II)-heavy meromyosin system was studied by measuring the ESR spectrum of Mn(II). The temperature dependence of the line width parameter W(1, t) of a freshly prepared sample changes at around 7-10 degrees C, where W(1, t) is the reciprocal of the peak-to-peak height of the lowest magnetic field component of the hyperfine structure. It is shown that the change in the slope of W(1, t) at 7-10 degrees C is due to a change in the structure of Mn(II)-heavy meromyosin or a change in the interaction between Mn(II) and heavy meromyosin without ATP. This result is in accord with the recently reported observations that heavy meromysin ATPase activity showed different temperature dependence above and below 10 degrees C in the presence of Mn(II). The characteristics of the spectrum of the Mn(II)-heavy meromyosin system in the liquid state between 2 degrees C and 20 degrees C are compared with those of a frozen sample of Mn(II)-heavy meromyosin in a low temperature region (-50-0 degrees C) and with those of the lyophilized material. The forbidden transitions are observed, and hence the zero field splitting parameter can be obtained. It is 115 +/- 15 gauss at -50 degrees C, and decreases with increase of the temperature to 70 +/- 15 gauss at 20 degrees C.     

Sludge-Derived Cu and Zn in a Humic-Gley Soil: Effect of Dissolved Metal-Organic Matter Complexes on Sorption and Partitioning

A sequential extraction scheme was combined with sorption isotherm analysis in order to investigate sorption of sewage sludge-derived Cu and Zn to the A-horizon of a humic-gley soil as a whole, and to the operationally defined exchangeable (1?M MgCl₂), carbonate (1?M NaOAc), Fe/Mn oxide (0.04?M NH₂OH.HCl), and organic (0.02?M HNO₃+30% H₂O₂) soil fractions. Sorption parameters were compared for a sample of sludge leachate (with 97.4% of Cu and 63.2% of Zn present as dissolved metal-organic matter complexes, as calculated by geochemical modeling involving MINTEQA2 and verified using an ion exchange resin method) with that of a reference solution exhibiting the same chemical characteristics as the leachate, except for the presence of dissolved organic material. Dissolved metal-organic matter complexes were found to significantly (P<0.05) depress sorption to the bulk soil and each fraction. The greatest depression of Cu and Zn sorption was observed for the exchangeable, carbonate, and Fe/Mn oxide fractions, while the organic fraction of the soil was the least affected. This reflects a greater affinity for the exchangeable, carbonate, and Fe/Mn oxide fractions by the free divalent metal (Cu²⁺, Zn²⁺), with sorption by these fractions attributed to cation exchange, chemisorption, and co-precipitation processes. The sorption characteristics of the organic fraction indicated that Cu and Zn sorption by soil organic matter mostly involved dissolved metal-organic matter complexes. This may be attributed to hydrophobic interactions between nonpolar regions of the dissolved metal-organic matter complexes and solid-phase soil organic matter.     

Experimental binding of lead to a low cost on biosorbent: Nopal (Opuntia streptacantha)

The use of nopal cladodes (Opuntia streptacantha) as raw material for Pb(2+) biosorption was investigated. Batch experiments were carried out to determine Pb(2+) sorption capacity and the efficiency of the sorption process under different pH, initial Pb(2+) and nopal biomass concentrations. The experimental data showed a good fit to Langmuir and Freundlich isotherms models. The maximum adsorption capacity for Pb(2+) was 0.14 mmol g(-1) with an efficiency higher than 94% (pH 5.0 and 2.5 g L(-1) nopal biomass). The Pb(2+) kinetics were best described by the pseudo-second-order rate model. The rate constant, the initial sorption rate and the equilibrium sorption capacity were determined. The practical implication of this study is the development of an effective and economic technology in which the nopal biomass did not undergo any chemical or physical pretreatment, which added to nopal abundance in Mexico and its low cost makes it a good option for Pb(2+) removal from contaminated waters.     

Interaction of calcium with bovine plasma protein C

The binding of 45Ca2+ to bovine plasma protein C (PC) and to activated bovine plasma protein C (APC) has been examined by equilibrium ultrafiltration at pH 7.4 and 25 degrees C. Under these conditions, PC possesses 16.0 plus or minus 2.0 equivalent Ca2+ binding sites, of average KD (8.7 plus or minus 1.5) x 10(-4) M, and APC contains 9.0 plus or minus 1.0 equivalent Ca2+ binding sites, with an average KD of (4.3 plus or minus 1.1) x 10(-4) M. Both Mn2+ and Sr2+ were capable of ready displacement of Ca2+ from a Ca2+-PC complex, while Mg2+ was less effective in this regard. The alpha-thrombin-catalyzed activation of PC was inhibited by the presence of Ca2+. A kinetic analysis of this effect demonstrated that it was, in large part, due to an increase in the Km of the reaction. Addition of other divalent cations, e.g. Mn2+, Sr2+, and Mg2+, in place of Ca2+ also resulted in inhibition of the alpha-thrombin-catalyzed activation of PC in a manner which paralleled their ability to displace Ca2+ from a Ca2+-PC complex. On the other hand, the activation of PC by the coagulant protein from Russell's Viper venom was augmented by the presence of Ca2+. Other divalent metal ions, such as Sr2+ and Mn2+, in the absence of Ca2+, also weakly stimulated this reaction. Mg2+ was without notable effect.     

Microbial Production of Pectin from Citrus Peel

A new method for the production of pectin from citrus peel was developed. For this purpose, a microorganism which produces a protopectin-solubilizing enzyme was isolated and identified as a variety of Trichosporon penicillatum. The most suitable conditions for the pectin production were determined as follows. Citrus (Citrus unshiu) peel was suspended in water (1:2, wt/vol), the organism was added, and fermentation proceeded over 15 to 20 h at 30°C. During the fermentation, the pectin in the peel was extracted almost completely without macerating the peel. By this method, 20 to 25 g of pectin was obtained per kg of peel. The pectin obtained was special in that it contained neutral sugar at high levels, which was determined to have a molecular weight suitable for practical applications.     

Mild, solvent-free omega-hydroxy acid polycondensations catalyzed by candida antarctica lipase B

Immobilized Candida antarctica Lipase B (Novozyme-435) was studied for bulk polyesterifications of linear aliphatic hydroxyacids of variable chain length. The products formed were not fractionated by precipitation. The relative reactivity of the hydroxyacids was l6-hydroxyhexadecanoic acid approximately 12-hydroxydodecanoic acid approximately 10-hydroxydecanoic acid (DPavg congruent with 120, Mw/Mn 6-hydroxyhexanoic acid (DPavg congruent with 80, Mw/Mn < or = 1.5, 48 h, 90 degrees C). Remarkable improvements in molecular-weight buildup resulted from leaving water in the reaction. By 4 h, without application of vacuum, the DPavg for 12- and 16-carbon hydroxyacids was about 90. In contrast, with identical substrates and water removal, the DPavg at 4 h was about 23. Large differences in the molecular-weight build up of 12-hydroxydodecanoic acid were observed for catalyst concentrations (%-by-wt relative to monomer) of 0.1, 0.5, 1, and 10. Nevertheless, by 24 h, with 1% catalyst containing 0.1% lipase, poly(12-hydroxydodecanoic acid) with Mn 17 600 was formed. For 12-hydroxydodecanoic acid polymerization at 90 degrees C, the catalyst activity decreased by 7, 18, and 25% at reaction times of 4, 24, and 48 h, respectively. Furthermore, the retention of catalyst activity was invariable as a function of the substrates used.     

Differential scanning calorimetry study of reversible, partial unfolding transitions in dodecameric glutamine synthetase from Escherichia coli.

Partial unfolding of dodecameric glutamine synthetase (GS) from Escherichia coli has been studied by differential scanning calorimetry (DSC). A single endotherm (tm = 51.6 +/- 0.1 degrees C and delta Hcal = 211 +/- 4 kcal/mol of enzyme) was observed in DSC experiments with Mn.GS in the presence of 1.0 mM free Mn2+ and 100 mM KCl at pH 7. The dodecameric structure of Mn.GS was retained throughout heating cycles, and thermal transitions were reversible as shown by rescans [with 6-18 mg of GS (Mr 622,000) from 15 to 68 degrees C at 20-60 degrees C/h] and by greater than 93% recovery of activity. A cooperative ratio delta Hcal/delta HvH of 1.6 +/- 0.1 and deconvolution analysis show two cooperative units (two-state transitions): t1 = 50.4 and t2 = 51.7 degrees C; the ratio of the relative sizes of thermally labile domains is approximately 1:2 as judged by delta H2/delta H1 approximately equal to 2. However, the thermally induced overall enthalpy change (0.34 cal/g) for GS dodecamer is only 5-10% of that for thermal unfolding of small globular proteins at 50 degrees C. The t1 and t2 values from deconvolutions of DSC data agree with t0.5 values previously calculated from spectral measurements of temperature-induced exposures of approximately 0.7 of 2 Trp and approximately 2 of 17 Tyr per subunit, respectively [Shrake et al. (1989) Biochemistry 28, 6281-6294], over a 14 degrees C temperature range using both stabilizing and destabilizing conditions for Mn.GS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Copper,Lead, Cadmium,and Zinc Sorption By Waterlogged and Air-Dry Soil

Competitive sorption of copper (Cu), lead (Pb), cadmium (Cd), and zinc (Zn) was studied in three soils of contrasting chemical and physical properties under air-dry and waterlogged conditions. Competitive sorption was determined using the standard batch technique using six solutions, each with Cu, Pb, Cd, and Zn concentrations of approximately 0, 2.5, 5, 10, 20, and 50?mg L^?1Waterlogged soils tended to sorb higher amounts of added Cu, Pb, Zn and Cd relative to soils in the air-dry condition; however, this increase in sorption was generally not statistically (p<0.05) significant. The magnitude of sorption under both waterlogged and air-dry conditions was affected by the type and amount of soil materials involved in metal sorption processes, and competition between other metals for the sorption sites. Metal sorption was closely correlated with soil properties such as cation exchange capacity, organic carbon, and Fe and Mn hydrous oxides. Exchangeable Al may have markedly reduced metal sorption due to its strong affinity for the sorption sites, while increases in exchangeable Mn may have enhanced Zn and Cd sorption. Heavy metal sorption was best described as a combination of both specific and nonspecific interactions. The extractability of Cu, Pb, Cd, and Zn under waterlogged and air-dry conditions was also studied. Three solutions containing these metals were mixed with each soil to achieve a final concentration of 0, 50, and 500?mg kg^?1. Each soil was extracted every 7 days using 1?M MgCl₂ (pH 7) to determine metal extractability. Metal extractability initially decreased then increased due to waterlogging. The increased extractability of added metals was closely related to increased solubility of Fe and Mn suggesting that dissolution of Fe and Mn, oxides under reducing conditions caused a release of previously sorbed Cu, Pb, Cd, and Zn.     

Optimized aminolysis conditions for cleavage of N-protected hydrophobic peptides from solid-phase resins.

Solid-phase synthesis and aminolysis cleavage conditions were optimized to obtain N- and C-terminally protected hydrophobic peptides with both high quality and yield. Uncharged 'WALP' peptides, consisting of a central (Leu-Ala)n repeating unit (where n = 5, 10.5 or 11.5) flanked on both sides by Trp 'anchors', and gramicidin A (gA) were synthesized using 9-fluorenylmethoxycarbonyl chemistry from either Wang or Merrifield resins. For WALP peptides, the N-terminal amino acid was capped by coupling N-acetyl- or N-formyl-Ala or -Gly to the peptide/resin or by formylation of the completed peptide/resin with para-nitrophenylformate (p-NPF). N-Terminal acetyl- or formyl-Ala racemized when coupled as an HOBt-ester to the resin-bound peptide, but not when the peptide was formylated with p-NPF. Racemization was avoided at the last step by completing the peptide with acetyl- or formyl-Gly. For both WALP peptides and gA, cleavage conditions using ethanolamine or ethylenediamine were optimized as functions of solvent, time, temperature and resin type. For WALP peptides, maximum yields of highly pure peptide were obtained by cleavage with 20% ethanolamine or ethylenediamine in 80% dichloromethane for 48 h at 24 degrees C. N-Acetyl-protected WALP peptides consistently gave higher yields than those protected with N-formyl. For gA, cleavage with 20% ethanolamine or ethylenediamine in 80% dimethylformamide for 48 h at 24 degrees C gave excellent results. For both WALP peptides and gA, decreasing the cleavage time to 4 h and increasing the temperature to 40-55 degrees C resulted in significantly lower yields. The inclusion of hexafluoroisopropanol in the cleavage solvent mixture did not improve yields for either gA or WALP peptides.     

β-Xylosidase and xylanase characterization and production by Streptomyces sp. CH-M-1035

Extracellular xylanase activity and cell-bound β-xylosidase production by a selected strain of Streptomyces sp. CH-M-1035 was characterized during growth on three xylans, sugar cane bagasse pith and lemon peel as sole carbon source. The cell-bound β-xylosidase and extracellular endoxylanase had pH optima of 6·0 and 5·0, and temperature optima of 50°C and 60°C, respectively. The highest level of β-xylosidase activity was obtained when Streptomyces sp. CH-M-1035 was grown on larchwood xylan, whereas the maximal endoxylanase production was found on lemon peel. Reducing sugars accumulated in the culture media when Streptomyces sp. CH-M-1035 was grown on xylans, but not on agroindustrial residues.