Developing HIV-1 Protease Inhibitors through Stereospecific Reactions in Protein Crystals.

Protease inhibitors are key components in the chemotherapy of HIV infection. However, the appearance of viral mutants routinely compromises their clinical efficacy, creating a constant need for new and more potent inhibitors. Recently, a new class of epoxide-based inhibitors of HIV-1 protease was investigated and the configuration of the epoxide carbons was demonstrated to play a crucial role in determining the binding affinity. Here we report the comparison between three crystal structures at near-atomic resolution of HIV-1 protease in complex with the epoxide-based inhibitor, revealing an in-situ epoxide ring opening triggered by a pH change in the mother solution of the crystal. Increased pH in the crystal allows a stereospecific nucleophile attack of an ammonia molecule onto an epoxide carbon, with formation of a new inhibitor containing amino-alcohol functions. The described experiments open a pathway for the development of new stereospecific protease inhibitors from a reactive lead compound.

Purification and characterization of a thermostable extracellular phytase from Bacillus licheniformis PFBL-03.

Extracellular phytase from Bacillus licheniformis PFBL-03 was purified in three steps by using ammonium sulfate precipitation, ion-exchange chromatography on a DEAE Sephadex A-50 column, and gel filtration chromatography on Sephadex G-100. The single protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suggested that the enzyme was homogeneous. The molecular mass determined from SDS-PAGE was 36 kD. The enzyme yield was 10% while the purification fold was 39. The purified phytase exhibited optimum activity at 55°C and was able to retain 55% of its original activity after 60 min of incubation at 80°C. The purified enzyme had optimum pH of 6.0 and was stable over a pH range of 4.0 to 7.5. The kinetic parameters K(m) and V(max) of the purified phytase for sodium phytate were 4.7 mM and 49.01 µmol/min. The activity of the enzyme was enhanced in the presence of Ca²âº but completely inhibited by Fe²âº, Mn²âº, and Cu²âº. The exhibited characteristics of the purified phytase from Bacillus licheniformis PFBL-03 show that the enzyme has potential for applications in the animal feed industry.

Bioremediation treatment improves water quality for Nile tilapia (Oreochromis niloticus) under crude oil pollution.

Despite favorable publicity of bioremediation as an affordable technology for cleanup of crude oil, public concerns on ecological safety in the presence of residual oil remain a global challenge. In this study, effects of crude oil exposure at sublethal concentration (0.25% v/v) and bioremediation treatment were investigated on histology and biochemical parameters of organs (gills, liver, kidney, and brain) of juvenile Nile tilapia (Oreochromis niloticus). Ten percent (10%) of mixed bacterial culture was used for bioaugmentation treatment. Ninety juvenile fish were used for study, and experiments were carried out in triplicates for three different groups. Malondialdehyde (MDA), an index of lipid peroxidation, was assayed as biomarker for oxidative stress. Activities of antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)), level of non-enzymatic antioxidant (reduced glutathione (GSH)), and activity of brain acetylcholinesterase (AChE) were assayed in selected fish organs as markers for environmental stressor. Histological examination of fish organs was done for all groups. Results of treated groups were compared with those of the control. Levels of MDA significantly increased with significant inhibition of antioxidant enzyme activities in the polluted group. Activities of SOD, CAT, and AChE and levels of GSH in fish organs in the bioaugmentation group were similar to results obtained in the control. Remarkably, the bioaugmentation group showed minimal or no lesions which suggested the efficacy of bioremediation technique in alleviating crude oil toxicity and preserving normal physiology of fish. This study provides deeper insights into the ecosafety of bioremediation treatment and can be extrapolated to other species of fish.

Characterization of free and immobilized laccase from Cyberlindnera fabianii and application in degradation of bisphenol A.

Recovery difficulty and inactivation of laccases are major challenges that hamper their application in biotechnology. In this study, laccase was purified from Cyberlindnera fabianii using ion-exchange and gel filtration chromatography with homogeneity confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Purified laccase of 52 kDa was immobilized on calcium and copper alginate beads by entrapment method. Free and immobilized enzymes were characterized, and efficiency of bisphenol A (BPA) degradation was determined. pH optima for free and immobilized laccases were 5.0 and 6.0, respectively. Ca and Cu alginate immobilized laccase (Ca-AIL and Cu-AIL) had optimum activity at 60 °C and 50 °C, respectively while free laccase (FL) was at 40 °C. Km and Vmax of FL, Ca-AIL and Cu-AIL were 0.032 mM and 15 mM/min, 0.078 mM and 6.98 mM/min, and 0.091 mM and 5.61 mM/min, respectively. Remarkably, immobilized laccases had higher operational stability than FL over 21 d at 4°C. Reusability of immobilized laccase was effective for 3 cycles with residual activity above 70%. Notably, Ca-AIL and Cu-AIL exhibited 71% and 65.5% BPA degradation efficiency on 14 d. Results reveal good kinetic parameters, improved thermal stability and enhanced reusability of immobilized laccase from C. fabianii with potentials for various industrial applications and bioremediation.

Biochemical characterization of an extremely stable pH-versatile laccase from Sporothrix carnis CPF-05.

Functionality of enzymes within narrow pH range and temperature is a major challenge which limits their industrial applications, hence, there is need to search for thermostable pH-versatile enzymes. Here, a novel thermostable pH-versatile laccase from Sporothrix carnis CPF-05 was purified by ion-exchange and gel filtration chromatography. Single protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) confirmed homogeneity of the enzyme with molecular weight of 56kDa. Enzyme yield was 3.9% and purification fold was 2.84. Purified laccase exhibited optimum activity at 50°C and retained 56% of its initial activity at 80°C after 180min of incubation with 2,2' azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as substrate. The enzyme had optimum pH of 7.0 and was stable over pH range of 3.0 to 11.0. Laccase activity was enhanced by Cu2+ and Mn2+ ions but inhibited by Ca2+, Mg2+, Ba2+and Hg2+ ions. Purified laccase was mildly inhibited by urea, sodium azide and surfactants while exhibiting tolerance to organic solvents. The enzyme demonstrated broad substrate specificity. Kinetic parameters, Km and Vmax of the purified laccase for ABTS were 0.0316mM and 7.940mM/min, respectively. Thermostability, pH-versatility and other characteristics of laccase from S. carnis CPF-05 indicate its suitability for variety of industrial processes.

Unravelling the Interactions between Hydrolytic and Oxidative Enzymes in Degradation of Lignocellulosic Biomass by Sporothrix carnis under Various Fermentation Conditions.

The mechanism underlying the action of lignocellulolytic enzymes in biodegradation of lignocellulosic biomass remains unclear; hence, it is crucial to investigate enzymatic interactions involved in the process. In this study, degradation of corn cob by Sporothrix carnis and involvement of lignocellulolytic enzymes in biodegradation were investigated over 240 h cultivation period. About 60% degradation of corn cob was achieved by S. carnis at the end of fermentation. The yields of hydrolytic enzymes, cellulase and xylanase, were higher than oxidative enzymes, laccase and peroxidase, over 144 h fermentation period. Maximum yields of cellulase (854.4 U/mg) and xylanase (789.6 U/mg) were at 96 and 144 h, respectively. Laccase and peroxidase were produced cooperatively with maximum yields of 489.06 U/mg and 585.39 U/mg at 144 h. Drastic decline in production of cellulase at 144 h (242.01 U/mg) and xylanase at 192 h (192.2 U/mg) indicates that they play initial roles in biodegradation of lignocellulosic biomass while laccase and peroxidase play later roles. Optimal degradation of corn cob (76.6%) and production of hydrolytic and oxidative enzymes were achieved with 2.5% inoculum at pH 6.0. Results suggest synergy in interactions between the hydrolytic and oxidative enzymes which can be optimized for improved biodegradation.

Production and Characterization of Highly Thermostable ß-Glucosidase during the Biodegradation of Methyl Cellulose by Fusarium oxysporum.

Production of ß-glucosidase from Fusarium oxysporum was investigated during degradation of some cellulosic substrates (Avicel, α-cellulose, carboxymethyl cellulose (CMC), and methylcellulose). Optimized production of ß-glucosidase using the cellulosic substrate that supported highest yield of enzyme was examined over 192 h fermentation period and varied pH of 3.0-11.0. The ß-glucosidase produced was characterized for its suitability for industrial application. Methyl cellulose supported the highest yield of ß-glucosidase (177.5 U/mg) at pH 6.0 and 30°C at 96 h of fermentation with liberation of 2.121 µmol/mL glucose. The crude enzyme had optimum activity at pH 5.0 and 70°C. The enzyme was stable over broad pH range of 4.0-7.0 with relative residual activity above 60% after 180 min of incubation. ß-glucosidase demonstrated high thermostability with 83% of its original activity retained at 70°C after 180 min of incubation. The activity of ß-glucosidase was enhanced by Mn(2+) and Fe(2+) with relative activities of 167.67% and 205.56%, respectively, at 5 mM and 360% and 315%, respectively, at 10 mM. The properties shown by ß-glucosidase suggest suitability of the enzyme for industrial applications in the improvement of hydrolysis of cellulosic compounds into fermentable sugars that can be used in energy generation and biofuel production.

Activity of the Antioxidant Defense System in a Typical Bioinsecticide-and Synthetic Insecticide-treated Cowpea Storage Beetle Callosobrochus maculatus F. (Coleoptera: Chrysomelidae).

The non-enzymatic and enzymatic antioxidant defense systems play a major role in detoxification of pro-oxidant endobiotics and xenobiotics. The possible involvement of beetle non-enzymatic [α-tocopherol, glutathione (GSH), and ascorbic acid] and enzymatic [catalase (CAT), superoxide dismutase (SOD), peroxidase (POX), and polyphenol oxidase (PPO)] antioxidant defense system on the insecticidal activity of synthetic insecticides (cypermethrin, 2,2-dicholorovinyl dimethyl phosphate, and λ-cyhalothrin) and ethanolic plant extracts of Tithonia diversifolia, Cyperus rotundus, Hyptis suaveolens leaves, and Jatropha Curcas seeds was investigated. 2,2-Dicholorovinyl dimethyl phosphate (DDVP; 200 ppm, LC50 = 13.24 ppm) and T. diversifolia (20,000 ppm) resulted in 100% beetle mortality at 96-hour post-treatment. The post-treatments significantly increased the beetle α-tocopherol and GSH contents. Activities of CAT, SOD, POX, and PPO were modulated by the synthetic insecticides and bioinsecticides to diminish the adverse effect of the chemical stresses. Quantitative and qualitative allelochemical compositions of bioinsecticides and chemical structure of synthetic insecticides possibly account and for modulation of their respective enzyme activities. Altogether, oxidative stress was enormous enough to cause maladaptation in insects. This study established that oxidative imbalance created could be the molecular basis of the efficacy of both insecticides and bio-insecticides. Two, there was development of functional but inadequate antioxidant defense mechanism in the beetle.

Impact of Stereochemistry on Ligand Binding: X-ray Crystallographic Analysis of an Epoxide-Based HIV Protease Inhibitor.

A new pseudopeptide epoxide inhibitor, designed for irreversible binding to HIV protease (HIV-PR), has been synthesized and characterized in solution and in the solid state. However, the crystal structure of the complex obtained by inhibitor-enzyme cocrystallization revealed that a minor isomer, with inverted configuration of the epoxide carbons, has been selected by HIV-PR during crystallization. The structural characterization of the well-ordered pseudopeptide, inserted in the catalytic channel with its epoxide group intact, provides deeper insights into inhibitor binding and HIV-PR stereoselectivity, which aids development of future epoxide-based HIV inhibitors.

Carbamylation of N-terminal proline.

Protein carbamylation is of great concern both in vivo and in vitro. Here, we report the first structural characterization of a protein carbamylated at the N-terminal proline. The unexpected carbamylation of the α-amino group of the least reactive codified amino acid has been detected in high-resolution electron density maps of a new crystal form of the HIV-1 protease/saquinavir complex. The carbamyl group is found coplanar to the proline ring with a trans conformation. The reaction of N-terminal with cyanate ion derived from the chaotropic agent urea was confirmed by mass spectra analysis on protease single crystals. Implications of carbamylation process in vitro and in vivo are discussed.