Browsing by Author "Agboola, Femi K."

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    Biochemical and biophysical characterisation of a small purified lipase from Rhizopus oryzae ZAC3
    (Taylor and Francis Online, 2021-02-08) Ayinla, Zainab Adenike; Ademakinwa, Adedeji Nelson; Gross, Richard A.; Agboola, Femi K.
    The characteristics of a purified lipase from Rhizopus oryzae ZAC3 (RoL-ZAC3) were investigated. RoL-ZAC3, a 15.8 kDa protein, which was optimally active at pH 8 and 55 °C had a half-life of 126 min at 60 °C. The kinetic parameters using p-nitrophenylbutyrate as substrate were 0.19 ± 0.02 mM, 126 ± 5.6 U/ml and 122 s−1 for Km , V max and k cat respectively. RoL-ZAC3 showed stability in methanol and isopropanol with Na+ enhancing the activity. p-nitrophenyloleate and castor oil were the best preferred substrates among the p-nitrophenyl esters and vegetable oils tested respectively. About 43% residual activity was observed after incubation for 30 min at 75 °C. Circular dichroism thermal scan showed that the lipase displayed intense negative ellipticities even at high temperature. Perturbation of the tertiary structure with increasing temperature caused the exposure of hydrophobic side chains to the aqueous environment as revealed by tryptophan fluorescence, with a t−Tm of 50 °C. Differential scanning calorimetry analysis showed melting temperature and calorimetric enthalpy of 55.5 °C and 444 kJ/mol respectively. Dynamic light scattering analysis indicated that the lipase was prone to aggregation upon unfolding at high temperature. It can be concluded that RoL-ZAC3 possesses promising potential for numerous biotechnological applications.
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    Biochemical characterization and kinetic studies on a purified yellow laccase from newly isolated Aureobasidium pullulans NAC8 obtained from soil containing decayed plant matter
    (Elsevier, 2016-06) Ademakinwa, Adedeji N.; Agboola, Femi K.
    The study investigated the biochemical characteristics and kinetic parameters of laccase from a newly isolated Aureobasidium pullulans NAC8 obtained from soil containing decay plant litters. This was with a view to identifying the type of laccase and its possible suitability for biotechnological applications. The fungal strain was identified as A. pullulans NAC8 by sequencing of its 5.8S rRNA and adjacent internally transcribed sequences (ITS) 1 and 2. A. pullulans NAC8 laccase was purified 2.0-fold with a yield of 59.3% and specific activity of 9.34 μmol/min/mg protein. The kinetic parameters KM, Vmax, kcat and kcat/KM for laccase with guaiacol as substrate were 1.05 ± 0.12 mM, 12.67 ± 0.55 μmol/ml/min, 25.3 × 10−1 s−1 and 2.4 × 103 M−1 s−1 respectively. Laccase exhibited maximum activity at 45 °C and optimum pH of 4.5. The enzyme showed stability at a temperature range of 45–55 °C after a 2 h incubation. The molecular weight determined on SDS–PAGE was 68.4 kDa. The enzyme was stable at 10% of all organic solvents used but displayed a loss of activity at 50%. 2.5 mM thioglycolic acid (TGA) and 0.05 mM sodium azide inactivated the enzyme. The substrate specificity was guaiacol > catechol > tannic acid > gallic acid. There was no peak observed at 610 nm and the ratio of absorbance at 280 nm and 610 was 26. This suggests a yellow laccase. The biochemical properties of A. pullulans NAC8 yellow laccase makes it potentially useful in several biotechnological applications
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    Optimization of Aqueous Two-Phase Partitioning of Aureobasidium pullulans α-Amylase via Response Surface Methodology and Investigation of its Thermodynamic and Kinetic Properties
    (Elsevier, 2019-08-29) Ademakinwa, Adedeji N.; Agunbiade, Mayowa O.; Ayinla, Zainab A.; Agboola, Femi K.
    Industrial enzymes such as α-amylase must be thermostable and also easily purified/concentrated. Hence, aqueous two-phase partitioning systems (ATPS) was exploited for the partitioning of α-amylase from Aureobasidium pullulans due to its numerous advantages over conventional purification strategy. A. pullulans α-amylase was partially purified using ATPS via response surface methodology (RSM). The potentials of the ATPS-purified enzyme for possible industrial application such as resistance to thermal inactivation was investigated in comparison with the crude enzyme. PEG-6000 was the polymer of choice for ATPS as it resulted in higher purification factor (PF), %yield (Y), and partition coefficient (PC). At optimum levels (% w/v) of 20, 12 and 7.5 for PEG-6000, sodium citrate and sodium chloride respectively, maximum PF, Y and PC of 4.2, 88%, and 9.9 respectively were obtained. The response model validation and reliability were established based on the closeness between the experimented and predicted values. The kinetic and thermodynamic parameters such as Q10, t1/2, kd, D − value, Ed, of the ATPS-purified α-amylase indicated that it was thermostable at 50 to 60 °C compared to the crude α-amylase. A thermodynamically stable and ATPS-purified α-amylase from A. pullulans has properties easily applicable for most industrial processes.
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    Purification, characterization and toxicity of a mannose-binding lectin from the seeds of Treculia africana plant
    (Taylor & Francis Group, 2009-10-01) Adeniran, Olukemi A.; Kuku, Adenike; Obuotor, Martins E.; Agboola, Femi K.; Famurewa, Akindele J.; Osasan, Stephen
    In this study, a mannose-specific, homodimeric lectin from the seeds of Treculia africana was purified, characterized and its adverse effects were investigated in mice. The purification protocol involved anionic exchange chromatography on DEAE-Cellulose followed by gel filtration on Sephadex G-100. The hemagglutinating activity of lectin towards human erythrocytes was sensitive to inhibition by D-mannose. Treatment of the protein with EDTA exerted no inhibitory effect; however, analysis of metal content by atomic absorption spectroscopy revealed the presence of Cu2+, Fe3+, and Mg2+. The results obtained showed that the lectin possesses maximum hemagglutinating activity towards human erythrocytes activity over the pH range 3–7.2 and is relatively thermostable up to 50°C. Periodic acid Schiff's (PAS) reagent staining showed that the protein was non-glycosylated while its amino acid composition analysis revealed that the protein contained 155 residues per subunit. The subunit had a minimal molecular weight of 22,139 Daltons, while the native molecular weight was estimated to be 41,000 Daltons. The lectin was found to be moderately toxic to mice with an LD50 of 47.21 µg g−1 body weight while, histopathological analysis showed no treatment related effects in any of the organs examined.