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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.150, #3)

A New Lipase Isolated from Oleaginous Seeds from Pachira aquatica (Bombacaceae) by Patricia Peres Polizelli; Fernanda Dell Antonio Facchini; Hamilton Cabral; Gustavo Orlando Bonilla-Rodriguez (pp. 233-242).

A new lipase from seeds of Pachira aquatica was purified to homogeneity by SDS-PAGE obtaining an enzyme with a molecular weight of approximately 55 kDa. The purified lipase exhibited maximum activity at 40°C and pH 8.0, for an incubation time of 90 min. Concerning temperature stability, at the range from 4 to 50°C, it retained approximately 47% of its original activity for 3 h. The enzyme activity increased in the presence of Ca⁺⁺ and Mg⁺⁺, but was inhibited by Hg⁺⁺, Mn⁺⁺, Zn⁺⁺, Al⁺⁺⁺ and various oxidizing and reducing agents. The lipase was highly stable in the presence of organic solvents, and its activity was stimulated by methanol. The values of K _m and V _max were 1.65 mM and 37.3 μmol mL⁻¹ min⁻¹, respectively, using p-nitrophenylacetate as substrate. The enzyme showed preference for esters of long-chain fatty acids, but demonstrated significant activity against a wide range of substrates.

Keywords: Enzyme; Lipase; Seeds; Pachira aquatica ; Hydrolysis

Non-Selective Toxicological Effects of the Insect Juvenile Hormone Analogue Methoprene. A Membrane Biophysical Approach by João P. Monteiro; Romeu A. Videira; Manuel J. Matos; Augusto M. Dinis; Amália S. Jurado (pp. 243-257).

The Gram-positive bacterium, Bacillus stearothermophilus, was used as a model organism to identify the non-selective toxic effects of the currently used insecticide methoprene (isopropyl(2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate). A significant decrease of the yield of bacterial cultures and a premature appearance of ultrastructural abnormalities in cells cultured in the presence of the insecticide were taken as indicators of cytotoxicity. A putative correlation of this cytotoxicity with methoprene-induced perturbations on membrane lipid organization was investigated, using differential scanning calorimetry and the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its propionic acid derivative (DPH–PA). The membrane physical effects depended on the lipid bilayer composition and packing. The most striking effect was a progressive broadening and shifting to lower temperatures, with increasing methoprene concentrations, of the main transition phase of the dimyristoyl- or dipalmitoylphosphatidylcholine bilayers and of the lateral phase separation of liposomes reconstituted with the lipid extracts of B. stearothermophilus.

Keywords: Methoprene; Lipid dynamics; Differential scanning calorimetry; Fluorescence polarization; Cell ultrastructure; Bacillus stearothermophilus

Phenol Inhibition and Restoration of the Bioactivity of Anaerobic Granular Sludge by Qun Yan; Dan Yu; Zhiliang Wang; Hua Zou; Wenquan Ruan (pp. 259-265).

Inhibition and restoration of different concentrations of phenol on the bioactivity of anaerobic granular sludge were investigated with laboratory-scale equipment. It indicated that phenol concentration lower than 50 mg/l show no inhibitory effect on bioactivities of granular sludge. However, methane productivity, extracellular polymeric substances (EPS) content, and coenzyme F₄₂₀ activity were decreased by varying degrees when phenol concentration adopted for inhibition ranged between 50 and 400 mg/l. Noticeably, methane productivity could be fully or partly restored in case the phenol was removed after 24 h of phenol inhibition.

Keywords: Anaerobic granular sludge; Phenol; Inhibition; Restoration; Bioactivity

Talaromyces thermophilus β-d-Xylosidase: Purification, Characterization and Xylobiose Synthesis by Mohamed Guerfali; Ali Gargouri; Hafedh Belghith (pp. 267-279).

When grown on wheat bran as the only carbon source, the filamentous fungus Talaromyces thermophilus produces large amounts of β-xylosidase activity. The presence of glucose drastically decreases the β-xylosidase production level. The β-xylosidase of T. thermophilus was purified by ammonium sulfate precipitation, DEAE–cellulose chromatography, and gel filtration (high-performance liquid chromatography). The molecular mass of the enzyme was estimated to be 97 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. The enzyme activity was optimum at 50 °C and pH 7. The apparent Michaelis constant K _m of the β-xylosidase was 2.37 mM for p-nitrophenyl-β-d-xylopyranoside, with a V _max of 0.049 μmol min⁻¹ per milligram protein. Enzyme activity was inhibited by Cu²⁺, Hg²⁺, and Zn²⁺ and activated by Ca²⁺, Mn²⁺, and Co⁺ at a concentration of 5 mM. At high xylose concentration, this enzyme catalyses the condensation reaction leading to xylobiose production.

Keywords: Talaromyces thermophilus ; β-xylosidase; Thermostability; Xylan; p-Nitropenyl β-d-xylopyranoside

Nitrite-Mediated Inactivation of Human Plasma Paraoxonase-1: Possible Beneficial Effect of Aromatic Amino Acids by Gamil M. Abd-Allah; Amr D. Mariee (pp. 281-288).

Paraoxonase-1 (PON-1) is a high-density-lipoprotein-bound enzyme, and its major function is to prevent oxidation of low-density lipoprotein. Atherogenesis could be related to decreased activity of this enzyme. Nitrites ( $${ ext{NO}}_2^ - $$ ), either present as a contaminant and/or the main metabolic end product of nitric oxide (NO) degradation, may trigger nitrative damage to PON-1 enzyme. Minimal information is available concerning the effect of nitrite on the enzyme activity and the mechanism which it exerts its effect. The aim of this study was to analyze whether nitrites could play a role in modifying human PON-1 activity. Our results revealed that PON-1 activity was inhibited by nitrite in dose- and time-dependent manner. Site-specific nitration focused on phenolic residues, particularly tyrosine residues of the enzyme, may result in modification of its biological functions. Nitration of phenolic residues occurs via peroxynitrite (ONOO⁻) formation, which requires peroxides and nitrite. Thus, we tested the presence of peroxides, which are found in all plasma samples regardless of nitrite concentration. The inhibition of PON-1 activity by nitrite was significantly reduced by tryptophan, reduced glutathione (GSH), and catalase additions. Therefore, we concluded that nitrites may have a role in the inactivation of PON-1, probably through nitration of enzyme phenyl residues, and additions of individual aromatic amino acids, with highlighting on tryptophan, could be of important value in minimizing the nitrite-induced inhibition of PON-1 enzyme.

Keywords: Nitrites; Paraoxonase; Peroxynitrite

Characterization of Surfactin Produced by Bacillus subtilis Isolate BS5 by Ahmad Mohammad Abdel-Mawgoud; Mohammad Mabrouk Aboulwafa; Nadia Abdel-Haleem Hassouna (pp. 289-303).

Physical and chromatographic characterization of the surfactin biosurfactant produced by Bacillus subtilis isolate BS5 has been conducted to study its potentiality for industrial application. The crude extract of test surfactin appeared as off-white to buff flake-like amorphous residue with bad odor similar to sour pomegranate. Test surfactin showed solubility in aqueous solution at pH > 5 with optimum solubility at pH 8–8.5. It was also soluble in organic solvents like ethanol, acetone, methanol, butanol, chloroform, and dichloromethane. Surfactin crystals appeared rectangular with blunt corners and were arranged perpendicular to each other making a plus sign. Extracted surfactin showed high surface activity, as it could lower the surface tension of water from about 70 to 36 mN/m at ~15.6 mg/l. Moreover, test surfactin exhibited excellent stabilities at high temperatures (100°C for up to 1 h at and autoclaving at 121°C for 10 min), salinities (up to 6% NaCl), and over a wide range of pH (5–13). Test surfactin in the cell-free supernatant or crude culture broth forms showed high emulsification indices against kerosene (62.5% and 59%, respectively), diesel (62.5% and 66%, respectively), and motor oil (62% and 66%, respectively). These characters can effectively make test surfactin, in its crude forms, a potential candidate for the use in bioremediation of hydrocarbon-contaminated sites or in the petroleum industry. Chromatographic characterization of test surfactin, using high-performance liquid chromatography technique, revealed that the extracted surfactin contained numerous isoforms, of which six were found in the standard surfactin preparation (Fluka). Additional peaks appeared in the test surfactin and not in the standard one. These peaks may correspond to new surfactin isoforms that may be present in the test surfactin produced by B. subtilis isolate BS5.

Keywords: Surfactin; Bacillus ; Characterization; Potential application

Optimization of Surfactin Production by Bacillus subtilis Isolate BS5 by A. Mohammad Abdel-Mawgoud; M. Mabrouk Aboulwafa; Nadia Abdel-Haleem Hassouna (pp. 305-325).

Bacillus subtilis BS5 is a soil isolate that produces promising yield of surfactin biosurfactant in mineral salts medium (MSM). It was found that cellular growth and surfactin production in MSM were greatly affected by the environmental fermentation conditions and the medium components (carbon and nitrogen sources and minerals). Optimum environmental conditions for high surfactin production on the shake flask level were found to be a slightly acidic initial pH (6.5–6.8), an incubation temperature of 30°C, a 90% volumetric aeration percentage, and an inoculum size of 2% v/v. For media components, it was found that the optimum carbon source was molasses (160 ml/l), whereas the optimum nitrogen source was NaNO₃ (5 g/l) and the optimum trace elements were ZnSO₄·7H₂O (0.16 g/l), FeCl₃·6H₂O (0.27 g/l), and MnSO₄·H₂O (0.017 g/l). A modified MSM (molasses MSM), combining the optimum medium components, was formulated and resulted in threefold increase in surfactin productivity that reached 1.12 g/l. No plasmid could be detected in the tested isolate, revealing that biosurfactant production by B. subtilis isolate BS5 is chromosomally mediated but not plasmid-mediated.

Keywords: Surfactin; Bacillus ; Production; Optimization

A Novel Fully Enzymatic Method for Determining Glucose and 1,5-Anhydro-d-Glucitol in Serum of One Cuvette by Feng Jing; Lu Jun; Yuping Wang; Mingan Zhu; Zhang Yong; Xie Fei; Jicai Zhang (pp. 327-335).

The aim of the study was to set up a novel fully enzymatic method for screening glucose and 1,5-anhydro-d-glucitol (1,5-AG) in one cuvette. We have determined glucose and 1,5-AG, based on glucokinase (GK) converting glucose to G6P, a compound that can be catalyzed ultimately into 6-PGA by G-6PD and its coenzyme NADP⁺, and then calculated glucose concentration according to absorbance variety. Furthermore, pyranose oxidase was used to oxidize 1,5-AG with the formation of 1, 5-anhydro-fructose and H₂O₂. Measurement was done according to Trinder’s reaction principle. The mean within-run and day-to-day precision (CV) of this method for glucose was 0.88% and 1.4%, and also that for 1,5-AG was 1.05% and 1.94%, respectively. The mean recovery rate of two targets was 100.2% and 101.6%, respectively. The correlation (R ²) between the results of 1,5-AG obtained with our proposed method (y) and those obtained with LanaAG method (x) was 0.999 (y = 1.002x − 0.675 μmol/l; n = 86), and the correlation (R ²) of glucose between the results obtained with our GK method (y) and those obtained with recommendatory hexokinase method (x) was 0.9999 (y = 1.0043x + 0.1229 mmol/l; n = 86). The reference range (95%) of serological glucose and 1,5-AG was 3.7 to 5.7 mmol/l (4.70 ± 0.51 mmol/l) and 83.1 to 240.7 μmol/l (161.9 ± 40.2 μmol/l), respectively; and there was no difference with age and sex (P > 0.05). This newly developed method was dependable and steady-going, with analysis automatization, and allows quicker and easier measurement of serum glucose and 1,5-AG in one identical reaction cuvette in-phase than previously described methods.

Keywords: Glucose; 1,5-Anhydro-d-glucitol; Assay

Activation Mechanisms of Butyrylcholinesterase by 2,4,6-Trinitrotoluene, 3,3-Dimethylbutyl-N-n-butylcarbamate, and 2-Trimethylsilyl-ethyl-N-n-butylcarbamate by Shyh-Ying Chiou; Yon-Gi Wu; Gialih Lin (pp. 337-344).

The goal of this work was to propose a possible mechanism for the butyrylcholinesterase activation by 2,4,6-trinitrotoluene (TNT), 3,3-dimethylbutyl-N-n-butylcarbamate (1), and 2-trimethylsilyl-ethyl-N-n-butylcarbamate (2). Kinetically, TNT, and compounds 1 and 2 were characterized as the nonessential activators of butyrylcholinesterase. TNT, and compounds 1 and 2 were hydrophobic compounds and were proposed to bind to the hydrophobic activator binding site, which was located outside the active site gorge of the enzyme. The conformational change from a normal active site gorge to a more accessible active site gorge of the enzyme was proposed after binding of TNT, and compounds 1 and 2 to the activator binding site of the enzyme. Therefore, TNT, and compounds 1 and 2 may act as the excess of butyrylcholine in the substrate activator for the butyrylcholinesterase catalyzed reactions.

Keywords: Butyrylcholinesterase; Enzyme kinetics; Activation; TNT; Carbamate

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.150, #3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.150, #3)