Ligand Binding to the FA3-FA4 Cleft Inhibits the Esterase-Like Activity of Human Serum Albumin

The hydrolysis of 4-nitrophenyl esters of hexanoate (NphOHe) and decanoate (NphODe) by human serum albumin (HSA) at Tyr411, located at the FA3-FA4 site, has been investigated between pH 5.8 and 9.5, at 22.0°C. Values of K _s, k ₊₂, and k ₊₂/K _s obtained at [HSA] ≥ 5×[NphOXx] and [NphOXx] ≥ 5×[HSA] (Xx is NphOHe or NphODe) match very well each other; moreover, the deacylation step turns out to be the rate limiting step in catalysis (i.e., k ₊₃ << k ₊₂). The pH dependence of the kinetic parameters for the hydrolysis of NphOHe and NphODe can be described by the acidic pK _a-shift of a single amino acid residue, which varies from 8.9 in the free HSA to 7.6 and 7.0 in the HSA:NphOHe and HSA:NphODe complex, respectively; the pK>_a-shift appears to be correlated to the length of the fatty acid tail of the substrate. The inhibition of the HSA-Tyr411-catalyzed hydrolysis of NphOHe, NphODe, and 4-nitrophenyl myristate (NphOMy) by five inhibitors (i.e., diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol) has been investigated at pH 7.5 and 22.0°C, resulting competitive. The affinity of diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol for HSA reflects the selectivity of the FA3-FA4 cleft. Under conditions where Tyr411 is not acylated, the molar fraction of diazepam, diflunisal, ibuprofen, and 3-indoxyl-sulfate bound to HSA is higher than 0.9 whereas the molar fraction of propofol bound to HSA is ca. 0.5.     

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.     

A novel fluorogenic substrate for dinuclear Zn(II)-containing metallo-β-lactamases

In an effort to prepare a fluorogenic substrate to be used in activity assays with metallo-β-lactamases, (6R,7R)-8-oxo-7-(2-oxo-2H-chromene-3-carboxamido)-3-((4-(2-oxo-2H-chromene-3-carboxamido)-phenylthio)methyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (CA) was synthesized and characterized. CA exhibited a fluorescence quantum yield (φ) of 0.0059, two fluorescence lifetimes of 3.63 × 10^?10 and 5.38 × 10^?9 s, and fluorescence intensity that is concentration-dependent. Steady-state kinetic assays revealed that CA is a substrate for metallo-β-lactamases (MβLs) L1 and CcrA, exhibiting K_m and k_cat values of 18 μM and 5 s^?1 and 11 μM and 17 s^?1, respectively.     

Carbonic anhydrase inhibitors. Characterization and inhibition studies of the most active β-carbonic anhydrase from Mycobacterium tuberculosis,Rv3588c

The Rv3588c gene product of Mycobacterium tuberculosis, a β-carbonic anhydrase (CA, EC 4.2.1.1) denominated here mtCA 2, shows the highest catalytic activity for CO₂ hydration (k_cat of 9.8 × 10⁵ s^?1, and k_cat/K_m of 9.3 × 10⁷ M^?1 s^?1) among the three β-CAs encoded in the genome of this pathogen. A series of sulfonamides/sulfamates was assayed for their interaction with mtCA 2, and some diazenylbenzenesulfonamides were synthesized from sulfanilamide/metanilamide by diazotization followed by coupling with amines or phenols. Several low nanomolar mtCA 2 inhibitors have been detected among which acetazolamide, ethoxzolamide and some 4-diazenylbenzenesulfonamides (K_Is of 9–59 nM). As the Rv3588c gene was shown to be essential to the growth of M. tuberculosis, inhibition of this enzyme may be relevant for the design of antituberculosis drugs possessing a novel mechanism of action.     

Inhibition studies of a β-carbonic anhydrase from Brucella suis with a series of water soluble glycosyl sulfanilamides

A β-carbonic anhydrase (CA, EC 4.2.1.1) from the bacterial pathogen Brucella suis, bsCA 1, has been cloned, purified characterized kinetically and for inhibition with a series of water soluble glycosylated sulfanilamides. bsCA 1 has appreciable activity as catalyst for the hydration of CO₂ to bicarbonate, with a k_cat of 6.4 × 10⁵ s^?1 and k_cat/K_m of 3.9 × 10⁷ M^?1 s^?1. All types of inhibitory activities have been detected, with K_Is in the range of 8.9–110 nM. The best bsCA 1 inhibitor were the galactose and ribose sulfanilamides, with inhibition constants of 8.9–9.2 nM. Small structural changes in the sugar moiety led to dramatic differences of enzyme inhibitory activity for this series of compounds. One of the tested glycosylsulfonamides and acetazolamide significantly inhibited the growth of the bacteria in cell cultures.     

Purification and characterization of an extracellular antifungal chitinase from Penicillium ochrochloron MTCC 517 and its application in protoplast formation

A highly chitinolytic strain Penicillium ochrochloron MTCC 517 was procured from MTCC, Chandigarh, India. Culture medium supplemented with 1% chitin was found to be suitable for maximum production of chitinase. Purification of extracellular chitinase was done from the culture medium by organic solvent precipitation and DEAE-cellulose column chromatography. The chitinase was purified 6.92-fold with 29.9% yield. Molecular mass of purified chitinase was found to be 64 kDa by SDS-PAGE. The chitinase showed optimum temperature 40 °C and pH 7.0. The enzyme activity was completely inhibited by Hg²⁺, Zn²⁺, K⁺ and NH₄⁺. The enzyme kinetic study of purified chitinase revealed the following characteristics, such as apparent K_m 1.3 mg ml^?1, V_max 5.523 × 10^?5 moles l^?1 min^?1 and K_cat 2.37 s^?1 and catalytic efficiency 1.82 s^?1 M^?1. The enzyme hydrolyzed colloidal chitin, glycol chitin, chitosan, glycol chitosan, N,N′-diacetylchitobiose, p-nitrophenyl N-acetyl-β-d-glucosaminide and 4-methylumbelliferyl N-acetyl-β-d-glucosaminide. The chitinase of P. ochrochloron MTCC 517 is an exoenzyme, which gives N-acetylglucosamine as the main hydrolyzate after hydrolysis of colloidal chitin. Protoplasts with high regeneration capacity were obtained from Aspergillus niger using chitinase from P. ochrochloron MTCC 517. Since it also showed antifungal activity, P. ochrochloron MTCC 517 seems to be a promising biocontrol agent.     

Kinetic and anion inhibition studies of a β-carbonic anhydrase (FbiCA 1) from the C4 plant Flaveria bidentis

Several β-carbonic anhydrases (CAs, EC 4.2.1.1) are present in all land plants examined thus far. Here we report the first detailed biochemical characterization of one such isoform, FbiCA 1, from the C₄ plant Flaveria bidentis, which was cloned, purified and characterized as recombinant protein. FbiCA 1 has an interesting CO₂ hydrase catalytic activity (k_cat of 1.2 × 10⁵ and k_cat/K_m of 7.5 × 10⁶ M^?1 × s^?1) and was moderately inhibited by most simple/complex inorganic anions. Potent FbiCA 1 inhibitors were also detected, such as trithiocarbonate, diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid (K_Is in the range of 4–60 μM). Such inhibitors may be used as tools to better understand the role of various β-CA isoforms in photosynthesis.     

Anion inhibition profiles of the complete domain of the η-carbonic anhydrase from Plasmodium falciparum

We have cloned, purified and investigated the catalytic activity and anion inhibition profiles of a full catalytic domain (358 amino acid residues) carbonic anhydrase (CA, EC 4.2.1.1) from Plasmodium falciparum, PfCAdom, an enzyme belonging to the η-CA class and identified in the genome of the malaria-producing protozoa. A truncated such enzyme, PfCA1, containing 235 residues was investigated earlier for its catalytic and inhibition profiles. The two enzymes were efficient catalysts for CO₂ hydration: PfCAdom showed a k_cat of 3.8 × 10⁵ s⁻¹ and k_cat/K_m of 7.2 × 10⁷ M⁻¹ × s⁻¹, whereas PfCA showed a lower activity compared to PfCAdom, with a k_cat of 1.4 × 10⁵ s⁻¹ and k_cat/K_m of 5.4 × 10⁶ M⁻¹ × s⁻¹. PfCAdom was generally less inhibited by most anions and small molecules compared to PfCA1. The best PfCAdom inhibitors were sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid, which showed K_Is in the range of 9–68 μM, followed by bicarbonate, hydrogensulfide, stannate and N,N-diethyldithiocarbamate, which were submillimolar inhibitors, with K_Is in the range of 0.53–0.97 mM. Malaria parasites CA inhibition was proposed as a new strategy to develop antimalarial drugs, with a novel mechanism of action.     

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.     

Kinetics parameter estimation for the binding process of salicylic acid to human serum albumin (HSA) with capacitive sensing technique

A novel method for real-time investigating the binding interaction between human serum albumin (HSA) and salicylic acid with capacitive sensing technique was successfully proposed. HSA was immobilized on the surface of a gold electrode modified with an insulating poly (o-phenylenediamine) (o-PD) film and colloid Au nanoparticles layers. The bioactivity of HSA was remained and major binding sites were available because of the excellent biocompatibility of gold nanoparticles. The capacitance and interfacial electron resistance of the sensor were altered, owing to the binding of HSA to salicylic acid. The time courses of the capacitance change were acquired with capacitive sensing technique during the binding process. Based on the capacitance response curves with time, the response model for the binding was derived in theory and the corresponding regression parameters were determined by fitting the real-time experimental data to the model. The binding and the dissociation rate constants (k₁ and k_− 1) were estimated to be 54.8 (mol l^− 1)^− 1 s^− 1 and 2.9 × 10^− 3 s^− 1, respectively. And the binding equilibrium constant (K_a) was calculated to be 1.89 × 10⁴ (mol l^− 1)^− 1.     

Carbonic anhydrase inhibitors. Inhibition of the β-class enzyme from the pathogenic yeast Candida glabrata with anions

A β-carbonic anhydrase (CA, EC 4.2.1.1), the protein encoded by the NCE103 gene of Candida glabrata which also present in Candida albicans and Saccharomyces cerevisiae, was cloned, purified, characterized kinetically and investigated for its inhibition by a series simple, inorganic anions such as halogenides, pseudohalogenides, bicarbonate, carbonate, nitrate, nitrite, hydrogen sulfide, bisulfite, perchlorate, sulfate and some isosteric species. The enzyme showed significant CO₂ hydrase activity, with a k_cat of 3.8 × 10⁵ s^?1 and k_cat/K_M of 4.8 × 10⁷ M^?1 s^?1. The Cà glabrata CA (CgCA) was moderately inhibited by metal poisons (cyanide, azide, cyanate, thiocyanate, K_Is of 0.60–1.12 mM) but strongly inhibited by bicarbonate, nitrate, nitrite and phenylarsonic acid (K_Is of 86–98 μM). The other anions investigated showed inhibition constants in the low millimolar range, with the exception of bromide and iodide (K_Is of 27–42 mM).     

Choline sulfatase from Ensifer (Sinorhizobium) meliloti: Characterization of the unmodified enzyme

Ensifer (Sinorhizobium) meliloti is a nitrogen-fixing α-proteobacterium able to biosynthesize the osmoprotectant glycine betaine from choline sulfate through a metabolic pathway that starts with the enzyme choline-O-sulfatase. This protein seems to be widely distributed in microorganisms and thought to play an important role in their sulfur metabolism. However, only crude extracts with choline sulfatase activity have been studied. In this work, Ensifer (Sinorhizobium) meliloti choline-O-sulfatase was obtained in a high degree of purity after expression in Escherichia coli. Gel filtration and dynamic light scattering experiments showed that the recombinant enzyme exists as a dimer in solution. Using calorimetry, its catalytic activity against its natural substrate, choline-O-sulfate, gave a k_cat=2.7×10^?1 s^?1 and a K_M=11.1 mM. For the synthetic substrates p-nitrophenyl sulfate and methylumbelliferyl sulfate, the k_cat values were 3.5×10^?2 s^?1 and 4.3×10^?2 s^?1, with K_M values of 75.8 and 11.8 mM respectively. The low catalytic activity of the recombinant sulfatase was due to the absence of the formylglycine post-translational modification in its active-site cysteine 54. Nevertheless, unmodified Ensifer (Sinorhizobium) meliloti choline-O-sulfatase is a multiple-turnover enzyme with remarkable catalytic efficiency.     

Structural and Mechanistic Analysis of the Choline Sulfatase from Sinorhizobium melliloti: A Class I Sulfatase Specific for an Alkyl Sulfate Ester

Hydrolysis of organic sulfate esters proceeds by two distinct mechanisms, water attacking at either sulfur (S–O bond cleavage) or carbon (C–O bond cleavage). In primary and secondary alkyl sulfates, attack at carbon is favored, whereas in aromatic sulfates and sulfated sugars, attack at sulfur is preferred. This mechanistic distinction is mirrored in the classification of enzymes that catalyze sulfate ester hydrolysis: arylsulfatases (ASs) catalyze S–O cleavage in sulfate sugars and arylsulfates, and alkyl sulfatases break the C–O bond of alkyl sulfates. Sinorhizobium meliloti choline sulfatase (SmCS) efficiently catalyzes the hydrolysis of alkyl sulfate choline-O-sulfate (k_cat/K_M = 4.8 × 10³ s^? 1 M^? 1) as well as arylsulfate 4-nitrophenyl sulfate (k_cat/K_M = 12 s^? 1 M^? 1). Its 2.8-Å resolution X-ray structure shows a buried, largely hydrophobic active site in which a conserved glutamate (Glu386) plays a role in recognition of the quaternary ammonium group of the choline substrate. SmCS structurally resembles members of the alkaline phosphatase superfamily, being most closely related to dimeric ASs and tetrameric phosphonate monoester hydrolases. Although > 70% of the amino acids between protomers align structurally (RMSDs 1.79–1.99 Å), the oligomeric structures show distinctly different packing and protomer–protomer interfaces. The latter also play an important role in active site formation. Mutagenesis of the conserved active site residues typical for ASs, H₂¹⁸O-labeling studies and the observation of catalytically promiscuous behavior toward phosphoesters confirm the close relation to alkaline phosphatase superfamily members and suggest that SmCS is an AS that catalyzes S–O cleavage in alkyl sulfate esters with extreme catalytic proficiency.     

Epoxidation of linear,branched and cyclic alkenes catalyzed by unspecific peroxygenase

Unspecific peroxygenases (EC 1.11.2.1) represent a group of secreted heme-thiolate proteins that are capable of catalyzing the mono-oxygenation of diverse organic compounds, using only H₂O₂ as a co-substrate. Here we show that the peroxygenase secreted by the fungus Agrocybe aegerita catalyzed the oxidation of 20 different alkenes. Five branched alkenes, among them 2,3-dimethyl-2-butene and cis-2-butene, as well as propene and butadiene were epoxidized with complete regioselectivity. Longer linear alkenes with a terminal double bond (e.g. 1-octene) and cyclic alkenes (e.g. cyclohexene) were converted into the corresponding epoxides and allylic hydroxylation products; oxidation of the cyclic monoterpene limonene yielded three oxygenation products (two epoxides and an alcohol). In the case of 1-alkenes, the conversion occurred with moderate stereoselectivity, in which the preponderance for the (S)-enantiomer reached up to 72% ee for the epoxide product. The apparent Michaelis–Menten constant (K_m) for the epoxidation of the model substrate 2-methyl-2-butene was 5 mM, the turnover number (k_cat) 1.3 × 10³ s^?1 and the calculated catalytic efficiency, k_cat/K_m, was 2.5 × 10⁵ M^?1 s^?1. As epoxides represent chemical building blocks of high relevance, new enzymatic epoxidation pathways are of interest to complement existing chemical and biotechnological approaches. Stable and versatile peroxygenases as that of A. aegerita may form a promising biocatalytic platform for the development of such enzyme-based syntheses.     

Sulfonamide inhibition studies of two β-carbonic anhydrases from the bacterial pathogen Legionella pneumophila

Two β-carbonic anhydrases (CAs, EC 4.2.1.1) were identified, cloned and purified in the pathogenic bacterium Legionella pneumophila, denominated LpCA1 and LpCA2. They efficiently catalyze CO₂ hydration to bicarbonate and protons, with k_cat in the range of (3.4–8.3) × 10⁵ s⁻¹ and k_cat/K_m of (4.7–8.5) × 10⁷ M⁻¹ s⁻¹, and are inhibited by sulfonamides and sulfamates. The best LpCA1 inhibitors were aminobenzolamide and structurally similar sulfonylated aromatic sulfonamides, as well as acetazolamide and ethoxzolamide(K_Is in the range of 40.3–90.5 nM). The best LpCA2 inhibitors belonged to the same class of sulfonylated sulfonamides, together with acetazolamide, methazolamide and dichlorophenamide (K_Is in the range of 25.2–88.5 nM). As these enzymes may be involved in pH regulation in the phagosome during Legionella infection, their inhibition may lead to antibacterials with a novel mechanism of action.     

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.     

The correction of reaction rates in continuous fluorometric assays of enzymes

The kinetic data obtained from the action of a cathepsin D-like enzyme from Biomphalaria glabrata hepatopancreas (digestive gland) on MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNp)-D–Arg-NH₂, was studied as a data prototype, generated by means of a fluorogenic substrate. An initial fluorescence, due to incomplete energy transfer, of about 8% of the values attained after complete substrate hydrolysis; a non-linear standard curve even at μM concentrations and an exponential decay of the steady state fluorescence of reaction product of the order of 10^− 4 × s^− 1 were the main analytical problems encountered. The standard curves for fluorescence of the substrate reaction product after 48 h of hydrolysis, and the reference compound MOCAc-Pro-Leu-Gly-NH₂, were fitted by polynomial approximation and the point derivates used as calibration factors. Time dependence of the calibration factor for the reaction product was − 2.96 × 10^− 4 a.u μM^− 1 × s^− 1 that is, in the same order of observed enzymic reaction rates. A mathematical treatment was devised for obtaining rates corrected for errors derived from the three analytical problems indicated. The method is of general application in continuous fluorometric assays, irrespective of the particular enzyme used, but of special value for substrates that present significant initial fluorescence. The reaction rates were 11% higher; as calculated by means of the calibration factor [substrate] ÷ (final − initial fluorescence intensities), which is the prevalent procedure in the literature; leading to underestimation of K_m and overestimation of V_max.     

Coupled effects of irradiance and iron on the growth of a harmful algal bloom-causing microalga Scrippsiella trochoidea

Effects of irradiance and iron on the growth of a typical harmful algal blooms (HABs) causative dinoflagellate, Scrippsiella trochoidea, were investigated under various irradiances (high light: 70 μmol m^?2 s^?1 and low light: 4 μmol m^?2 s^?1) and iron concentrations (low iron: 0.063 mg L^?1, medium iron: 0.63 mg L^?1 and high iron: 6.3 mg L^?1), and evaluated by the parameters of algal cell density, specific growth rate, optical density and chlorophyll a content. The results indicated that there was significant difference in the cell density of dinoflagellate S. trochoidea between high light and low light intensity treatments across the entire experiments, 7-fold higher at high irradiance as compared with low irradiance, which was further enhanced by the iron concentration. It was found that the maximum cell density of 25 × 10⁴ cell mL^?1 occurred under the combination of high light intensity and high iron concentration, followed by 23 × 10⁴ cell mL^?1 in the combination of high light and medium iron, and 20 × 10⁴ cell mL^?1 in the combination of high light and low iron. There was no significant effect of iron concentration on the cell density under low light intensity. The cell density maintained about 3 × 10⁴ cell mL^?1 across all combinations of iron concentrations and low light in the end of experiments. Such interactive effects of light intensity and iron level dependent were also observed for the specific growth rate, OD₆₈₀ and chlorophyll a content of S. trochoidea. The maximum values of specific growth rate, OD₆₈₀ and chlorophyll a content peaked at the condition of high irradiance and high iron, which were 0.22 d^?1, 0.282 and 0.673 mg L^?1, respectively. In general, their values increased significantly with the increasing of iron concentration at high irradiance, whereas no significant difference was observed among three iron concentrations at low irradiance, all remaining approximately 0.06 d^?1, 0.03 and 0.050 mg L^?1, respectively. Those results suggest that there may be a strong interactive effect between irradiance and iron on microalgal growth and their physiological characteristics. The combination of high light and high iron concentration may accelerate algal cell growth and pigment biosynthesis, thus leading to massive occurrence of HABs.     

Extraction of hemoglobin with calixarenes and biocatalysis in organic media of the complex with pseudoactivity of peroxidase

The present work involves the use of p-tert-butylcalix[4,6,8]arene carboxylic acid derivatives (^tButyl[4,6,8]CH₂COOH) for selective extraction of hemoglobin. All three calixarenes extracted hemoglobin into the organic phase, exhibiting extraction parameters higher than 0.90. Evaluation of the solvent accessible positively charged amino acid side chains of hemoglobin (PDB entry 1XZ2) revealed that there are 8 arginine, 44 lysine and 30 histidine residues on the protein surface which may be involved in the interactions with the calixarene molecules. The hemoglobin–^tButyl[6]CH₂COOH complex had pseudoperoxidase activity which catalysed the oxidation of syringaldazine in the presence of hydrogen peroxide in organic medium containing chloroform. The effect of pH, protein and substrate concentrations on biocatalysis was investigated using the hemoglobin–^tButyl[6]CH₂COOH complex. This complex exhibited the highest specific activity of 9.92 × 10^?2 U mg protein^?1 at an initial pH of 7.5 in organic medium. Apparent kinetic parameters (V′_max, K′_m, k′_cat and k′_cat/K′_m) for the pseudoperoxidase activity were determined in organic media for different pH values from a Michaelis–Menten plot. Furthermore, the stability of the protein–calixarene complex was investigated for different initial pH values and half-life (t_1/2) values were obtained in the range of 1.96 and 2.64 days. Hemoglobin–calixarene complex present in organic medium was recovered in fresh aqueous solutions at alkaline pH, with a recovery of pseudoperoxidase activity of over 100%. These results strongly suggest that the use of calixarene derivatives is an alternative technique for protein extraction and solubilisation in organic media for biocatalysis.     

Anion inhibition study of the β-class carbonic anhydrase (PgiCAb) from the oral pathogen Porphyromonas gingivalis

The oral pathogenic bacterium Porphyromonas gingivalis, encodes for two carbonic anhydrase (CA, EC 4.2.1.1) one belonging to the β-class (PgiCAb) and another one to the γ-class (PgiCA). This last enzyme has been characterized earlier for its inhibition profile with various classes of CA inhibitors, such as the sulfonamides and anions, whereas for PgiCAb such data were not yet reported. Here we show that PgiCAb has a good catalytic activity for the CO₂ hydration reaction, with k_cat 2.8 × 10⁵ s⁻¹ and k_cat/K_m of 1.5 × 10⁷ M⁻¹ × s⁻¹, being inhibited by cyanate and diethyldithiocarbamate in the submillimolar range (K_Is of 0.23–0.76 mM) and more efficiently by sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid (K_Is of 60–78 μM). The anion inhibition profile of the two P. gingivalis enzymes is very different. Identification of selective inhibitors of PgiCAb/PgiCA may lead to pharmacological tools useful for understanding the physiological role(s) of these enzymes, since this bacterium is the main causative agent of periodontitis and few treatment options are presently available.