Swainsonine,a novel fungal metabolite: optimization of fermentative production and bioreactor operations using evolutionary programming

The optimization of bioreactor operations towards swainsonine production was performed using an artificial neural network coupled evolutionary program (EP)-based optimization algorithm fitted with experimental one-factor-at-a-time (OFAT) results. The effects of varying agitation (300–500 rpm) and aeration (0.5–2.0 vvm) rates for different incubation hours (72–108 h) were evaluated in bench top bioreactor. Prominent scale-up parameters, gassed power per unit volume (P _g/V _L, W/m³) and volumetric oxygen mass transfer coefficient (K _L a, s^?1) were correlated with optimized conditions. A maximum of 6.59 ± 0.10 μg/mL of swainsonine production was observed at 400 rpm-1.5 vvm at 84 h in OFAT experiments with corresponding P _g/V_L and K _L a values of 91.66 W/m³ and 341.48 × 10^?4 s^?1, respectively. The EP optimization algorithm predicted a maximum of 10.08 μg/mL of swainsonine at 325.47 rpm, 1.99 vvm and 80.75 h against the experimental production of 7.93 ± 0.52 μg/mL at constant K _L a (349.25 × 10^?4 s^?1) and significantly reduced P _g/V _L (33.33 W/m³) drawn by the impellers.     

Multivariate spectrochemical analysis of interactions of three common Isatin derivatives to calf thymus DNA in vitro

Interactions of Isatin and its derivatives, Isatin-3-isonicotinylhydrazone (IINH) and Isatin-β-thiosemicarbazone (IBT), with calf thymus DNA (ctDNA) have been investigated to delineate pharmaceutical-physicochemical properties using UV–Vis/fluorescence/circular dichroism (CD) spectroscopy, viscosity measurements, and multivariate chemometrics. IINH and IBT molecules intercalate between base pairs of DNA, hypochromism in UV absorptions, increase in the CD positive band, sharp increase in specific viscosity, and the displacement of the methylene blue and Neutral Red dye in complexes with ctDNA, by the IINH and IBT molecules, respectively. The observed intrinsic binding constants (K_{b[IBT–ctDNA]?}=?1.03 × 10⁵ and K_{b[IINH–ctDNA]?}=?1.09 × 10⁵ L mol^?1) were roughly comparable to other intercalators. In contrast, Isatin binds with ctDNA via groove mode (K_{b[Isatin–ctDNA]?}=?7.32 × 10⁴ L mol^?1) without any significant enhancement in ctDNA viscosity. The fluorescence quenching of Isatin by ctDNA was observed as static. CD spectra indicated that Isatin effectively absorbs into grooves of ctDNA, leading to transition from B to C form. Thermodynamic parameters like enthalpy changes (?H < 0) and entropy changes (?S > 0) were calculated according to Van’t Hoff’s equation, indicating the spontaneous interactions. The common soft/hard chemometric methods were used not only to resolve pure concentration and spectral profiles of components using the acquired spectra but also to calculate Stern–Volmer quenching constants, binding stoichiometry, apparent binding constants (K_a), binding constants (K_b), and thermodynamic parameters. The K_b values for Isatin, IINH, and IBT were calculated as 9.18 × 10³, 1.53 × 10⁵, and 2.45 × 10⁴ L mol^?1, respectively. The results obtained from experimental-spectroscopic analyses showed acceptable agreement with chemometric outlines.     

Production,characteristics and applications of phytase from a rhizosphere isolated <Emphasis Type="Italic">Enterobacter</Emphasis> sp. ACSS

Optimization of process parameters for phytase production by Enterobacter sp. ACSS led to a 4.6-fold improvement in submerged fermentation, which was enhanced further in fed-batch fermentation. The purified 62 kDa monomeric phytase was optimally active at pH 2.5 and 60 °C and retained activity over a wide range of temperature (40–80 °C) and pH (2.0–6.0) with a half-life of 11.3 min at 80 °C. The kinetic parameters K _m, V _max, K _cat, and K _cat/K _m of the pure phytase were 0.21 mM, 131.58 nmol mg^?1 s^?1, 1.64 × 10³ s^?1, and 7.81 × 10⁶ M^?1 s^?1, respectively. The enzyme was fairly stable in the presence of pepsin under physiological conditions. It was stimulated by Ca⁺², Mg⁺² and Mn⁺², but inhibited by Zn⁺², Cu⁺², Fe⁺², Pb⁺², Ba⁺² and surfactants. The enzyme can be applied in dephytinizing animal feeds, and the baking industry.     

Spectroscopic Studies on the Interaction of Fluorine Containing Triazole with Bovine Serum Albumin

The binding of one fluorine including triazole (C₁₀H₉FN₄S, FTZ) to bovine serum albumin (BSA) was studied by spectroscopic techniques including fluorescence spectroscopy, UV–Vis absorption, and circular dichroism (CD) spectroscopy under simulative physiological conditions. Fluorescence data revealed that the fluorescence quenching of BSA by FTZ was the result of forming a complex of BSA–FTZ, and the binding constants (K _a) at three different temperatures (298, 304, and 310 K) were 1.516?×?10⁴, 1.627?×?10⁴, and 1.711?×?10⁴?mol L^?1, respectively, according to the modified Stern–Volmer equation. The thermodynamic parameters ΔH and ΔS were estimated to be 7.752 kJ mol^?1 and 125.217 J?mol^?1?K^?1, respectively, indicating that hydrophobic interaction played a major role in stabilizing the BSA–FTZ complex. It was observed that site I was the main binding site for FTZ to BSA from the competitive experiments. The distance r between donor (BSA) and acceptor (FTZ) was calculated to be 7.42 nm based on the Förster theory of non-radioactive energy transfer. Furthermore, the analysis of fluorescence data and CD data revealed that the conformation of BSA changed upon the interaction with FTZ.     

Cloning, Expression, and Enzymatic Characterization of Isocitrate Dehydrogenase from Helicobacter pylori

Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate with NAD(P) as a cofactor in the tricarboxylic acid cycle. As a housekeeping protein in Helicobacter pylori, IDH was considered as a possible candidate for serological diagnostics and detection. Here, we identified a new icd gene encoding IDH from H. pylori strain SS1. The recombinant H. pylori isocitrate dehydrogenase (HpIDH) was cloned, expressed, and purified in E. coli system. The enzymatic characterization of HpIDH demonstrates its activity with k _cat of 87 s^?1, K _m of 124 μM and k _cat/K _m of 7 × 10⁵ M^?1s^?1 toward isocitrate, k _cat of 80 s^?1, K _m of 176 μM and k _cat/K _m of 4.5 × 10⁵ M^?1s^?1 toward NADP. The optimum pH of the enzyme activity is around 9.0, and the optimum temperature is around 50 °C. This current work is expected to help better understand the features of HpIDH and provide useful information for H. pylori serological diagnostics and detection.     

Abscisic Acid-Induced Starch Accumulation in Bioenergy Crop Duckweed <Emphasis Type="Italic">Spirodela polyrrhiza</Emphasis>

Spirodela polyrrhiza, a fast-growing duckweed with high starch and low lignin content, shows promise as a feedstock for bioenergy. Abscisic acid (ABA) is a biological hormone that controls plant growth and stress response. The effects of different ABA concentrations (0, 1.0 × 10^?5, 1.0 × 10^?4, 1.0 × 10^?3, 1.0 × 10^?2, and 1.0 × 10^?1 mg/L) on duckweed biomass growth, carbon dioxide fixation, formation of photosynthetic pigments (Chlorophyll a (Chla), Chlorophyll b (Chlb), and carotenoids), the activities of soluble starch synthase (SSS) and starch branching enzyme (SBE), and the starch content of biomass were investigated in this study. ABA at concentrations lower than 1.0 × 10^?3 mg/L promoted carbon dioxide fixation, whereas it inhibited carbon dioxide fixation at concentrations over 1.0 × 10^?3 mg/L. ABA enhanced SSS and SBE activities at concentrations lower than 1.0 × 10^?2 mg/L. ABA treatment increased the content of Chla, Chlb, and carotenoids and resulted in the enhancement of starch content. Chla content gradually increased with the increasing concentration of ABA (1.0 × 10^?5 to 1.0 × 10^?2 mg/L). After culturing for 10 days, starch content in 1.0 × 10^?2 mg/L ABA medium reached 35.3% of dry weight (DW), which was the highest level in this study. This suggests that there is a great potential to develop a technology to increase starch accumulation in duckweed which can be used as an alternative to corn, sugarcane, or other food crops as a starch source.     

A chimeric α-amylase engineered from <Emphasis Type="Italic">Bacillus acidicola</Emphasis> and G<Emphasis Type="Italic">eobacillus thermoleovorans</Emphasis> with improved thermostability and catalytic efficiency

The α-amylase (Ba-amy) of Bacillus acidicola was fused with DNA fragments encoding partial N- and C-terminal region of thermostable α-amylase gene of Geobacillus thermoleovorans (Gt-amy). The chimeric enzyme (Ba-Gt-amy) expressed in Escherichia coli displays marked increase in catalytic efficiency [K _cat: 4 × 10⁴ s^?1 and K _cat/K _m: 5 × 10⁴ mL^?1 mg^?1 s^?1] and higher thermostability than Ba-amy. The melting temperature (T _m) of Ba-Gt-amy (73.8 °C) is also higher than Ba-amy (62 °C), and the CD spectrum analysis revealed the stability of the former, despite minor alteration in secondary structure. Langmuir–Hinshelwood kinetic analysis suggests that the adsorption of Ba-Gt-amy onto raw starch is more favourable than Ba-amy. Ba-Gt-amy is thus a suitable biocatalyst for raw starch saccharification at sub-gelatinization temperatures because of its acid stability, thermostability and Ca²⁺ independence, and better than the other known bacterial acidic α-amylases.     

Sulfonamide inhibition profile of the γ-carbonic anhydrase identified in the genome of the pathogenic bacterium Burkholderia pseudomallei the etiological agent responsible of melioidosis

A new γ-carbonic anhydrase (CA, EC 4.1.1.1) was cloned and characterized kinetically in the genome of the bacterial pathogen Burkholderia pseudomallei, the etiological agent of melioidosis, an endemic disease of tropical and sub-tropical regions of the world. The catalytic activity of this new enzyme, BpsCAγ, is significant with a k_cat of 5.3 × 10⁵ s^?1 and k_cat/K_m of 2.5 × 10⁷ M^?1 × s^?1 for the physiologic CO₂ hydration reaction. The inhibition constant value for this enzyme for 39 sulfonamide inhibitors was obtained. Acetazolamide, benzolamide and metanilamide were the most effective (K_Is of 149–653 nM) inhibitors of BpsCAγ activity, whereas other sulfonamides/sulfamates such as ethoxzolamide, topiramate, sulpiride, indisulam, sulthiame and saccharin were active in the micromolar range (K_Is of 1.27–9.56 μM). As Burkholderia pseudomallei is resistant to many classical antibiotics, identifying compounds that interfere with crucial enzymes in the B. pseudomallei life cycle may lead to antibiotics with novel mechanisms of action.     

Influence of mixing and shear stress on Chlorella vulgaris, Scenedesmus obliquus, and Chlamydomonas reinhardtii

Photosynthetic activity (PA) and growth of different microalgae species (Chlorella vulgaris, Scenedesmus obliquus, and Chlamydomonas reinhardtii) depends in addition to other factors on mixing (tip speed) and shear stress (friction velocity) and was studied in a stirring tank (microcosm). In order to detect cause–effect relationships for an increase in photosynthetic activity, experiments were conducted under different pH values (6.0–8.5) and CO₂ concentrations (0.038 and 4 % (v/v)). The PA was determined as the effective quantum yield by pulse amplitude modulation during a stepwise increase of the tip speed from 0 to 589 cm s^?1 (friction velocity: 0–6.05 cm s^?1) in short-term experiments. The increase caused a distinctive pattern of PA of each species. Compared to 0 cm s^?1, C. vulgaris and S. obliquus showed a 4.0 and 4.8 % higher PA at the optimum tip speed of 126 cm s^?1 (friction velocity of 2.09 cm s^?1) and a 48 and 71 % higher growth, respectively. At 203 cm s^?1, the PA dropped to the value of the unstirred control, while at 589 cm s^?1, the PA decreased of up to 7 and 8 %. In contrast, C. reinhardtii showed 7 % stronger growth at 126 cm s^?1, while the PA decreased about 15 % at an increase of tip speed to 589 cm s^?1. For all investigated microalgae, the pattern of PA and higher growth was not only explained by the main contributing factors like light supply, nutrient supply, and overcoming diffusion gradients. The results indicate that hydrodynamic forces have a stimulating effect on the physiological processes within the cells.     

The Interaction of Chromium(VI) with Urease in Solution

The interaction between K₂Cr₂O₇ and urease was investigated using fluorescence, UV-vis absorption, and circular dichroism (CD) spectroscopy. The experimental results showed that the fluorescence quenching of urease by K₂Cr₂O₇ was a result of the formation of K₂Cr₂O₇–urease complex. The apparent binding constant K _A between K₂Cr₂O₇ and urease at 295, 302, and 309 K were obtained to be 2.14?×?10⁴, 1.96?×?10⁴, and 1.92?×?10⁴ L mol^?1, respectively. The thermodynamic parameters, ΔH° and ΔS° were estimated to be ?5.90 kJ mol^?1, 43.67 J mol^?1 K^?1 according to the Van’t Hoff equation. The electrostatic interaction played a major role in stabilizing the complex. The distance r between donor (urease) and acceptor (K₂Cr₂O₇) was 5.08 nm. The effect of K₂Cr₂O₇ on the conformation of urease was analyzed using UV-vis absorption, CD, synchronous fluorescence spectroscopy, and three-dimensional fluorescence spectra, the environment around Trp and Tyr residues were altered.     

On enzymatic clotting processes. II. The colloidal instability of chymosin-treated casein micelles.

The enzymatic clotting of casein micelles dispersed in 0.01 M CaCl₂ was monitored by turbidimetry and electrophoresis. The relation between the duration of the lag phase and the enzyme concentration, (e), can be represented by t = K(e)^?γ, where K is a constant and the exponent γ is found to vary between 0.92 and 1.00. This result is interpreted in terms of a flocculation rate constant increasing with the concentration of the enzyme. It is shown that the colloidal instability of chymosin-treated casein micelles cannot be explained on the basis of the well-known theory of the stability of lyophobic colloids, but that clotting is achieved through short-range interactions. The short-range effects that most probably account for the clotting are: hydrophobic bond formation, Ca-bridgas and electrostatic interactions. Under typica'. experimental conditions (33°C; maximum rate of enzymatic product formation about 1.8 × 10^?10 mol ml^?1 s^?1) the flocculation rate constant of clotting micelles was found to be 5 × 10⁵ mlmol^?1s^?1. Various factors, which could be responsible for this low value, are discussed. In the initial stages of the clotting process the turbidity of the system passes through a shallow minimum, which is ascribed to the cleavage of a macropeptide from K-casein by the clotting enzyme. The condition for the minimum has been derived.     

Characterization of EstB,a novel cold-active and organic solvent-tolerant esterase from marine microorganism Alcanivorax dieselolei B-5(T)

A novel esterase gene, estB, was cloned from the marine microorganism Alcanivorax dieselolei B-5(T) and overexpressed in E. coli DE3 (BL21). The expressed protein EstB with a predicted molecular weight of 45.1 kDa had a distinct catalytic triad (Ser²¹¹-Trp³⁵³-Gln³⁸⁵) and the classical consensus motif conserved in most lipases and esterases Gly²⁰⁹-X-Ser²¹¹-X-Gly²¹³. EstB showed very low similarity to any known proteins and displayed the highest similarity to the hypothetical protein (46 %) from Rhodococcus jostii RHA1. EstB showed the optimal activity around pH 8.5 and 20 °C and was identified to be extremely cold-adaptative retaining more than 95 % activity between 0 and 10 °C. The values of kinetic parameters on p-NP caproate (K _m, K _cat and K _cat/K _m) were 0.15 mM, 0.54 × 10³ s^?1 and 3.6 × 10³ s^?1 mM^?1, respectively. In addition, EstB showed remarkable stability in several studied organic solvents and detergents of high concentrations with the retention of more than 70 % activity after treatment for 30 min. The cold activity and its tolerance towards organic solvents made it a promising biocatalyst for industrial applications under extreme conditions.     

A remarkable activity of human leukotriene A4 hydrolase (LTA4H) toward unnatural amino acids

Leukotriene A₄ hydrolase (LTA4H––EC 3.3.2.6) is a bifunctional zinc metalloenzyme, which processes LTA₄ through an epoxide hydrolase activity and is also able to trim one amino acid at a time from N-terminal peptidic substrates via its aminopeptidase activity. In this report, we have utilized a library of 130 individual proteinogenic and unnatural amino acid fluorogenic substrates to determine the aminopeptidase specificity of this enzyme. We have found that the best proteinogenic amino acid recognized by LTA4H is arginine. However, we have also observed several unnatural amino acids, which were significantly better in terms of cleavage rate (k _cat/K _m values). Among them, the benzyl ester of aspartic acid exhibited a k _cat/K _m value that was more than two orders of magnitude higher (1.75 × 10⁵ M^?1 s^?1) as compared to l-Arg (1.5 × 10³ M^?1 s^?1). This information can be used for design of potent inhibitors of this enzyme, but may also suggest yet undiscovered functions or specificities of LTA4H.     

Purification and characterization of laccase secreted by Phellinus linteus MTCC-1175 and its role in the selective oxidation of aromatic methyl group

A laccase from the culture filtrate of Phellinus linteus MTCC-1175 has been purified to homogeneity. The method involved concentration of the culture filtrate by ammonium sulphate precipitation and an anion exchange chromatography on DEAE-cellulose. The SDS-PAGE and native-PAGE gave single protein band indicating that the enzyme preparation was pure. The molecular mass of the enzyme determined from SDS-PAGE analysis was 70 kDa. Using 2.6-dimethoxyphenol, 2.2′[azino-bis-(3-ethylbonzthiazoline-6-sulphonic acid) diammonium salt] (ABTS) and 4-hydroxy-3,5-dimethoxybenzaldehyde azine as the substrates, the K _m, k _cat and k _cat/K _m values of the laccase were found to be 160 μM, 6.85 s^?1, 4.28 × 10⁴ M^?1 s^?1, 42 μM, 6.85 s^?1, 16.3 × 10⁴ M^?1 s^?1 and 92 μM, 6.85 s^?1, 7.44 × 10⁴ M^?1 s^?1, respectively. The pH and the temperature optima of the P. linteus MTCC-1175 laccase were 5.0 and 45°C, respectively. The activation energy for thermal denaturation of the enzyme was 38.20 kJ/mole/K. The enzyme was the most stable at pH 5.0 after 1 h reaction. In the presence of ABTS as the mediator, the enzyme transformed toluene, 3-nitrotoluene and 4-chlorotoluene to benzaldehyde, 3-nitrobenzaldehyde and 4-chlorobenzaldehyde, respectively.     

Light Intensity Alters the Extent of Arsenic Toxicity in Helianthus annuus L. Seedlings

The present study is aimed at assessing the extent of arsenic (As) toxicity under three different light intensities—optimum (400 μmole photon m^?2 s^?1), sub-optimum (225 μmole photon m^?2 s^?1), and low (75 μmole photon m^?2 s^?1)—exposed to Helianthus annuus L. var. DRSF-113 seedlings by examining various physiological and biochemical parameters. Irrespective of the light intensities under which H. annuus L. seedlings were grown, there was an As dose (low, i.e., 6 mg kg^?1 soil, As₁; and high, i.e., 12 mg kg^?1 soil, As₂)-dependent decrease in all the growth parameters, viz., fresh mass, shoot length, and root length. Optimum light-grown seedlings exhibited better growth performance than the sub-optimum and low light-grown seedlings; however, low light-grown plants had maximum root and shoot lengths. Accumulation of As in the plant tissues depended upon its concentration used, proximity of the plant tissue, and intensity of the light. Greater intensity of light allowed greater assimilation of photosynthates accompanied by more uptake of nutrients along with As from the medium. The levels of chlorophyll a, b, and carotenoids declined with increasing concentrations of As. Seedlings acquired maximum Chl a and b under optimum light which were more compatible to face As₁ and As₂ doses of As, also evident from the overall status of enzymatic (SOD, POD, CAT, and GST) and non-enzymatic antioxidant (Pro).     

Feeding efficiency of Chaoborus flavicans (Insecta, Diptera) under turbulent conditions

Turbulence can affect predator–prey interactions. The effect of turbulence on the feeding efficiency of an ambush predator was tested with laboratory experiments. The experiments were conducted in 100-L aquaria in which ten individuals of fourth instar Chaoborus flavicans larvae were placed as predators. Two prey densities (3 and 10 ind. of Daphnia pulex L^?1) and two durations (30 and 120 min) were tested in a nonturbulent treatment and five different turbulence levels [average root-mean-square (RMS) velocities ranging from 0 to 7.3 cm s^?1, corresponding dissipation rates from 7.2 × 10^?7 to 1.3 × 10^?3 m² s^?3]. We hypothesized that the feeding rate of C. flavicans would be enhanced by turbulence due to increasing encounter rates up to a turbulence level above which a disturbance in post-encounter processes would lead to reduced feeding efficiency. However, the results showed no significant increase in the feeding rate of C. flavicans at intermediate turbulence. At high turbulence we found the expected significant negative response in the feeding rate of Chaoborus larvae. The feeding rate declined below the rates at nonturbulent and intermediate turbulence conditions as the average RMS velocity exceeded 3.1 cm s^?1 (dissipation rate 9.9 × 10^?5 m² s^?3, respectively).     

Can uranium follow the iron-acquisition pathway? Interaction of uranyl-loaded transferrin with receptor 1

Transferrin receptor 1 (R_D) binds iron-loaded transferrin and allows its internalization in the cytoplasm. Human serum transferrin also forms complexes with metals other than iron, including uranium in the uranyl form (UO₂ ²⁺). Can the uranyl-saturated transferrin (TUr₂) follow the receptor-mediated iron-acquisition pathway? In cell-free assays, TUr₂ interacts with R_D in two different steps. The first is fast, direct rate constant, k ₁ = (5.2 ± 0.8) × 10⁶ M^?1 s^?1; reverse rate constant, k _?1 = 95 ± 5 s^?1; and dissociation constant K ₁ = 18 ± 6 μM. The second occurs in the 100-s range and leads to an increase in the stability of the protein–protein adduct, with an average overall dissociation constant K _d = 6 ± 2 μM. This kinetic analysis implies in the proposed in vitro model possible but weak competition between TUr₂ and the C-lobe of iron-loaded transferrin toward the interaction with R _D.     

Uridine-cytidine kinase. Kinetic studies and reaction mechanism.

The initial velocity pattern has been determined for uridine-cytidine kinase purified from the murine mast cell neoplasm P815. With either uridine or cytidine as phosphate acceptor, and ATP as phosphate donor, the pattern observed was one of intersecting lines, ruling out a ping-pong reaction mechanism, and suggesting that the reaction probably proceeds by the sequential addition of both substrates to the enzyme to form a ternary complex, followed by the sequential release of the two products. This pattern was obtained whether the reaction was run in 0.01 m potassium phosphate buffer, pH 7.5, or in 0.1 m Tris-HCl, pH 7.2. When analyzed by the Sequen computer program, the data indicated an apparent K_m of the enzyme for uridine of 1.5 × 10^?4m, an apparent K_m for cytidine of 4.5 × 10^?5m, and a K_m for ATP, with uridine or cytidine as phosphate acceptor, of 3.6 × 10^?3m or 2.1 × 10^?3m, respectively. The V was 1.83 μmol phosphorylated/min/mg enzyme protein for the uridine kinase reaction and 0.91 μmol for the cytidine kinase reaction.     

Constant surface tension molecular dynamics simulations of lipid bilayers with trehalose

Surface areas and fluctuations evaluated from 50 ns molecular dynamics simulations of fully hydrated dipalmitoylphosphatidylcholine (DPPC) bilayers in a 1:2 trehalose:lipid ratio carried out at surface tensions 10, 17 and 25 dyn/cm/leaflet are compared with those of pure bilayers under the same conditions. Trehalose increases the surface area, as consistent with the surface tension lowering observed in simulations at constant area. The system bulk elastic modulus K _b  = 1.5 ± 0.3 × 10¹⁰ dyn/cm². It is independent of bilayer surface area and trehalose content within statistical error. In contrast, the area elastic modulus K _a shows a strong area dependence. At 64 Å²/lipid (the experimental surface area), K _a  = 138 ± 26 dyn/cm for a pure DPPC bilayer and 82 ± 10 dyn/cm for one with trehalose; i.e. trehalose increases fluidity of the bilayer surface at this area per lipid.