Search

Aureobasidium pullulans α-amylase (ApAmy) mixed with melted agar-agar solution and drop-wisely added to a mixture of organic solvent solution allowed for the entrapment of the α-amylase in the agar-agar organic matrix as beads. The immobilized ApAmy’s characteristics and wash performance were elucidated in comparison with the soluble ApAmy. Agar-agar at 2.0 % (w/v) and toluene: chloroform at 3:1 resulted in the highest immobilization yield retaining about 98% residual activity after ten catalytic cycles. The optimum temperature and pH for the immobilized enzyme were 60ºC and 6.5 respectively. The immobilized ApAmy hydrolysed branched and linear substrates thus establishing its broad substrate specificity. Relatively, the immobilized ApAmy (iApAmy) was more tolerant to organic solvents than the free enzyme. The iApAmy was mildly inhibited by cobalt but metals such as zinc, manganese, calcium and sodium enhanced the free and immobilized ApAmy activity. The iApAmy had a higher washing efficiency (77%) in the presence of detergents than the free enzyme (68%) and control (36%). The iApAmy showed good potentials as a detergent additive and from its characteristics, it could be useful in other industrial applications.

A low molecular weight intracellular and extracellular fructosyltransferase was purified from the wild (Wt) and improved mutant-type (Mt) Aureobasidium pullulans and its characteristics and thermod...

The study aimed at evaluating the phytochemical composition, antioxidant potentials and the levels of trace elements in the fruit extract of Kigelia africana obtained by different extraction solvents in order to ascertain its numerous pharmacological activities and identify the different chemical compounds responsible for these activities. The crude extract in ethanol and four other solvent fractions (hexane, ethylacetate, butanol and aqueous) were obtained for phytochemical screening. Antioxidant potentials of K. africana fruit were investigated spectrophotometrically using hydroxyl ion scavenging (OH−) activity, metal ion chelating activity, anti-lipid peroxidation activity as well as total antioxidant capacity assays. Trace element (Mn, Zn, Cd, Ni, Cu, Pb, Cr, Co and Fe) levels were measured using a plasma-emission spectrometer that has an auto sampler AS 93-plus and coupled with Nebulizer CETAC U-6000AT+ after microwave acid digestion of the fruit extracts. Chemical identification was performed using ultra-high-pressure liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-qTOF-MS2). Kigelia africana fruit extracts obtained showed a variety of bioactive phytochemical compounds including phenolic acids, flavonoids, saponins, tannins and glycosides. The total antioxidant capacity activities of the aqueous, butanol, ethanol, hexane and ethylacetate extracts are 15.04, 52.11, 44.95, 79.27 and 175.20 mg AAE/g. Metal ion chelating activity showed significant correlation with lipid peroxidation inhibition activity at p ≤ 0.01 and with OH− scavenging activity at p ≤ 0.05. PCA analysis revealed that all the extract/fractions have higher total antioxidant activities compared to aqueous extract with hexane extract exhibiting the highest radical scavenging potential. HCA showed similarities with three well-defined clusters and PLS regression was used to predict total antioxidant activity. High sensitivity by low values of limits of detection and quantification was observed ranging from 0.021 to 0.085 mg/ml and 0.063 to 0.258 mg/ml for Zn and Fe respectively. Ethylacetate extract had high concentration of Fe (0.5656 mg/kg). For the standardization of the K. africana fruit extract, 244 chemical compounds were identified by measuring m/z values with threshold override of 100,000 and analysing mass spectrometer fragmentation behaviour while 16 of these were confirmed. Kigelia africana fruit extract is a good source of antioxidant and possess maximum accepted concentration of trace elements according to European legislation (1881/2006/EC). The metabolites identified exhibited numerous pharmacological activities. The method and results suggest the applicability for commercial use of this K. africana fruit in the treatment of oxidative-related diseases.

The production of cell-wall degrading enzymes (CWDE) such as cellulase and pectinase by Aureobasidium pullulans NAC8 through induction using orange peels was investigated for the potential ...

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.

Strain 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

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, Δ H d ∗ , Δ G d ∗ and Δ S d ∗ 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.

The 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. Key words: Cyanide, detoxification, tilapia, liver, rhodanese.

The purification and characterization of rhodanese, an enzyme that catalyses the detoxification of cyanide, from the gut of the variegated grasshopper (Zonocerus variegatus L.) were carried out to understand the biochemical basis of the survival of this grasshopper living on cyanogenic plants such as cassava. All experiments, including enzyme assay, were carried out at room temperature and all buffers contained 10mM sodium thiosulphate to stabilize the enzyme. Grasshoppers were caught alive from a cassava farm within the locality and kept frozen until analysis. Each grasshopper was dissected and the gut was removed quickly. Approximately 102g of the gut were homogenized in three volumes of 0.1 M acetate glycine buffer (pH 7.8) containing e-amino-n-caproic acid. The supernatant was collected by centrifugation at 12,000 rpm, for 30 min at 40 °C. The enzyme was purified to homogeneity by a combination of procedures such as ammonium sulphate precipitation, ion-exchange chromatography (CM-Sephadex and DEAE-Sephadex), gel filtration (Sephadex G-75) and Agarose-Blue affinity chromatography. Sodium dodecyl sulphate Polyacrylamide gel electrophoresis (SDS-PAGE) and non-SDS-PAGE were used to ascertain the purity of the enzyme. The native and subunit molecular weights of the enzyme were determined by gel filtration on a Bio-Gel P-200 column and SDS-PAGE, respectively. Kinetic parameters were determined by using varying concentrations of one of the substrates at a fixed concentration of the other, and vice versa. Furthermore, the effects of temperature, pH and cations on the activity of the enzyme were investigated. The purified enzyme had a specific activity of 51.7 μmol thiocyanate formed/ml/min/mg protein (U/mg protein) with a yield of about 29%. The apparent molecular weight of the enzyme estimated by Sephadex G-75 gel filtration was 35,400 ± 482 Da and its subunit molecular weight determined by SDS-PAGE was 33,000 ± 212 Da. The Km values of KCN and Na2S2O3 were found to be 29.63 ± 02.87 and 26 ± 03.04mM, respectively. An optimum pH and temperature of 7.0 and 35 °C, respectively, were obtained for the enzyme. The results of enzyme inhibition showed that the activity of the enzyme was not affected by NH4Cl, MgCl2, CoCl2, CaCl2, MnCl2, NiCl2 and SnCl2, but inhibited by ZnCl2 and BaCl2. In conclusion, these results suggest that the survival of Z. variegatus depends on the presence of the enzyme rhodanese, which shows high activity and has suitable kinetic properties in the gut of the grasshopper that feeds mainly on cassava leaves which are cyanogenic.

Amylases, a-amylase (EC 3.2.1.1, α-1, 4-glucan-4-glucanohydrolase) and glucoamylase (EC 3.2.1.3, α-1, 4; α-1, 6-glucan glucohydrolase; amyloglucosidase), extracted and partially purified from sorghum malt were used to hydrolyze maize starch. The process and products of the enzymatic hydrolysis were also compared with that of acid hydrolysis using hydrochloric acid. Maize grains were dry milled into flour as starch used for the hydrolytic procedures. Green (for glucoamylase) and dry (for a-amylase) sorghum malts were homogenized and the amylases were extracted in appropriate buffer solutions, precipitated by ammonium sulphate and desalted by column chromatography. Acid hydrolysis was investigated under various temperature, acid concentrations and starch concentrations. The combination of acid–enzyme hydrolyses involved the use of 0.1 M HCl followed by the addition of the extracted a-amylase and glucoamylase and enzyme-enzyme (a- amylase and glucoamylase) hydrolyses were also investigated. The results of the acid hydrolysis showed that the concentration of reducing sugar produced increased with temperature of hydrolysis and the concentrations of the acid. However, the products decreased with increasing starch granule concentration. The overall study showed that enzyme–enzyme hydrolysis has the highest dextrose equivalent of 47.0% followed by acid– enzyme with 43.0% and the acid hydrolysis with 27.0%. © 2009 International Formulae Group. All rights reserved. Keywords: a-amylase, glucoamylase, maize (Zea may), Sorghum bicolor (Sorghum).

1. 1. Pyruvate kinase from Africa land tortoise (Kinixys erosa) skeletal muscle was isolated and purified to homogeneity. 2. 2. The mol. wt of the enzyme was estimated to be 212,333 ± 2887 with four subunits of 49,680 ± 526 . 3. 3. The enzyme, denatured by 4M guanidine-HCl, regained a maximum of 80–87% of its original activity upon dilution at 20°C and at a protein concentration of 80 μg/ml in appropriate buffer containing 10 mM PEP and 1 mM l -valine. The kinetics of renaturation was first order. 4. 4. The catalytically active renatured enzyme was a dimer even though it was kinetically similar to the tetrameric native enzyme.

Two forms of rhodanese were purified from the liver of Clarias gariepinus Burchell, designated catfish rhodanese I (cRHD I) and rhodanese II (cRHD II), by ion-exchange chromatography on a CM-Sepharose CL-6B column and gel filtration through a Sephadex G-75 column. The apparent molecular weight obtained for cRHD I and cRHD II was 34,500 +/- 707 and 36,800 +/- 283 Da, respectively. The subunit molecular weight determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis was 33,200 +/- 283 and 35,100 +/- 141 Da for cRHD I and cRHD II, respectively. Atomic absorption spectrophotometric analysis revealed that cRHD II contained a high level of iron (Fe), which presumably was responsible for the brownish colour of the preparation. In contrast, no Fe was identified in cRHD I, and its preparation was colourless. Further characterization of cRHD II gave true Michaelis-Menten constant (K(m)) values of 25.40 +/- 1.70 and 18.60 +/- 1.68 mM for KCN and Na(2)S(2)O(3), respectively, an optimum pH of 6.5 and an optimum temperature of 40 degrees C. The Arrhenius plot of the effects of temperature on the reaction rate consisted of two linear segments with a break occurring at 40 degrees C. The apparent activation energy values from these slopes were 7.3 and 72.9 kcal/mol. Inhibition studies on the cRHD II enzyme showed that the activity of the enzyme was not affected by Mn(2+), Co(2+), Sn(2+), Ni(2+) and NH(4) (+), but Zn(2+) inhibited the enzyme considerably.

The kinetic and physicochemical properties of glycogen phosphorylase b from the breast muscle of the fruit bat, Eidolon helvum Kerr were investigated in order to obtain some information about the possible physiological role of the enzyme in meeting the energy requirements of the bat muscle either at the initiation of or during flight. Glycogen phosphorylase b was purified from fruit bat breast muscle to apparent homogeneity with specific activity of approximately 37 units/mg of protein and a yield of 4 %. The enzyme was completely dependent on AMP for activity; hence it was designated phosphorylase b. The native and subunit molecular weights of the enzyme as determined by gel filtration on Sephadex G-200 and SDS-polyacrylamide gel electrophoresis were 187,000  12,600 Da and 90,500  1,200 Da respectively, thus implying that it is a dimeric protein. The Michealis-Menten constants, Km, of the enzyme for glycogen and glucose-1-phosphate were 0.06 mg/ml and 6.94 mM respectively, while apparent Km for AMP was 0.08 mM. The turnover number, kcat, was 65.56 s-1. Although sodium fluoride (NaF) and sodium sulphate (Na2SO4) on their own activated the enzyme, it was observed that in the presence of AMP, only sodium sulphate (Na2SO4) caused activation at low concentration. The optimum pH for the fruit bat muscle phosphorylase b activity was 6.4. In conclusion, the overall results of this study showed that the fruit bat muscle glycogen phosphorylase b is physically and catalytically similar to the enzyme from other mammalian sources. International Journal of Biological & Chemical Sciences Vol. 1 (2) 2007: pp. 99-107

β-Cyanoalanine synthase (β-cyano- l -alanine synthase; l -cysteine: hydrogen sulphide lyase (adding hydrogen cyanide (HCN)); EC 4. 4.1.9) was purified from the cytosolic fraction of the gut of grasshopper Zonocerus variegatus (L.) by ion-exchange chromatography on DEAE-Cellulose and gel filtration on Sephadex G-100 columns. The crude enzyme had a specific activity of 2.16 nmol H2S/min/mg. A purified enzyme with a specific activity, which was seventeen times higher than that of the crude extract, was obtained. A molecular weight of about 55.23±1.00 Kd was estimated from its elution volume on Sephadex G-100. The fraction when subjected to sodium dodecyl sulphate–polyacrylamide elel electrophoresis revealed the presence of a protein band with Mr of 23.25±0.25 Kd. The enzyme exhibited Michaelis–Menten kinetics having Km of 0.38 mM for l -cysteine and Km of 6.25 mM for cyanide. The optimum temperature and pH for activity were determined to be at 30 °C and pH 9.0, respectively. This enzyme might be responsible for the ability to detoxify cyanide in this insect pest and hence its tolerance of the cyanogenic cassava plant. Biophysical, biochemical and kinetic properties of this enzyme, which will reveal how this ability can possibly be compromised by enzyme inhibition, may lead, in the long term, to the potential use of this enzyme as drug target for pest control.