Binding of biogenic and synthetic polyamines to β-lactoglobulin

The bindings of biogenic polyamines spermine (spm), spermidine (spmd) and synthetic polyamines 3,7,11,15-tetrazaheptadecane·4HCl (BE-333) and 3,7,11,15,19-pentazahenicosane·5HCl (BE-3333) with β-lactoglobulin (β-LG) were determined in aqueous solution. FTIR, UV-vis, CD and fluorescence spectroscopic methods as well as molecular modeling were used to determine the polyamine binding sites and the effect of polyamine complexation on protein stability and secondary structure. Structural analysis showed that polyamines bind β-LG via both hydrophilic and hydrophobic contacts. Stronger polyamine-protein complexes formed with synthetic polyamines than biogenic polyamines, with overall binding constants of K_spm-β-LG = 3.2(±0.6) × 10⁴ M⁻¹, K_spmd-β-LG = 1.8(±0.5) × 10⁴ M⁻¹, K_BE-333-β-LG = 5.8(±0.3) × 10⁴ M⁻¹ and K_{BE-3333-β-LG} = 6.2(±0.05) × 10⁴ M⁻¹. Molecular modeling showed the participation of several amino acids in the polyamine complexes with the following order of polyamine-protein binding affinity: BE-3333 > BE-333 > spermine > spermidine, which correlates with their positively charged amino group content. Alteration of protein conformation was observed with a reduction of β-sheet from 57% (free protein) to 55-51%, and a major increase of turn structure from 13% (free protein) to ∼21% in the polyamine-β-LG complexes, indicating a partial protein unfolding.     

Ca significantly enhanced development and salt-secretion rate of salt glands of Limonium bicolor under NaCl treatment

The divalent cation, Ca²⁺, plays crucial roles in plant growth, development and stress resistance. Limonium bicolor seedlings were treated with 200 mM NaCl combined with three levels of Ca²⁺ (0 mM, 5 mM and 20 mM) for 15 days to study the effects of Ca²⁺ on development and salt-secretion rates of salt glands. It was shown that the 4th leaf areas of L. bicolor seedlings under 20 mM Ca²⁺ treatment were significantly higher than those under 0 mM and 5 mM Ca²⁺ treatments. The total number and the densities of salt glands per leaf increased markedly with increased Ca²⁺ concentrations. The diameters of salt glands increased by 59% and 63% as Ca²⁺ concentration increased from zero to 5 mM and 20 mM, respectively. Under 20 mM Ca²⁺ treatment, the salt-secretion rate per leaf was obviously higher than that treated with 5 mM Ca²⁺, but there was no significant difference in the salt-secretion rates per salt gland between the two groups. Under 0 mM Ca²⁺ treatment, leaf-cell membrane permeability increased significantly, which led to serious leakage of ions and a significant increase in Na⁺ loss rate. The results showed that the increase of Ca²⁺ concentration markedly enhanced development and salt-secretion rates of salt glands in the leaves of L. bicolor, the increase of salt secretion per leaf is due to the efficiency of the secretion process per salt gland and the number of salt glands, the salt-secretion rates per salt gland have a relationship with the diameters of salt glands.     

Effects of lime-induced inorganic carbon reduction on the growth of three aquatic macrophyte species

Lime application to aquatic systems may be an effective means of stressing macrophyte growth and promoting changes in species assemblage by inducing temporary dissolved inorganic carbon (DIC) limitation of productivity. Shoot and root growth response to lime (as Ca(OH)₂) application was investigated for three macrophyte species (Elodea canadensis, Stuckenia pectinata, and Vallisneria americana) grown in experimental outdoor mesocosms. Lime was applied to mesocosms at three treatment levels to maintain pH for 1 week at 9.8–10.0 (1.64 mM), 10.3–10.5 (at the bicarbonate–carbonate equivalence point; 3.00 mM), and 10.8–11.0 (4.34 mM). pH recovered to control levels in all treated mesocosms 20 days after lime application. After treatment, HCO₃⁻ and DIC declined by 66, 93, and 93% and 60, 89, and 87%, respectively, versus increasing lime application. Concentrations remained lower in treated mesocosms versus the control throughout post-treatment. Differential growth response was observed in the 1.64 and 3.00 mM treatments, suggesting species-specific tolerances to both DIC concentration and form. V. americana was most sensitive to lime as the 1.64 mM treatment resulted in 54% shoot growth suppression versus the control and shoot plus root biomass loss in the 3.00 and 4.34 mM treatments. S. pectinata and E. canadensis exhibited net shoot and root growth (although significantly lower than controls) in both the 1.64 and 3.00 mM treatments and complete growth suppression in the 4.34 mM treatment. Selective control and shifts in species assemblage may be possible by adjusting lime concentration in relation to compensation point and needs to be investigated under field conditions.     

Effect of polyamines on synthesis and degradation of guanosine 5′-diphosphate 3′-diphosphate

The effect of polyamines on the in vitro and in vivo synthesis and degradation on guanosine 5′-diphosphate 3′-diphosphate (ppGpp) has been studied in Escherichia coli. The presence of 2 mM spermidine lowered the optimal Mg²⁺ concentration for ppGpp formation from 17 mM to 11 mM. The formation of ppGpp in the presence of 2 mM spermidine and 11 mM Mg²⁺ was about 15% greater than that in the presence of 17 mM Mg²⁺. At a concentration of less than 11 mM Mg²⁺, spermidine was found to stimulate ppGpp formation greatly. Putrescine did not cause any effect. When a polyamine-requiring mutant of E. coli (EWH319) was starved for an amino acid by the addition of valine, spermidine stimulated ppGpp formation. the degradation of ppGpp was not influenced significantly by polyamines.     

Lactococcus lactis as a cell factory: a twofold increase in phosphofructokinase activity results in a proportional increase in specific rates of glucose uptake and lactate formation

Despite the fact that the area of glycolysis in Lactococcus lactis has been intensively studied, only a limited number of studies have been focused on the regulation of uptake of glucose itself. Using the tool of the glucostat fed-batch mode of culture, it was demonstrated in our earlier work that the concentration of glucose regulates its uptake rate and that the control of the glycolytic flux resides to a large extent in processes outside the pathway itself, like glucose transport and the ATP consuming reactions, while allosteric properties of key enzymes like phosphofructokinase (PFK) have a significant influence on the control. Extending our work, we report here the results of fermentations with engineered L. lactis strains with altered PFK activity in which the pfkA gene from Aspergillus niger, and its truncated version pfk13 that encodes a shorter PFK1 fragment were cloned. The results in this study suggest that, under the optimum for the microorganism applied microaerobic conditions, the glycolytic capacity of L. lactis was significantly increased in engineered strains with increased PFK activity. The transformant strain in which the truncated pfk13 gene of A. niger was expressed performed more efficiently as it was able to grow successfully in glucostat cultures with 277 mM glucose - while the optimum glucose concentration for the parental strain was 55 mM. The present work demonstrates the direct effect of PFK activity on the glycolytic flux in L. lactis since a twofold increase in specific PFK activity (from 7.1 to 14.5 U/OD₆₀₀) resulted in a proportional increase of the maximum specific rates of glucose uptake (from 0.8 to 1.7 μM s⁻¹ g CDW⁻¹) and lactate formation (from 15 to 22.8 g lactate (g CDW)⁻¹ h⁻¹).     

Examination of microbial fuel cell start-up times with domestic wastewater and additional amendments

Rapid startup of microbial fuel cells (MFCs) and other bioreactors is desirable when treating wastewaters. The startup time with unamended wastewater (118 h) was similar to that obtained by adding acetate or fumarate (110-115 h), and less than that with glucose (181 h) or Fe(III) (353 h). Initial current production took longer when phosphate buffer was added, with startup times increasing with concentration from 149 h (25 mM) to 251 h (50 mM) and 526 h (100 mM). Microbial communities that developed in the reactors contained Betaproteobacteria, Acetoanaerobium noterae, and Chlorobium sp. Anode biomass densities ranged from 200 to 600 μg/cm² for all amendments except Fe(Ш) (1650 μg/cm²). Wastewater produced 91 mW/m², with the other MFCs producing 50 mW/m² (fumarate) to 103 mW/m² (Fe(III)) when amendments were removed. These experiments show that wastewater alone is sufficient to acclimate the reactor without the need for additional chemical amendments.     

Production,partial purification and characterization of organic solvent tolerant lipase from Burkholderia multivorans V2 and its application for ester synthesis

Burkholderia multivorans V2 (BMV2) isolated from soil was found to produce an extracellular solvent tolerant lipase (6.477 U/mL). This lipase exhibited maximum stability in n-hexane retaining about 97.8% activity for 24 h. After performing statistical optimization of medium components for lipase production, a 2.2-fold (14 U/mL) enhancement in the lipase production was observed. The crude lipase from BMV2 was partially purified by ultrafiltration and gel permeation chromatography with 24.64-fold purification. The K_m and V_max values for partially purified BMV2 lipase were found to be 1.56 mM and 5.62 μmoles/mg min. The metal ions Ca²⁺, Mg²⁺ and Mn²⁺ had stimulatory effect on lipase activity, whereas Cu²⁺, Fe²⁺ and Zn²⁺ strongly inhibited the lipase activity. EDTA and PMSF at 10 mM concentration strongly inhibited the lipase activity. Non-ionic and anionic surfactants stimulated the lipase activity. BMV2 lipase was proved to be efficient in synthesis of ethyl butyrate ester under non-aqueous environment.     

Enzyme kinetics of ginsenosidase type IV hydrolyzing 6-O-multi-glycosides of protopanaxatriol type ginsenosides

In this work, the kinetics of ginsenosidase type IV hydrolyzing the 6-O-multi-glycosides of protopanaxatriol type ginsenosides (PPT) from Aspergillus sp.39g strain were investigated. The enzyme molecular weight was about 56 kDa. The enzyme hydrolyzes the 6-O-α-l-(1 → 2)-rhamnoside of ginsenoside Re and 6-O-β-d-(1 → 2)-xyloside of R1 into Rg1, and subsequently hydrolyzes 6-O-β-d-glucoside of Rg1 into F1. The enzyme hydrolyzes 6-O-α-l-(1 → 2)-rhamnoside of Rg2 and 6-O-β-d-(1 → 2)-glucoside of Rf into Rh1, and subsequently hydrolyzes 6-O-β-d-glucoside of Rh1 into its aglycone. The enzyme K_m and V_max for Re were 22.2 mM, and 7.94 mM/h; the K_m and V_max for R1 were 7.06 mM and 1.61 mM/h; the enzyme transformation velocity (V₀) at 5 mM substrate was 1.46 mM/h for Re, and 0.67 mM/h for R1. Therefore, the enzyme hydrolysis on the Re rhamnoside was faster than that on R1 xyloside. The enzyme V₀ on Rg1 was 0.05 mM/h that indicated the enzyme hardly hydrolyzed the 6-O-β-d-glucoside of Rg1. The enzyme kinetic parameters of Rg2 and Rf were 5.74 and 9.43 mM for K_m; 2.70 and 2.84 mM/h for V_max; 1.26 and 0.98 mM/h for V₀ at 5 mM substrate, respectively. Thus the enzyme hydrolysis on Rg2 rhamnoside was faster than that on the glucoside of Rf.     

Structure–activity relationships of analogs of 3,4,5-trimethylfuran-2(5H)-one with germination inhibitory activities

Smoke-derived butenolide compounds have, in recent years, been shown to be important germination signaling molecules, which also affect seedling growth. The butenolide 3,4,5-trimethylfuran-2(5H)-one was previously isolated from plant-derived smoke and was found to significantly reduce the effect on germination by the highly active promotor karrikinolide (KAR₁, 3-methyl-2H-furo[2,3-c]pyran-2-one), another smoke-derived compound. In this study, 11 analogs of 3,4,5-trimethylfuran-2(5H)-one were synthesized and their effect on the germination of light-sensitive ‘Grand Rapids’ lettuce seeds (Lactua sativa cv. ‘Grand Rapids’) were evaluated. A concentration series (1 mM–1 μM) of the analogs were tested alone, or in combination with 0.01 μM KAR₁. Only two compounds were found to reduce the germination promotory effect of 0.01 μM KAR₁ in a similar manner as observed with 3,4,5-trimethylfuran-2(5H)-one, with activity ranging from 1 mM to 10 μM. Four compounds were found to have inhibitory activity at 1 mM and 100 μM. The retention of activity by some of the analogs may be useful for designing novel compounds with improved activity. Furthermore, understanding the structure–activity relationships of these compounds may be helpful in synthesizing molecular probes that can be used to further investigate the mechanism of action of these compounds in regulating seed germination.     

Effects of amines and aminoalcohols on bovine intestine alkaline phosphatase activity

Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay (EIA). In this study, we evaluated the effects of various aminoalcohols and amines on the activity of BIALP in the hydrolysis of p-nitrophenyl phosphate (pNPP) at pH 9.8, at 20 °C. The k_cat values at 0.05 M diethanolamine, 0.1 M triethanolamine, and 0.2 M N-methylethanolamine were 190 ± 10, 840 ± 30, and 500 ± 10 s⁻¹, respectively. The k_cat values increased with increasing concentrations of diethanolamine, triethanolamine, and N-methylethanolamine and reached 1240 ± 60, 1450 ± 30, and 2250 ± 80 s⁻¹, respectively, at 1.0 M. On the other hand, the k_cat values at 0.05-1.0 M ethanolamine, ethylamine, methylamine, and dimethylamine were in the range of 100-600 s⁻¹. These results indicate that diethanolamine, triethanolamine and N-methylethanolamine highly activate BIALP and might be suitable as a dilution buffer of BIALP in EIA. Interestingly, the K_m values increased with increasing concentrations of diethanolamine and N-methylethanolamine, but not triethanolamine: the K_m value at 1.0 M diethanolamine (0.83 ± 0.15 mM) was 12-fold higher than that at 0.05 M (0.07 ± 0.01 mM), and that at 1.0 M N-methylethanolamine (2.53 ± 0.20 mM) was 14-fold higher than that at 0.2 M (0.18 ± 0.02 mM), while that at 1.0 M triethanolamine (0.31 ± 0.01 mM) was similar as that at 0.2 M (0.25 ± 0.01 mM), suggesting that the mechanisms of BIALP activation are different between the aminoalcohols.     

Biochemical characterization of pea ornithine-δ-aminotransferase: Substrate specificity and inhibition by di- and polyamines

Ornithine-δ-aminotransferase (OAT, EC 2.6.1.13) catalyzes the transamination of l-ornithine to l-glutamate-γ-semialdehyde. The physiological role of OAT in plants is not yet well understood. It is probably related to arginine catabolism resulting in glutamate but the enzyme has also been associated with stress-induced proline biosynthesis. We investigated the enzyme from pea (PsOAT) to assess whether diamines and polyamines may serve as substrates or they show inhibitory properties. First, a cDNA coding for PsOAT was cloned and expressed in Escherichia coli to obtain a recombinant protein with a C-terminal 6xHis tag. Recombinant PsOAT was purified under native conditions by immobilized metal affinity chromatography and its molecular and kinetic properties were characterized. Protein identity was confirmed by peptide mass fingerprinting after proteolytic digestion. The purified PsOAT existed as a monomer of 50 kDa and showed typical spectral properties of enzymes containing pyridoxal-5′-phosphate as a prosthetic group. The cofactor content of PsOAT was estimated to be 0.9 mol per mol of the monomer by a spectrophotometric analysis with phenylhydrazine. l-Ornithine was the best substrate (K_m = 15 mM) but PsOAT also slowly converted N_α-acetyl-l-ornithine. In these reactions, 2-oxoglutarate was the exclusive amino group acceptor (K_m = 2 mM). The enzyme had a basic optimal pH of 8.8 and displayed relatively high temperature optimum. Diamines and polyamines were not accepted as substrates. On the other hand, putrescine, spermidine and others represented weak non-competitive inhibitors. A model of the molecular structure of PsOAT was obtained using the crystal structure of human OAT as a template.     

Distinct actions of strontium on mineral formation in matrix vesicles

Matrix vesicles (MVs) are involved in the initial step of mineralization in skeletal tissues and provide an easily model to analyze the hydroxyapatite (HA) formation. Sr stimulates bone formation and its effect was tested on MVs. Sr²⁺ (15-50 μM) in the mineralization medium containing MVs, 2 mM Ca²⁺ and 3.42 mM P_i, retarded HA formation. Sr²⁺ (1-5 mM) in the same medium-induced other types of mineral than HA and cancelled the ATP-, ADP- or PP_i-induced retardation in the mineral formation. Our findings suggest that the beneficial effect of Sr²⁺ at a low dose (15-50 μM) is rather an inhibitor of bone resorption than an activator of mineral formation, while at high Sr²⁺ concentration (1-5 mM), mineral formation, especially other types of mineral than HA, is favored.     

The relationship between single-channel and whole-cell conductance in the T-type Ca2+ channel CaV3.1

In T-type Ca²⁺ channels, macroscopic I_Ba is usually smaller than I_Ca, but at high Ca²⁺ and Ba²⁺, single-channel conductance (γ) is equal. We investigated γ as a function of divalent concentration and compared it to macroscopic currents using Ca_V3.1 channels studied under similar experimental conditions (TEA_o and K_i). Single-channel current-voltage relationships were nonlinear in a way similar to macroscopic open-channel I/Vs, so divalent γ was underestimated at depolarized voltages. To estimate divalent γ, concentration dependence, i_Div, was measured at voltages <−50 mV. Data were well described by Langmuir isotherms with γ_max(Ca²⁺) of 9.5 ± 0.4 pS and γ_max(Ba²⁺) of 10.3 ± 0.5 pS. Apparent K_M was lower for Ca²⁺ (2.3 ± 0.7 mM) than for Ba²⁺ (7.9 ± 1.3 mM). A subconductance state with an amplitude 70% that of the main state was observed, the relative occupancy of which increased with increasing Ca²⁺. As predicted by γ, macroscopic G_maxCa was larger than G_maxBa at 5 mM (G_maxCa²⁺/Ba:²⁺1.43 ± 0.14) and similar at 60 mM (G_maxCa²⁺/Ba:²⁺1.10 ± 0.02). However, over the range of activation, I_Ca was larger than I_Ba under both conditions. This was a consequence of the fact that V_rev was more negative for I_Ba than for I_Ca, so that the driving force determining I_Ba was smaller than that determining I_Ca over the range of potentials in standard current-voltage relationships.     

β-Galactosidase from Lactobacillus plantarum WCFS1: biochemical characterization and formation of prebiotic galacto-oligosaccharides

Recombinant β-galactosidase from Lactobacillus plantarum WCFS1, homologously over-expressed in L. plantarum, was purified to apparent homogeneity using p-aminobenzyl 1-thio-β-d-galactopyranoside affinity chromatography and subsequently characterized. The enzyme is a heterodimer of the LacLM-family type, consisting of a small subunit of 35 kDa and a large subunit of 72 kDa. The optimum pH for hydrolysis of its preferred substrates o-nitrophenyl-β-d-galactopyranoside (oNPG) and lactose is 7.5 and 7.0, and optimum temperature for these reactions is 55 and 60 °C, respectively. The enzyme is most stable in the pH range of 6.5-8.0. The K_m, k_cat and k_cat/K_m values for oNPG and lactose are 0.9 mM, 92 s⁻¹, 130 mM⁻¹ s⁻¹ and 29 mM, 98 s⁻¹, 3.3 mM⁻¹ s⁻¹, respectively. The L. plantarum β-galactosidase possesses a high transgalactosylation activity and was used for the synthesis of prebiotic galacto-oligosaccharides (GOS). The resulting GOS mixture was analyzed in detail, and major components were identified by using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as well as capillary electrophoresis. The maximal GOS yield was 41% (w/w) of total sugars at 85% lactose conversion (600 mM initial lactose concentration). The enzyme showed a strong preference for the formation of β-(1→6) linkages in its transgalactosylation mode, while β-(1→3)-linked products were formed to a lesser extent, comprising ∼80% and 9%, respectively, of the newly formed glycosidic linkages in the oligosaccharide mixture at maximum GOS formation. The main individual products formed were β-d-Galp-(1→6)-d-Lac, accounting for 34% of total GOS, and β-d-Galp-(1→6)-d-Glc, making up 29% of total GOS.     

Measuring Ca binding to short chain fatty acids and gluconate with a Ca electrode: Role of the reference electrode

Many organic anions bind free Ca²⁺, the total concentration of which must be adjusted in experimental solutions. Because published values for the apparent dissociation constant (K_app) describing the Ca²⁺ affinity of short chain fatty acids (SCFAs) and gluconate are highly variable, Ca²⁺ electrodes coupled to either a 3 M KCl or a Na⁺ selective electrode were used to redetermine K_app. All solutions contained 130 mM Na⁺, whereas the concentration of the studied anion was varied from 15 to 120 mM, replacing Cl⁻ that was decreased concomitantly to maintain osmolarity. This induces changes in the liquid junction potential (LJP) at the 3 M KCl reference electrode, leading to a systematic underestimation of K_app if left uncorrected. Because the Na⁺ concentration in all solutions was constant, a Na⁺ electrode was used to directly measure the changes in the LJP at the 3 M KCl reference, which were under 5 mV but twice those predicted by the Henderson equation. Determination of K_app either after correction for these LJP changes or via direct reference to a Na⁺ electrode showed that SCFAs do not bind Ca²⁺ and that the K_app for the binding of Ca²⁺ to gluconate at pH 7.4, ionic strength 0.15 M, and 23 °C was 52.7 mM.     

Conformational stability of α-amylase from malted sorghum (Sorghum bicolor): Reversible unfolding by denaturants

α-Amylase from Sorghum bicolor, is reversibly unfolded by chemical denaturants at pH 7.0 in 50 mM Hepes containing 13.6 mM calcium and 15 mM DTT. The isothermal equilibrium unfolding at 27 °C is characterized by two state transition with ΔG (H₂O) of 16.5 kJ mol⁻¹ and 22 kJ mol⁻¹, respectively, at pH 4.8 and pH 7.0 for GuHCl and ΔG (H₂O) of 25.2 kJ mol⁻¹ at pH 4.8 for urea. The conformational stability indicators such as the change in excess heat capacity (ΔC_p), the unfolding enthalpy (H_g) and the temperature at ΔG = 0 (T_g) are 17.9 ± 0.7 kJ mol⁻¹ K⁻¹, 501.2 ± 18.2 kJ mol⁻¹ and 337.3 ± 6.9 K at pH 4.8 and 14.3 ± 0.5 kJ mol⁻¹ K⁻¹, 509.3 ± 21.7 kJ mol⁻¹ and 345.4 ± 4.8 K at pH 7.0, respectively. The reactivity of the conserved cysteine residues, during unfolding, indicates that unfolding starts from the ‘B’ domain of the enzyme. The oxidation of cysteine residues, during unfolding, can be prevented by the addition of DTT. The conserved cysteine residues are essential for enzyme activity but not for the secondary and tertiary fold acquired during refolding of the denatured enzyme. The pH dependent stability described by ΔG (H₂O) and the effect of salt on urea induced unfolding confirm the role of electrostatic interactions in enzyme stability.     

Effects of polyamines on tyrosine hydroxylase activity in adrenals

The ability of polyamines (putrescine, spermidine, and spermine) to modify tyrosine hydroxylase (TH) activity was examined in crude or purified enzyme preparation and in adrenal tissue slices. Polyamines showed biphasic effects on TH activity in vitro at physiological pH 7.0, with an inhibitory effect at low concentrations (<1 mM) and a stimulatory effect at high concentrations. The degree of both inhibition and stimulation produced by polyamines at low and high concentrations, respectively, were proportional to the number of the amino group in the polyamines (putrescine < spermidine < spermine). The degree of inhibition by polyamines was much greater with purified enzyme than with crude enzyme preparations. Tyrosine hydroxylation in situ in adrenal tissue slices was stimulated by polyamines without inhibition at any concentrations tested. This evidence suggests that TH molecules in vivo could interact with polyamines or polyamine-like substances which inhibit the TH activity at physiological concentrations less than 1 mM.