Predictive modeling of β-carotene accumulation by Dunaliella salina as a function of pH,NaCI, and irradiance

Predictive modeling of β-carotene accumulation by Dunaliella salina as a function of NaCI, pH, and irradiance was studied. Modified Logistic, Gompertz, Schnute, Richards, and Stannard models were fitted to describe β-carotene accumulation by the alga under various environmental conditions. Lag time (λ, days), maximum accumulation (A, pg/cell), and the maximum production rate (μ, 1/day) for β-carotene accumulation were calculated by modified Logistic and Gompertz models. Values of λ, A, and μ for β-carotene accumulation varied between 0.26 and 20.14 days, 57.48 to 198.76 pg β-carotene/cell, and 1.80 to 3.68 1/day, respectively. Results revealed that Logistic and Gompertz models could be used to describe the accumulation of β-carotene by D. salina as a function of salt concentrations, pH, and irradiance. The highest asymptotic value was predicted from Logistic and Gompertz models at pH 9.0, 48 kerg/(cm² s) light intensity, and 20% NaCl concentration.     

The role of surface electrostatics on the stability,function and regulation of human cystathionine β-synthase,a complex multidomain and oligomeric protein

Human cystathionine β-synthase (hCBS) is a key enzyme of sulfur amino acid metabolism, controlling the commitment of homocysteine to the transsulfuration pathway and antioxidant defense. Mutations in hCBS cause inherited homocystinuria (HCU), a rare inborn error of metabolism characterized by accumulation of toxic homocysteine in blood and urine. hCBS is a complex multidomain and oligomeric protein whose activity and stability are independently regulated by the binding of S-adenosyl-methionine (SAM) to two different types of sites at its C-terminal regulatory domain. Here we study the role of surface electrostatics on the complex regulation and stability of hCBS using biophysical and biochemical procedures. We show that the kinetic stability of the catalytic and regulatory domains is significantly affected by the modulation of surface electrostatics through noticeable structural and energetic changes along their denaturation pathways. We also show that surface electrostatics strongly affect SAM binding properties to those sites responsible for either enzyme activation or kinetic stabilization. Our results provide new insight into the regulation of hCBS activity and stability in vivo with implications for understanding HCU as a conformational disease. We also lend experimental support to the role of electrostatic interactions in the recently proposed binding modes of SAM leading to hCBS activation and kinetic stabilization.     

Methodology for identification of pore forming antimicrobial peptides from soy protein subunits β-conglycinin and glycinin

Antimicrobial peptides (AMPs) inactivate microbial cells through pore formation in cell membrane. Because of their different mode of action compared to antibiotics, AMPs can be effectively used to combat drug resistant bacteria in human health. AMPs can also be used to replace antibiotics in animal feed and immobilized on food packaging films. In this research, we developed a methodology based on mechanistic evaluation of peptide-lipid bilayer interaction to identify AMPs from soy protein. Production of AMPs from soy protein is an attractive, cost-saving alternative for commercial consideration, because soy protein is an abundant and common protein resource. This methodology is also applicable for identification of AMPs from any protein. Initial screening of peptide segments from soy glycinin (11S) and soy β-conglycinin (7S) subunits was based on their hydrophobicity, hydrophobic moment and net charge. Delicate balance between hydrophilic and hydrophobic interactions is necessary for pore formation. High hydrophobicity decreases the peptide solubility in aqueous phase whereas high hydrophilicity limits binding of the peptide to the bilayer. Out of several candidates chosen from the initial screening, two peptides satisfied the criteria for antimicrobial activity, viz. (i) lipid-peptide binding in surface state and (ii) pore formation in transmembrane state of the aggregate. This method of identification of antimicrobial activity via molecular dynamics simulation was shown to be robust in that it is insensitive to the number of peptides employed in the simulation, initial peptide structure and force field. Their antimicrobial activity against Listeria monocytogenes and Escherichia coli was further confirmed by spot-on-lawn test.     

Biodegradation of cellulose by β-glucosidase and cellulase immobilized on a pH-responsive copolymer

Biodegradation of cellulose involves synergistic action of the endoglucanases, exoglucanases and β-glucosidases in cellulase. However, the yield of glucose is limited by the lack of β-glucosidase to hydrolyze cellobiose into glucose. In this study, β-glucosidase as a supplemental enzyme along with cellulase are co-immobilized on a pHresponsive copolymer, poly (MAA-co-DMAEMA-co-BMA) (abbreviated P_MDB, where MAA is α-methacrylic acid, DMAEMA is 2-dimethylaminoethyl methacrylate and BMA is butyl methacrylate). The thermal and storage stabilities of PMDB with immobilized enzymes are improved greatly, compared with those of free cellulase. Biodegradation of cellulose is carried out in a pH-responsive recyclable aqueous two-phase system composed of poly (AA-co- DMAEMA-co-BMA) (abbreviated P_{ADB 3.8}, where AA is acrylic acid) and P_MDB. Insoluble substrate and P_MDB with immobilized cellulase and β-glucosidase (Celluclast 1.5L FG and Novozyme 188, respectively) were biased to the bottom phase, while the product was partitioned to the top phase in the presence of 40 mM (NH₄)₂SO₄. When the degradation reaction of cellulose is carried out with PMDB containing immobilized cellulase and β-glucosidase, the concentration of glucose reaches 4.331 mg/mL after 108 h. The yield of glucose is 50.25% after P_MDB containing the immobilized enzymes is recycled five times.     

Differential scanning calorimetry and fluorescence study of lactoperoxidase as a function of guanidinium–HCl,urea, and pH

The stability of bovine lactoperoxidase to denaturation by guanidinium–HCl, urea, or high temperature was examined by differential scanning calorimetry (DSC) and tryptophan fluorescence. The calorimetric scans were observed to be dependent on the heating scan rate, indicating that lactoperoxidase stability at temperatures near T_m is controlled by kinetics. The values for the thermal transition, T_m, at slow heating scan rate were 66.8, 61.1, and 47.2 °C in the presence of 0.5, 1, and 2 M guanidinium–HCl, respectively. The extrapolated value for T_m in the absence of guanidinium–HCl is 73.7 °C, compared with 70.2 °C obtained by experiment; a lower experimental value without a denaturant is consistent with distortion of the thermal profile due to aggregation or other irreversible phenomenon. Values for the heat capacity, C_p, at T_m and E_a for the thermal transition decrease under conditions where T_m is lowered. At a given concentration, urea is less effective than guanidinium–HCl in reducing T_m, but urea reduces C_p relatively more. Both fluorescence and DSC indicate that thermally denatured protein is not random coil. A change in fluorescence around 35 °C, which was previously reported for EPR and CD measurements (Boscolo et al. Biochim. Biophys. Acta 1774 (2007) 1164–1172), is not seen by calorimetry, suggesting that a local and not a global change in protein conformation produces this fluorescence change.     

Molecular evolution of glycinin and β-conglycinin gene families in soybean (Glycine max L. Merr.)

There are two main classes of multi-subunit seed storage proteins, glycinin (11S) and β-conglycinin (7S), which account for approximately 70% of the total protein in a typical soybean seed. The subunits of these two protein classes are encoded by a number of genes. The genomic organization of these genes follows a complex evolutionary history. This research was designed to describe the origin and maintenance of genes in each of these gene families by analyzing the synteny, phylogenies, selection pressure and duplications of the genes in each gene family. The ancestral glycinin gene initially experienced a tandem duplication event; then, the genome underwent two subsequent rounds of whole-genome duplication, thereby resulting in duplication of the glycinin genes, and finally a tandem duplication likely gave rise to the Gy1 and Gy2 genes. The β-conglycinin genes primarily originated through the more recent whole-genome duplication and several tandem duplications. Purifying selection has had a key role in the maintenance of genes in both gene families. In addition, positive selection in the glycinin genes and a large deletion in a β-conglycinin exon contribute to the diversity of the duplicate genes. In summary, our results suggest that the duplicated genes in both gene families prefer to retain similar function throughout evolution and therefore may contribute to phenotypic robustness.     

The rate of ATP synthesis as a function of ΔpH in normal and dithiothreitol-modified chloroplasts

The rate of ATP synthesis catalyzed by normal and by dithiothreitol-modified ATPases is investigated as a function of ΔpH in spinach chloroplasts at constant pH_out. The transmembrane ΔpH was generated by an acid-base transition and the reaction time was limited to 150 ms by using a rapidly mixing quenched-flow apparatus. The result was that the functional dependence of the rate on ΔpH is shifted to lower ΔpH values and that the shape of this curve is altered after dithiothreitol modification. The maximal rate (400 ATP / CF₁ per s) is the same under both conditions.     

Tissue-specific and temporal regulation of a β-conglycinin gene: roles of the RY repeat and other cis-acting elements

Upstream regulatory sequences (URS) of the gene that encodes the subunit of -conglycinin, the 7S soybean seed storage protein, includes two RY repeat elements. The role of RY elements and sequences that bind soybean embryo factors 3 and 4 (SEF3 and SEF4; Allen et al., Plant Cell 1 (1989) 623–631) in regulating expression of the promoter was studied following site directed mutagenesis. Specific mutations introduced into these sequences abolished the in vitro binding activities of SEF3 and SEF4. The biological activities resulting from the mutations were determined in transgenic plants using two chimeric promoters comprising sequences from the CaMV 35S promoter and the subunit promoter. The uidA reporter gene was used to assess the levels of gene expression in transgenic plants. The mutations in the RY element and SEF3 and SEF4 binding sites had little effect on expression of the promoter. By contrast, mutations in the RY element had significant effect on gene expression when the URS from the promoter was ligated upstream of the core 35S promoter. Mutations in the RY element abolished the seed specific enhancing activity of the URS and caused expression of the chimeric promoter in leaves. These results indicate that the RY element plays a key role in seed-specific gene regulation in coordination with other cis-acting elements.     

Discovery of aminoheterocycles as a novel β-secretase inhibitor class: pH dependence on binding activity part 1

We have developed a novel series of heteroaromatic BACE-1 inhibitors. These inhibitors interact with the enzyme in a unique fashion that allows for potent binding in a non-traditional paradigm. In addition to the elucidation of their binding profile, we have discovered a pH dependent effect on the binding affinity as a result of the intrinsic pK_a of these inhibitors and the pH of the BACE-1 enzyme binding assay.     

Adsorption of tetracyanoquinodimethane and tetrathiafulvalene on aluminium (100) surface – a first principle study of structural and electronic properties

Possible adsorption configurations and electronic properties, such as charge analysis, density of states, work function and Schottky barrier height of tetracyanoquinodimethane (TCNQ) and tetrathiafulvalene (TTF) on Aluminium (100) surface is studied by using density functional theory methods with local density approximation (LDA), generalised gradient approximation (GGA), PBE and PBE-D2 methods. TCNQ is strongly adsorbed on Al(100) with adsorption energy of ?3.66?eV. The charge is transferred from Al(100) to TCNQ and charge transfer occurs mainly through cyano group of TCNQ. Adsorption on Al(100) surface leads to downshift in energy difference between highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of TCNQ by 2.5?eV as result of hybridisation of p orbital of carbon and nitrogen atoms of TCNQ and p orbital of surface Al atoms. Compared to TCNQ adsorption, TTF adsorption on Al(100) surface is having less adsorption energy and type of interaction is physisorption. The charge transfer from TTF to Al(100) surface leads to decrease in work function of Al by 0.24?eV. The n-type Schottky barrier height of TCNQ/Al(100) and p-type Schottky barrier height of TTF/Al(100) is 0.68eV and 1.97?eV, respectively showing that TCNQ and Al(100) are suitable for organic photovoltaic and electrochemical applications.     

Binding,folding and insertion of a β-hairpin peptide at a lipid bilayer surface: Influence of electrostatics and lipid tail packing

Antimicrobial peptides (AMPs) act as host defenses against microbial pathogens. Here we investigate the interactions of SVS-1 (KVKVKVKV^dP^lPTKVKVKVK), an engineered AMP and anti-cancer β-hairpin peptide, with lipid bilayers using spectroscopic studies and atomistic molecular dynamics simulations. In agreement with literature reports, simulation and experiment show preferential binding of SVS-1 peptides to anionic over neutral bilayers. Fluorescence and circular dichroism studies of a Trp-substituted SVS-1 analogue indicate, however, that it will bind to a zwitterionic DPPC bilayer under high-curvature conditions and folds into a hairpin. In bilayers formed from a 1:1 mixture of DPPC and anionic DPPG lipids, curvature and lipid fluidity are also observed to promote deeper insertion of the fluorescent peptide. Simulations using the CHARMM C36m force field offer complementary insight into timescales and mechanisms of folding and insertion. SVS-1 simulated at an anionic mixed POPC/POPG bilayer folded into a hairpin over a microsecond, the final stage in folding coinciding with the establishment of contact between the peptide's valine sidechains and the lipid tails through a “flip and dip” mechanism. Partial, transient folding and superficial bilayer contact are seen in simulation of the peptide at a zwitterionic POPC bilayer. Only when external surface tension is applied does the peptide establish lasting contact with the POPC bilayer. Our findings reveal the influence of disruption to lipid headgroup packing (via curvature or surface tension) on the pathway of binding and insertion, highlighting the collaborative effort of electrostatic and hydrophobic interactions on interaction of SVS-1 with lipid bilayers.     

Adsorption of rutin with a novel β-cyclodextrin polymer adsorbent: Thermodynamic and kinetic study

The adsorption properties toward rutin of a cyclodextrin polymer adsorbent CroCD-TuC 3 have been studied. The adsorption capacity is reduced as temperature and pH of solution rises, but increases with the increase of solvent polarity. Compared with Sephadex? G-15 dextran gel beads, CroCD-TuC 3 shows dramatically higher isosteric enthalpy due to a significant contribution of rutin/β-cyclodextrin inclusion complex formation in CroCD-TuC 3 skeleton. A highlight in our study is that the pore diffusion model has been employed to describe the mass transfer inside the adsorbent pores. It reveals that the diffusion inside the pores is the rate-restricting step in the whole adsorption process. The effective pore diffusivity of rutin in CroCD-TuC 3 calculated is much lower than the diffusivity in diluted solution. The pore diffusion model is an available tool to investigate the profile of mass transfer inside the pores, and provides an effective method to describe adsorption kinetics.     

The effects of surface and macromolecular interactions on the kinetics of inactivation of trypsin and α-chymotrypsin

The autolysis of trypsin and α-chymotrypsin is accelerated in the presence of colloidal silica and glass surfaces. It is proposed that adsorption of the enzymes (favoured by electrostatic factors) results in a conformational change that renders the adsorbed enzyme more susceptible to proteolytic attack. Although the adsorbed enzymes are more susceptible to proteolysis, their activity towards low-molecular-weight substrates is not affected, indicating a relatively minor conformational change on adsorption. The rates of autolysis in solution (i.e. in `inert' vessels) are second-order for both trypsin and α -chymotrypsin, with rate constants of 13.0mol<sup>−1</sup>·dm<sup>3</sup>·s<sup>−1</sup> for trypsin (in 50mm-NaCl at pH8.0 at 25°C) and 10.2mol<sup>−1</sup>·dm<sup>3</sup>·s<sup>−1</sup> for α-chymotrypsin (in 0.1m-glycine at pH9.2 at 30°C). In glass vessels or in the presence of small areas of silica surface (as colloidal silica particles), the autolysis of both trypsin and α-chymotrypsin can show first-order kinetics. Under these conditions, saturation of the surface occurs and the fast surface proteolytic reaction controls the overall kinetic order. However, when greater areas of silica surface are present, saturation of the surface does not occur, and, since for a considerable portion of the adsorption isotherm the amount adsorbed is approximately proportional to the concentration in solution, second-order kinetics are again observed. A number of negatively charged macromolecules have been shown similarly to increase the rate of autolysis of trypsin: thus this effect, observed initially with glass and silica surfaces, is of more general occurrence when these enzymes adsorb on or interact with negatively charged surfaces and macromolecules. These observations explain the confusion in the literature with regard to the kinetics of autolysis of α-chymotrypsin, where first-order, second-order and intermediate kinetics have been reported. A further effect of glass surfaces and negatively charged macromolecules is to shift the pH–activity curve of trypsin to higher pH values, as a consequence of the effective decrease in pH in the `microenvironment' of the enzyme associated with the negatively charged surface or macromolecule.     

Ultrastructural localization of glycinin and β-conglycinin in Glycine max (soybean) cv. Maple Arrow by the immunogold method

Summary -Conglycinin (7S globulin) and glycinin (11S globulin) are the major reserve proteins of soybean. They were localized by the protein A immunogold method in thin sections of glycine max (soybean) cv. Maple Arrow. In cotyledons, both globulins were simultaneously present in all protein bodies. Statistical analysis of marking intensities indicated no correlation between globulin concentration and size of protein bodies. The immunogold method failed to detect either globulin in the embryonic axis and in cotyledons of four-day-old seedlings. Similar observations were made with cotyledons of two soy varieties lacking either the lectin or the Kunitz trypsin inhibitor. In another variety (T-102) lacking the lectin, the 7S globulin could not be detected.     

Effect of pH, light intensity, salt and nitrogen concentrations on growth and β-carotene accumulation by a new isolate of Dunaliella sp

Summary In the present study Dunaliella sp. that could grow in the Johnson medium was isolated from hypersaline Lake Tuz and its β-carotene production was studied in a batch system, in order to determine the optimal conditions required for the highest β-carotene accumulation. In the experiments with light intensity, the cell numbers and β-carotene content were maximum at pH 9, with 20% of NaCl concentration and 48 kerg cm⁻² s⁻¹ light intensity. At this light intensity, the β-carotene content of Dunaliella sp. ranged between 0.177 and 1.095 mg/ml for the culture broth and 0.119 to 0.261 ng/cell on a per cell basis under the nitrogen limitation. At the end of the experiments, the maximum β-carotene accumulation and the cell number were obtained at pH 7, 5 mM NaNO₃ and 20% NaCl concentrations as 0.261 ng/cell, 4.2×10⁶ cell/ml, respectively.     

Screening of β-fructofuranosidase-producing microorganisms and effect of pH and temperature on enzymatic rate

Seventeen different strains of filamentous fungi were grown in batch cultures to compare their abilities for the production of β-fructofuranosidase. Three of them, Aspergillus oryzae IPT-301, Aspergillus niger ATCC 20611 and strain IPT-615, showed high production with total fructosyltransferase activity higher than 12,500 units l⁻¹. In addition, the β-fructofuranosidases of those strains have a high fructosyltransferase activity-to-hydrolytic activity ratio. The temperature and pH effects on the sucrose-β-fructofuranosidase reaction rate were studied using a 2² factorial experimental design. The comparative analysis of the tested variable coefficients shows that the variable pH contributes mostly to the changes in the fructosyltransferase and hydrolytic rates and in the V _t/V _h ratio. At 40 and 50°C, there were no significant differences between the fructosyltransferase and hydrolytic velocities of these enzymes.     

Reductive methylation as a probe of the heat-labile α-bungarotoxin-acetylcholine receptor membrane complex: Evidence for surface interactions

α-Bungarotoxin (α-Bgt) is a potent postsynaptic neurotoxin which blocks neurotransmission by binding very tightly to the acetylcholine-receptor (AcChR) protein. We have previously shown (P. Calvo-Fernandez, and M. Martinez-Carrion (1981) Arch. Biochem. Biophys., 208, 154–159) that α-Bgt free in its native solution conformation incorporates 12 methyl groups when reductively methylated using formaldehyde and sodium cyanoborohydride. We now show that when the α-Bgt molecule is bound to the AcChR contained in native membranes prepared from Torpedo californica electroplax, the number of accessible methylation sites is significantly reduced. This favors a model of α-Bgt-AcChR interaction involving significant numbers of lysyl moieties distributed over a reasonably large surface of the toxin molecule. In addition, this paper presents a novel procedure for the rapid and nondestructive dissociation of the toxin-AcChR membrane complex which takes advantage of the thermal instability of the complex.