Biodegradation of cyanide in cassava wastewater using a novel thermodynamically-stable immobilized rhodanese

Staff PublicationExtracellular rhodanese obtained from Aureobasidium pullulans was employed in both free and immobilized forms for the biodegradation of cyanide present in cassava processing mill effluent (CPME). Crosslinking with glutaraldehyde (at an optimum concentration of 5% v/v) before entrapment in alginate beads resulted in the highest immobilization yield of 94.5% and reduced enzyme leakage of 1.8%. Rhodanese immobilized by cross-linking before entrapment (cbe) retained about 46% of its initial activity after eight cycles of catalysis compared to the entrapment in alginate alone (eaa) which lost more than 79% after the fifth catalytic cycle. A cross-examination of thermodynamic (DG d, DS d, DH d) kinetic (kd, t1=2, D and z values) parameters at 30–70 C showed that cbe displayed a higher resistance to thermal inactivation when compared to the free enzyme (fe) and (eaa). The efficiency of cyanide biodegradation from the CPME by the fe, eaa and cbe were 55, 62, and 74% respectively after 6 h. Biodegradation of cyanide using the cbe was monitored using FTIR spectroscopy. Rhodanese immobilized via cbe had a higher resistance to thermal denaturation over other enzyme forms. Hence, this makes cbe adaptable for large-scale detoxification of cyanide from CPME

Strain improvement and statistical optimization as a combined strategy for improving fructosyltransferase production by Aureobasidium pullulans NAC8

Staff PublicationStrain improvement of a low fructosyltransferase-producing Aureobasidium pullulans NAC8 (Accession No. KX023301) was carried out using chemical mutagens such as ethidium bromide and ethyl methane sulfonate. The wild-type and mutant strain were distinguished using Random amplified polymorphic DNA PCR and DNA fingerprinting analysis. Plackett-Burman and Box Behnken design were statistical tools used to determine important media parameters and optimization, respectively. Phenotypically and genetically, the new improved strain was different from the wild-type. The most important media parameters from PDB influencing fructosyltransferase production were ammonium chloride, sucrose and yeast extract at p<0.05. Some significant parameters obtained with the BBD exhibited quadratic effects on FTase. The F values (35.37 and 32.11), correlation coefficient (0.98 and 0.97) and the percent coefficient of variation (2.53% and 2.40%) were obtained for extracellular and intracellular FTase respectively. The validation of the model in the improved strain resulted in an overall 6.0 and 2.0-fold increase in extracellular and intracellular FTase respectively compared to the wild-type. A relatively low FTase-producing strain of Aureobasidium pullulans NAC8 was enhanced for optimum production using a two-pronged approach involving mutagenesis and statistical optimization. The improved mutant strain also had remarkable biotechnological properties that make it a suitable alternative than the wild-typ

Trilepisium madagascariense fruit-wastes as cheap feedstock for bioethanol production

Trilepisium madagascariense fruits are carbohydrate-rich and this study directly fermented the fruit wastes into bioethanol without the need for nutrient supplementation. The total reducing sugar (TRS) present in the mesocarp and seed of T. madagascariense fruit wastes (Tmfw) was fermented to bioethanol using Aureobasidium pullulans. Bioethanol production by A. pullulans was also optimized using Box-Behnken response surface methodology (RSM). The TRS in the mesocarp and seed of Tmfw were 11.2 ± 0.8 and 17.1 ± 1.2 g/L, respectively and further hydrolysis with cellulase resulted in increased TRS indicating the presence of cellulose. Pre-optimization, the bioethanol yield (Yps) and volumetric productivity (Qp) obtained from the fermentation of the seed by A. pullulans were 0.57 ± 0.03 g/g and 0.21 ± 0.02 g/L-1h-1, respectively. The optimum conditions for maximum bioethanol production were pH (5.95), time (24 h) and substrate concentration (5 g/L) resulting in Yps, Qp of 0.66 ± 0.06 g/g and 0.27 + 0.01 g/L-1h-1, respectively after model validation. Tmfw served as a suitable, cheap, non-toxic and readily available substrate especially in Nigeria to produce bioethanol while A. pullulans is a fungus that might be utilized for large-scale industrial bioethanol production

Purification and Physicochemical Properties of Rhodanese from Liver of Goat, Capra Aegagrus Hircus

Staff PublicationRhodanese from the liver of domestic goat (Capra aegagrus hircus) was extracted, purified and the catalytic as well as physicochemical properties determined in order to gain an insight into how domestic goats are able to feed and thrive successfully on cyanogenic plants such as sorghum, millet and cassava. A domestic goat was purchased from a freehold rearing place in Ile-Ife, Osun State, Nigeria, where it was slaughtered and the liver excised. It was rinsed with normal saline (0.9 % NaCl, pH 7.4) to remove blood and other impurities. Approximately 95 g of the liver was homogenized in 3 volumes of 0.1 M acetate glycine buffer, pH 7.8 containing 1 mM ԑ-amino-n-caprioc acid and 10 mM sodium thiosulphate in a blender, after which it was centrifuged for 15 min at 12,000 rpm at 4℃. A specific activity of 1.55 micromole thiocyanate formed per millilitre per minute (Rhodanese Unit; RU) per mg of protein (U/mg) with a purification fold of 1.88 and 36 % yield was obtained from the rhodanese extracted from the liver of goat rhodanese after CM-Sephadex, Sephacryl S-400 and Reactive Blue 2- agarose column chromatography. An apparent molecular weight of 36 kDa was obtained on Sephacryl S-400 and a subunit molecular weight of 19 kDa was obtained from Sodium dodecyl sulphate polyacylamide gel electrophoresis (SDS-PAGE). A Km value of 0.034±0.007 mM and Vmax of 16.70±3.36 units/mL was obtained for Na2S2O3 while a Km value of 0.038±0.004 mM and Vmax of 18.77±1.58 units/mL was obtained for KCN. An optimal pH of 8.5, 9.5 and 7.0 were obtained using citrate-phosphate-borate, Tris-HCl and citrate-phosphate buffers respectively. Goat liver rhodanese showed an optimal temperature of 40℃. All the chloride salts used which included KCl, MgCl2, CaCl2, MnCl2, AlCl3 and NH4Cl inhibited rhodanese activity, with the highest inhibition was observed with MnCl2 and least inhibition observed with KCl. Goat liver rhodanese was inhibited by all active site inhibitors, with the the highest inhibition observed with arsenic acid. It can therefore be concluded that domestic goats (Capara aegagrus hircus) are able to feed and survive on cyanogenic plants due to the presence of the cyanide detoxyfying enzyme, rhodanese, present in their liver at high activity with suitable kinetic properties

Marine Actinobacteria Bioflocculant: A Storehouse of Unique Biotechnological Resources for Wastewater Treatment and Other Applications

Staff PublicationThe bioactive compounds produced by actinobacteria have played a major role in antimicrobials, bioremediation, biofuels, enzymes, and anti-cancer activities. Biodegradable microbial flocculants have been produced by bacteria, algae, and fungi. Microbial bioflocculants have also attracted biotechnology importance over chemical flocculants as a result of degradability and environmentally friendly attributes they possess. Though, freshwater actinobacteria flocculants have been explored in bioflocculation. Yet, there is a paucity of information on the application of actinobacteria flocculants isolated from the marine environment. Similarly, marine habitats that supported the biodiversity of actinobacteria strains in the field of biotechnology have been underexplored in bioflocculation. Hence, this review reiterates the need to optimize culture conditions and other parameters that a ect bioflocculant production by using a response surface model or artificial neural network

Properties of Rhodanese from the Liver of Tilapia Oreochromis Niloticus, in Asejire Lake, Nigeria

Staff PublicationThe study investigates the purification and characterisation of rhodanese from the liver of the tilapia fish (Oreochromis niloticus) collected from Asejire Lake in Nigeria. This was with a view to understanding the biochemical basis of the survival of the fish in cyanide polluted water. Rhodanese was isolated and purified from liver tissue homogenate of tilapia using CM-Sephadex ion exchange chromatography and Sephadex G-75 gel filtration. The specific activity of the enzyme was 56.86 U/mg. The Km values for KCN and Na2S2O3 as substrates were 0.1240 ± 0.0021 mM and 0.0516 ± 0.0097 mM, respectively. The apparent molecular weight was estimated by gel filtration on a Sephacyl S-400 column to be 35,460 Da. The optimal activity was found at pH 6.5 and the temperature optimum was 40°C. The rhodanese enzyme showed that the activity of the enzyme was not affected by MgCl2, KCl, NH4Cl, MnCl2 and CaCl2 while AlCl3, inhibited the enzyme