Folasade M. Olajuyigbe

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

Production of β-glucosidase from Fusarium oxysporum was investigated during degradation of some cellulosic substrates (Avicel, α-cellulose, carboxymethyl cellulose (CMC), and methylcellulose). Optimized production of β-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 β-glucosidase produced was characterized for its suitability for industrial application. Methyl cellulose supported the highest yield of β-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. β-glucosidase demonstrated high thermostability with 83% of its original activity retained at 70°C after 180 min of incubation. The activity of β-glucosidase was enhanced by Mn2+ and Fe2+ 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 β-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.

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 Km and Vmax 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 Ca2+ but completely inhibited by Fe2+, Mn2+, and Cu2+. 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.

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.

Assessment of crude oil degradation efficiency of newly isolated actinobacteria reveals untapped bioremediation potentials

ABSTRACT Bioremediation is gaining favorable attention as a more economical and environmentally friendly technique for the remediation of crude oil hydrocarbons. This makes the search for crude oil–degrading microbes very crucial. In this study, the isolation and identification of actinobacteria in soil samples from a selected crude oil spill site were carried out. Eighteen isolates from different soil depths (20–120 cm) were screened for their ability to grow on crude oil–based medium (COBM). Actinomyces naeslundii, Actinomyces viscosus, Actinomyces israelii, Actinomyces meyeri, and Nocardia formicae from a 20 cm soil depth exhibited higher growth profiles on COBM than on glucose-based medium (GBM). A. viscosus and A. isrealii exhibited 5- and 3-fold increase in growth over GBM and were selected for biodegradation studies. Growth kinetics and residual crude oil were used to measure the degradation efficiency of A. viscosus and A. israeli over varying crude oil concentrations. Surprisingly, A. viscosus and A. isrealii achieved 98% degradation of 10 g/L crude oil in 12 days and 97% degradation of 30, 50, and 75 g/L in 16 and 18 days, respectively. Specific activity of total peroxidase was assayed over the biodegradation period. Peroxidase activity increased with degradation efficiency of A. viscosus and A. isrealii, suggesting that peroxidases play a key role in the crude oil biodegradation process. The unique tolerance exhibited by A. viscosus and A. israelii to crude oil and high crude oil degradation efficiencies indicate their promising potential for bioremediation applications.

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.

An evaluation of glutathione transferase associated with Dichlorvos degradation in African palm weevil (Rynchophorus phoenicis) larva

Abstract This study was conducted to investigate the metabolic defensive mechanism in the larvae of African palm weevil (Rynchophorus phoenicis) administered with dichlorvos (2,2-dichlorovinyl dimethylphosphate) solution. Bioassay experiment with dichlorvos was conducted on the larva and glutathione-utilizing enzyme activities were determined in the major organs: fat body, gut, and head of R. phoenicis larva 48 h after treatment with 0–0.060 μg g−1 body weight dichlorvos solution. Glutathione transferase was purified from the gut of larvae by ion-exchange chromatography on diethylaminoethyl-Sephadex A50 and affinity chromatography on glutathione-Sepharose 4B columns. The purified enzyme was homogenous as revealed by sodium dodecylsulfate polyacrylamide gel electrophoresis. Initial velocity studies were carried out on the purified enzyme using standard procedures. Bioassay experiment indicated alterations of glutathione peroxidase, glutathione reductase, and glutathione transferase activities in the major organs of larva caused by dichlorvos. Glutathione transferase activity in the gut of larva was three times higher than that of glutathione peroxidase and glutathione reductase activities, an indication of possible detoxification role of glutathione transferase in the organ. A 49.7 kDa homodimeric glutathione transferase was identified from the gut of larva and was tagged rplGSTc. Mechanism of action of rplGSTc with 1-chloro-2,4-dinitrobenzene, and glutathione as substrates conformed to the random sequential mechanism. These results confirmed the presence of GST associated with the degradation of dichlorvos in the gut of R. phoenicis larva.

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.

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.

Preliminary Studies on the Expression and Purification of Functionally Active Recombinant Plasmepsin 9 from Plasmodium falciparum

Aim: Plasmepsins, a group of homologous aspartic proteinases are attractive drug targets against malaria. Plasmepsin 9 (PM9) expressed in the blood stage of malaria life cycle with unknown function has been stconsidered as a potential target. However, recombinant expression of active PM9 for biochemical and structure -activity analysis of the enzyme has been very challenging. This paper presents preliminary report on the expression and purification of act ive recombinant plasmepsin 9 fromPlasmodium