Structural characterization of thermostable,solvent tolerant,cytosafe tannase from Bacillus subtilis PAB2

Tannase production by Bacillus subtilis PAB2, was investigated under solid state fermentation using tamarind seed as sole carbon source and it was found as the highest titer (73.44 U/gds). The enzyme was purified to homogeneity, which showed the molecular mass around 52 kDa (K_m = 0.445 mM, V_max = 125.8 mM/mg/min and K_cat = 2.88 min^–1). The enzyme was found stable in a range of pH (3.0–8.0) and temperature (30–70 °C) with an optimal activity at pH 5.0, pI of 4.4 and at 40 °C temperature. It exhibited half-life (t_1/2) of 4.5 h at 60 °C. The enzyme comprised a typical secondary structure containing α-helix (9.3%), β-pleated sheet (33.6%) and β-turn (17.2%). The native conformation of the enzyme was alike a 44 nm spherical nanoparticle upon aggregation. Thermodynamic parameters of tannase revealed that it was stable at 40 °C and showed Q₁₀, ΔG_d and ΔS_d values of 2.08, 99.37 KJ/mol and 252.38 J mol⁻¹ K⁻¹, respectively. Organic solvents were stimulatory with regard to enzyme activity. Moreover, the altered enzyme activity was determined to be correlated with the changes in structural conformation in presence of inducer and inhibitor. Tannase was explored to have no cytotoxicity on Vero cell line as well as rat model study.     

Interaction kinetics of liposome-incorporated unsaturated fatty acids with fatty acid-binding protein 3 by surface plasmon resonance

The role of heart-type fatty acid-binding protein (FABP3) in human physiology as an intracellular carrier of fatty acids (FAs) has been well-documented. In this study, we aimed to develop an analytical method to study real-time interaction kinetics between FABP3 immobilized on the sensor surface and unsaturated C18 FAs using surface plasmon resonance (SPR). To establish the conditions for SPR experiments, we used an FABP3-selective inhibitor 4-(2-(1-(4-bromophenyl)-5-phenyl-1H-pyrazol-3-yl)-phenoxy)-butyric acid. The affinity index thus obtained was comparable to that reported previously, further supporting the usefulness of the SPR-based approach for evaluating interactions between FABPs and hydrophobic ligands. A pseudo-first-order affinity of FABP3 to K⁺ petroselinate (C18:1 Δ6 cis), K⁺ elaidate (C18:1 Δ9 trans), and K⁺ oleate (C18:1 Δ9 cis) was characterized by the dissociation constant (K_d) near micromolar ranges, whereas K⁺ linoleate (C18:2 Δ9,12 cis/cis) and K⁺ α-linolenate (C18:3 Δ9,12,15 cis/cis/cis) showed a higher affinity to FABP3 with K_d around 1 × 10⁻⁶ M. Interactions between FAPB3 and C18 FAs incorporated in large unilamellar vesicles consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and FAs (5:1 molar ratio) were also analysed. Control DMPC liposomes without FA showed only marginal binding to FABP3 immobilized on a sensor chip while liposome-incorporated FA revealed significant responses in sensorgrams, demonstrating that the affinity of FAs to FABP3 could be evaluated by using the liposome-incorporated analytes. Significant affinity to FABP3 was observed for monounsaturated fatty acids (K_d in the range of 1 × 10⁻⁷ M). These experiments demonstrated that highly hydrophobic compounds in a liposome-incorporated form could be subjected to SPR experiments for kinetic analysis.     

The stability and thermodynamic parameters of a very thermostable and calcium-independent α-amylase from a newly isolated bacterium,Anoxybacillus beppuensis TSSC-1

Anoxybacillus beppuensis TSSC-1 (GenBank Number, EU710556), a thermophilic bacterium isolated from a hot spring reservoir, was found to optimally secrete a monomeric α-amylase at 55 °C and pH 7. The enzyme was purified to homogeneity by a single-step purification on phenyl sepharose 6FF, achieving a 58% yield, 10,000 U/mg specific activity and 19.5 fold purification. The molecular weight, K_m and V_max were 43 kD, 0.5 mg ml^?1 and 3571.42 μmol ml^?1 m^?1, respectively. The enzymatic catalysis of soluble starch was optimum at 80 °C and pH 7. The thermodynamic parameters, K_d, t_1/2, ΔH*, ΔS*, E and ΔG*, were consistent. The very compact structure of the enzyme and the transitional enzyme–substrate complex resisted denaturation at extreme temperatures and alkaline pH. The K_d and t_1/2 measurements were consistent with the high thermostability and pH tolerance observed. The structural stability of the enzyme was also reflected by the values of ΔH*, ΔS*, E and ΔG*. While the enzyme did not exhibit metal ion dependency, it was resistant to chemical denaturation. The broad thermo- and pH-tolerance of this enzyme suggests potential commercial opportunities.     

Purification,kinetic and thermodynamic characterization of soluble acid invertase from sugarcane (Saccharum officinarum L.)

We report for the first time kinetic and thermodynamic properties of soluble acid invertase (SAI) of sugarcane (Saccharum officinarum L.) salt sensitive local cultivar CP 77-400 (CP-77). The SAI was purified to apparent homogeneity on FPLC system. The crude enzyme was about 13 fold purified and recovery of SAI was 35%. The invertase was monomeric in nature and its native molecular mass on gel filtration and subunit mass on SDS-PAGE was 28 kDa. SAI was highly acidic having an optimum pH lower than 2. The acidic limb was missing. Proton transfer (donation and receiving) during catalysis was controlled by the basic limb having a pKa of 2.4. Carboxyl groups were involved in proton transfer during catalysis. The kinetic constants for sucrose hydrolysis by SAI were determined to be: k_m = 55 mg ml^?1, k_cat = 21 s^?1, k_cat/k_m = 0.38, while the thermodynamic parameters were: ΔH* = 52.6 kJ mol^?1, ΔG* = 71.2 kJ mol^?1, ΔS* = ?57 J mol^?1 K^?1, ΔG*_E–S = 10.8 kJ mol^?1 and ΔG*_E–T = 2.6 kJ mol^?1. The kinetics and thermodynamics of irreversible thermal denaturation at various temperatures 53–63 °C were also determined. The half -life of SAI at 53 and 63 °C was 112 and 10 min, respectively. At 55 °C, surprisingly the half -life increased to twice that at 53 °C. ΔG*, ΔH* and ΔS* of irreversible thermal stability of SAI at 55 °C were 107.7 kJ mol^?1, 276.04 kJ mol^?1 and 513 J mol^?1K^?1, respectively.     

Optimizing isothermal titration calorimetry protocols for the study of 1:1 binding: Keeping it simple

BackgroundSuccessful ITC experiments require conversion of cell reagent (titrand M) to product and production or consumption of heat. These conditions are quantified for 1:1 binding, M + X ⇔ MX.MethodsNonlinear least squares is used in error-propagation mode to predict the precisions with which the key quantities — binding constant K, reaction enthalpy ΔH°, and stoichiometry number n — can be estimated over a wide range of the dimensionless quantity that governs isotherm shape, c = K[M]₀. The measurement precision σ_q is estimated from analysis of water–water blanks.ResultsWhen the product conversion exceeds 90%, the parameter relative standard errors are proportional to σ_q/q_tot, where the total heat q_tot ≈ ΔH° [M]₀ V₀. Specifically, σ_K/K × q_tot/σ_q ≈ 25 for c = 10 ^− 3 − 10, ≈ 11 c^1/3 for c = 10 − 10⁴. For c > 1, n and ΔH° are more precise than K; this holds also at smaller c for the product n × ΔH° and for ΔH° when n can be held fixed. Use of as few as 10 titrant injections can outperform the customary 20–40 while also improving productivity.ConclusionThese principles are illustrated in experiment design using the program ITC-PLANNER15.General significanceSimple quantitative guidelines replace the “c rules” that have dominated the literature for decades. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Effects of boldine on tyrosinase: Inhibition kinetics and computational simulation

Boldine is one of the most potent natural antioxidants and displays some important pharmacological activities, such as cytoprotective and anti-inflammatory activities. Based on its antioxidant properties, we studied the effects of boldine on l-DOPA oxidation by evaluating the inhibitory kinetics and a computational simulation between boldine and tyrosinase. Boldine reversibly inhibited tyrosinase from mushroom (Agaricus bisporus) in a mixed-type manner, with a K_i = 7.203 ± 0.933 mM. To gain insight into the inactivation process, we computed the kinetics via time-interval measurements and continuous substrate reactions. The results indicated that the inactivation induced by boldine was a first-order reaction with biphasic processes and that the substrate can promote the inactivation process. To gain further insight, we performed computational docking and molecular dynamics simulations, and the results showed that boldine can interact with several residues near the tyrosinase active site. Our study provides insight into the inhibition of tyrosinase in response to alkaloids. Based on its tyrosinase-inhibiting effect and low toxicity, boldine is a potential natural anti-pigmentation agent.     

The effect of PI3 kinase inhibitor LY294002 on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

AimsWe examined the effect of LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, on voltage-dependent K⁺ (Kv) channels.Main methodsElectrophysiological recordings were performed in freshly isolated rabbit coronary arterial smooth muscle cells.Key findingsThe Kv current amplitude was inhibited by LY294002 in a dose-dependent manner, with a K_d value of 1.48 μM. Without alteration of the kinetics of activation, LY294002 accelerated the decay rate of Kv channel inactivation. The rate constants of association and dissociation for LY294002 were 1.83 ± 0.01 μM^? 1 s^? 1 and 2.59 ± 0.14 s^? 1, respectively. Application of LY294002 had no significant impact on the steady-state activation or inactivation curves. In the presence of LY294002, the recovery time constant from inactivation was increased, and Kv channel inhibition increased under train pulses (1 or 2 Hz). This indicates that LY294002-induced Kv channel inhibition is use-dependent. Furthermore, pretreatment with another PI3K inhibitor, wortmannin (10 μM), did not affect the Kv current, and did not change the inhibitory effect of LY294002.SignificanceBased on these results, we suggest that LY294002 directly blocks Kv current irrespective of PI3K inhibition.     

Kinetic analysis for suicide-substrate inactivation of microperoxidase-11: A modified model for bisubstrate enzymes in the presence of reversible inhibitors

Kinetics of microperoxidase-11 (MP-11) as a heme–peptide enzyme model in oxidation reaction of guaiacol (AH) by hydrogen peroxide was studied in the presence of amino acids, taking into account the inactivation of MP-11 during reaction by its suicide substrate, H₂O₂. Reliability of the kinetic equation was evaluated by non-linear mathematical fitting. Fitting of experimental data into a new integrated kinetic relation showed a close match between the kinetic model and the experimental data. Indeed, it was found that the mechanism of suicide-peroxide inactivation of MP-11 in the presence of amino acids is different from MP-11 and/or horseradish peroxidase. In this mechanism, amino acids compete with hydrogen peroxide for the sixth co-ordination position of iron atom in the heme group through a competitive inhibition mechanism.The proposed model can successfully determine the kinetic parameters including inactivation by hydrogen peroxide as well as the inhibitory rate constants by the amino acid inhibitor.Kinetic parameters of inactivation including the initial activity of MP-11, α₀, the apparent inactivation rate constant, k_i and the apparent inhibition rate constant for cysteine, k_I were obtained 0.282 ± 0.006 min^?1, 0.497 ± 0.013 min^?1 and 1.374 ± 0.007 min^?1 at [H₂O₂] = 1.0 mM, 27 °C, phosphate buffer 5.0 mM, pH 7.0. Results showed that inactivation and inhibition of microperoxidase as a peroxidase model enzyme occurred simultaneously even at low concentrations of hydrogen peroxide (0.4 mM). This kinetic analysis based on the suicide-substrate inactivation of microperoxidase-11, provides a tool and model for studying peroxidase models in the presence of reversible inhibitors. The introduced inhibition procedure can be used in designing activity tunable and specific protected enzyme models in the hidden and reversibly inhibited forms, which do not undergo inactivation.     

Carbonic anhydrase inhibitors: Inhibition of the β-class enzyme from the yeast Saccharomyces cerevisiae with sulfonamides and sulfamates

The protein encoded by the Nce103 gene of Saccharomyces cerevisiae, a β-carbonic anhydrase (CA, EC 4.2.1.1) designated as scCA, has been cloned, purified, characterized kinetically and investigated for its inhibition with a series of sulfonamides and one sulfamate. The enzyme showed high CO₂ hydrase activity, with a k_cat of 9.4 × 10⁵ s^?1, and k_cat/K_M of 9.8 × 10⁷ M^?1 s^?1. Simple benzenesulfonamides substituted in 2-, 4- and 3,4-positions of the benzene ring with amino, alkyl, halogeno and hydroxyalkyl moieties were weak scCA inhibitors with K_Is in the range of 0.976–18.45 μM. Better inhibition (K_Is in the range of 154–654 nM) was observed for benzenesulfonamides incorporating aminoalkyl/carboxyalkyl moieties or halogenosulfanilamides; benzene-1,3-disulfonamides; simple heterocyclic sulfonamides and sulfanilyl-sulfonamides. The clinically used sulfonamides/sulfamate (acetazolamide, ethoxzolamide, methazolamide, dorzolamide, topiramate, celecoxib, etc.) generally showed effective scCA inhibitory activity, with K_Is in the range of 82.6–133 nM. The best inhibitor (K_I of 15.1 nM) was 4-(2-amino-pyrimidin-4-yl)-benzenesulfonamide. These inhibitors may be useful to better understand the physiological role of β-CAs in yeast and some pathogenic fungi which encode orthologues of the yeast enzyme and eventually for designing novel antifungal therapies.     

Molecular characterization of a novel Na+/H+ antiporter cDNA from Eucalyptus globulus

Environmental stress factors such as salt, drought and heat are known to affect plant productivity. However, high salinity is spreading throughout the world, currently affecting more than 45 million ha. One of the mechanisms that allow plants to withstand salt stress consists on vacuolar sequestration of Na⁺, through a Na⁺/H⁺ antiporter. We isolated a new vacuolar Na⁺/H⁺ antiporter from Eucalyptus globulus from a cDNA library. The cDNA had a 1626 bp open reading frame encoding a predicted protein of 542 amino acids with a deduced molecular weight of 59.1 KDa. Phylogenetic and bioinformatic analyses indicated that EgNHX1 localized in the vacuole. To assess its role in Na⁺ exchange, we performed complementation studies using the Na⁺ sensitive yeast mutant strain Δnhx1. The results showed that EgNHX1 partially restored the salt sensitive phenotype of the yeast Δnhx1 strain. However, its overexpression in transgenic Arabidopsis confers tolerance in the presence of increasing NaCl concentrations while the wild type plants exhibited growth retardation. Expression profiles of Eucalyptus seedlings subjected to salt, drought, heat and ABA treatment were established. The results revealed that Egnhx1 was induced significantly only by drought. Together, these results suggest that the product of Egnhx1 from E. globulus is a functional vacuolar Na⁺/H⁺ antiporter.     

Two functional sites of phosphatidylglycerol for regulation of reaction of plastoquinone QB in photosystem II

Functional roles of an anionic lipid phosphatidylglycerol (PG) were studied in pgsA-gene-inactivated and cdsA-gene-inactivated/phycobilisome-less mutant cells of a cyanobacterium Synechocystis sp. PCC 6803, which can grow only in PG-supplemented media. 1) A few days of PG depletion suppressed oxygen evolution of mutant cells supported by p-benzoquinone (BQ). The suppression was recovered slowly in a week after PG re-addition. Measurements of fluorescence yield indicated the enhanced sensitivity of Q_B to the inactivation by BQ. It is assumed that the loss of low-affinity PG (PG_L) enhances the affinity for BQ that inactivates Q_B. 2) Oxygen evolution without BQ, supported by the endogenous electron acceptors, was slowly suppressed due to the direct inactivation of Q_B during 10 days of PG depletion, and was recovered rapidly within 10 h upon the PG re-addition. It is concluded that the loss of high-affinity PG (PG_H) displaces Q_B directly. 3) Electron microscopy images of PG-depleted cells showed the specific suppression of division of mutant cells, which had developed thylakoid membranes attaching phycobilisomes (PBS). 4) Although the PG-depletion for 14 days decreased the chlorophyll/PBS ratio to about 1/4, florescence spectra/lifetimes were not modified indicating the flexible energy transfer from PBS to different numbers of PSII. Longer PG-depletion enhanced allophycocyanin fluorescence at 683 nm with a long 1.2 ns lifetime indicating the suppression of energy transfer from PBS to PSII. 5) Action sites of PG_H, PG_L and other PG molecules on PSII structure are discussed.     

Application of seaweeds for the removal of lead from aqueous solution

Ten different seaweed species were compared on the basis of lead uptake at different pH conditions. The brown seaweed, Turbinaria conoides, exhibited maximum lead uptake (at pH 4.5) and hence was selected for further studies. Sorption isotherms, obtained at different pH (4–5) and temperature (25–35 °C) conditions were fitted using Langmuir and Sips models. According to the Langmuir model, the maximum lead uptake of 439.4 mg/g was obtained at optimum pH (4.5) and temperature (30 °C). The Sips model better described the sorption isotherms with high correlation coefficients at all conditions examined. Various thermodynamic parameters such as ΔG°, ΔH° and ΔS° were calculated indicating that the present system was a spontaneous and endothermic process. Through potentiometric titrations, number of binding sites (carboxyl groups) and pK₁ were determined as 4.1 mmol/g and 4.4, respectively. The influence of co-ions (Na⁺, K⁺, Mg²⁺ and Ca²⁺) on lead uptake was well pronounced in the case of divalent ions compared to monovalent ions. The solution of 0.1 M HCl successfully eluted all lead ions from lead-loaded T. conoides biomass. The regeneration experiments revealed that the alga could be successfully reused for five cycles without any loss in lead biosorption capacity. A glass column (2 cm i.d. and 35 cm height) was used to study the continuous lead biosorption performance of T. conoides. At 25 cm (bed height), 5 ml/min (flow rate) and 100 mg/l (initial lead concentration), T. conoides exhibited lead uptake of 220.1 mg/g. The column was successfully eluted using 0.1 M HCl, with elution efficiency of 99.7%.     

Partitioning of latent heat flux at a northern peatland

The partitioning of latent heat flux (Q_E) to vascular plant and moss surface components was assessed for a Sphagnum-dominated bog with a hummock–hollow surface having a sparse canopy of low shrubs. Results from porometry and eddy covariance measurements of Q_E showed evaporation from the moss surface ranged from greater than 50% of total Q_E early in the growing season to less than 20% after a dry period toward the end of the growing season. Both soil moisture and vapour pressure deficit (D_a) affected this partitioning with drier moss and peat, lower water table, and smaller D_a all reducing moss Q_E. Daily maximum moss Q_E ranged from greater than 200 W m⁻² early in the growing season to less than 100 W m⁻² during a dry period. In contrast, vascular contribution to total Q_E increased over the season from a daily maximum of about 150 W m⁻² to 250 W m⁻² due to increase in leaf area by leaf replacement and emergence and to drying of the moss surface. Porometry results showed average daily maximum conductance from bog shrubs was near 8 mm s⁻¹. These conductance values were smaller than those reported for vascular plants from more nutrient-rich wetlands. The effect of increases in D_a on vascular Q_E were moderated by decreases in stomatal conductance. At constant available energy, vascular leaf conductance was reduced by as much as 2 mm s⁻¹ and moss surface conductance was enhanced by up to 3 mm s⁻¹ by large D_a. Considering vascular and non-vascular water transport characteristics and frequency of water table position and given the observed variations of Q_E partitioning with water table location and moss and peat water content, it is suggested that modelling efforts focus on how dry hummocks and wet hollows each contribute to Q_E, especially as related to D_a and soil moisture dynamics.     

Pyrrolidine analogs of GZ-793A: Synthesis and evaluation as inhibitors of the vesicular monoamine transporter-2 (VMAT2)

Central heterocyclic ring size reduction from piperidinyl to pyrrolidinyl in the vesicular monoamine transporter-2 (VMAT2) inhibitor GZ-793A and its analogs resulted in novel N-propane-1,2(R)-diol analogs 11a–i. These compounds were evaluated for their affinity for the dihydrotetrabenazine (DTBZ) binding site on VMAT2 and for their ability to inhibit vesicular dopamine (DA) uptake. The 4-difluoromethoxyphenethyl analog 11f was the most potent inhibitor of [³H]-DTBZ binding (K_i = 560 nM), with 15-fold greater affinity for this site than GZ-793A (K_i = 8.29 μM). Analog 11f also showed similar potency of inhibition of [³H]-DA uptake into vesicles (K_i = 45 nM) compared to that for GZ-793A (K_i = 29 nM). Thus, 11f represents a new water-soluble inhibitor of VMAT function.     

Inhibition of monoamine oxidase B by N-methyl-2-phenylmaleimides

Based on a recent report that 1-methyl-3-phenylpyrrolyl analogues are moderately potent reversible inhibitors of the enzyme monoamine oxidase B (MAO-B), a series of structurally related N-methyl-2-phenylmaleimidyl analogues has been prepared and evaluated as inhibitors of MAO-B. In general, the maleimides were more potent competitive inhibitors than the corresponding pyrrolyl analogues. N-Methyl-2-phenylmaleimide was found to be the most potent inhibitor with an enzyme–inhibitor dissociation constant (K_i value) of 3.49 μM, approximately 30-fold more potent than 1-methyl-3-phenylpyrrole (K_i = 118 μM). This difference in activities may be dependent upon the ability of the maleimidyl heterocyclic system to act as a hydrogen bond acceptor. This is in correspondence with literature reports which suggest that hydrogen bond formation is involved in stabilizing inhibitor–MAO-B complexes. Also reported here is a brief kinetic study of the hydrolysis of the N-methyl-2-phenylmaleimidyl analogues in aqueous solution. The findings of the inhibition studies are discussed with reference to the rate and extent of hydrolysis.     

Comparison of thermotolerance of sun-exposed peel and shaded peel of ‘Fuji’ apple

The thermotolerance of the sun-exposed peel and the shaded peel of ‘Fuji’ apple (Malus domestica Borkh.) fruit was evaluated by measuring pigments, chlorophyll a fluorescence transients and O₂ evolution or uptake after exposure to 25, 35, 40, 42, 44, 46 or 48 °C for 30 min in the dark. A major effect of heat stress at 46–48 °C on the chlorophyll a fluorescence transients was the appearance of a very clear K step at 200–300 μs for both peel types. The K step was slightly more pronounced in the sun-exposed peel than in the shaded peel, suggesting that the resistance of oxygen-evolving complex to heat stress is slightly lower in the sun-exposed peel than in the shaded peel. Minimal fluorescence (F_O), relative to the value at 25 °C, increased to a greater extent in the shaded peel than in the sun-exposed peel after exposure to 46–48 °C, but the temperature dependencies of F_O changes were similar for both peel types. Maximum quantum yield of PSII (F_V/F_M) decreased to a similar extent in the sun-exposed peel and the shaded peel as temperature rose from 25 to 44 °C, but the sun-exposed peel reached slightly lower values at 46–48 °C. Correspondingly, gross O₂ evolution rate, relative to that at 25 °C, was also slightly lower in the sun-exposed peel than in the shaded peel at 46–48 °C. In response to heat stress, the ratio of Q_A-reducing reaction centers (RCs) to total RCs and the ratio of Q_B-reducing RCs to Q_A-reducing RCs decreased, but both of them decreased to lower values in the sun-exposed peel than in the shaded peel at 46–48 °C, indicating that the capacity of electron transfer between P₆₈₀⁺ and Q_B via Q_A was damaged to a greater extent in the sun-exposed peel than in the shaded peel. At each given temperature, dark respiration was similar between the two peel types. Overall, it appears that the exposure to higher surface temperature under high light does not make the sun-exposed peel more tolerant of heat stress than the shaded peel of apple fruit.     

The role of a conserved tyrosine in the 49-kDa subunit of complex I for ubiquinone binding and reduction

Iron–sulfur cluster N2 of complex I (proton pumping NADH:quinone oxidoreductase) is the immediate electron donor to ubiquinone. At a distance of only ～ 7 Å in the 49-kDa subunit, a highly conserved tyrosine is found at the bottom of the previously characterized quinone binding pocket. To get insight into the function of this residue, we have exchanged it for six different amino acids in complex I from Yarrowia lipolytica. Mitochondrial membranes from all six mutants contained fully assembled complex I that exhibited very low dNADH:ubiquinone oxidoreductase activities with n-decylubiquinone. With the most conservative exchange Y144F, no alteration in the electron paramagnetic resonance spectra of complex I was detectable. Remarkably, high dNADH:ubiquinone oxidoreductase activities were observed with ubiquinones Q₁ and Q₂ that were coupled to proton pumping. Apparent K_m values for Q₁ and Q₂ were markedly increased and we found pronounced resistance to the complex I inhibitors decyl-quinazoline-amine (DQA) and rotenone. We conclude that Y144 directly binds the head group of ubiquinone, most likely via a hydrogen bond between the aromatic hydroxyl and the ubiquinone carbonyl. This places the substrate in an ideal distance to its electron donor iron–sulfur cluster N2 for efficient electron transfer during the catalytic cycle of complex I.     

Purification and characterization of a novel proteinase A inhibitor from Ganoderma lucidum by submerged fermentation

A novel proteinase A inhibitor was purified from Ganoderma lucidum. The purification was carried out by ethanol precipitation (50–80%), ACA44 gel filtration and Source 30Q anion exchange, respectively. The molecular mass of the inhibitor was 38 kDa as estimated via SDS-PAGE and gel filtration. Its carbohydrate content was up to 70%. β-Elimination revealed that the linkage between the glycan and the core protein backbone might be O-linkage. This inhibitor showed a remarkable heat stability. By investigating the interaction between this inhibitor and a variety of proteinases, it is indicated that the inhibitor was more specific against yeast proteinase A than other proteinases. The dissociation constants (Ki) and concentration required for 50% inhibition (IC₅₀) for proteinase A were 2.7 × 10⁻⁶ M and 0.16 mg/ml, respectively.     

Dihydropyrano [2,3-c] pyrazole: Novel in vitro inhibitors of yeast α-glucosidase

Inhibition of α-glucosidase enzyme activity is a reliable approach towards controlling post-prandial hyperglycemia associated risk factors. During the current study, a series of dihydropyrano[2,3-c] pyrazoles (1–35) were synthesized and evaluated for their α-glucosidase inhibitory activity. Compounds 1, 4, 22, 30, and 33 were found to be the potent inhibitors of the yeast α-glucosidase enzyme. Mechanistic studies on most potent compounds reveled that 1, 4, and 30 were non-competitive inhibitors (K_i = 9.75 ± 0.07, 46 ± 0.0001, and 69.16 ± 0.01 μM, respectively), compound 22 is a competitive inhibitor (K_i = 190 ± 0.016 μM), while 33 was an uncompetitive inhibitor (K_i = 45 ± 0.0014 μM) of the enzyme. Finally, the cytotoxicity of potent compounds (i.e. compounds 1, 4, 22, 30, and 33) was also evaluated against mouse fibroblast 3T3 cell line assay, and no toxicity was observed. This study identifies non-cytotoxic novel inhibitors of α-glucosidase enzyme for further investigation as anti-diabetic agents.     

Long chain fatty acids degradation in anaerobic digester: Thermodynamic equilibrium consideration

The biological oxidations and reductions occurring in batch anaerobic digesters may approach equilibrium. The approach depends strongly on the activities of the micro-organisms present. The thermodynamics of linear long chain fatty acid degradation in a batch reactor was modeled. The substrates considered are the saturated fatty acids from acetic acid to stearic acid. From the thermodynamic perspective, the fermentation (acidogenesis and acetogenesis), decomposing the long chain saturated fatty acids to acetic acid though shorter chain acids, could not proceed spontaneously (ΔH ≫ 0 and ΔG ≫ 0). However the model suggests that the major driving force for the fermentation may be found in the methanogenesis. The model results show two distinct cases: (ΔS > 0 and ΔH > 0) and (ΔS < 0 and ΔH < 0), relating to spontaneous but endothermic and non-spontaneous but exothermic processes respectively. Where, spontaneous digestion is associated with high initial concentrations of LCFA and endotherm. This implies that the digestion of this type of substrate might be better facilitated by the supply of supplemental heat. The digestion of very low concentrations of LCFA is found to be non-spontaneous due in large part to the solubility of carbon dioxide. This implies that the digestion of this type of substrate might be enabled by selectively removing carbon dioxide.