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Elucidation of lipase-substrate interactions will guide the proper industrial use and applicability of the enzyme. The aim of this study was to predict the 3 D structure of

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 mi...

This study utilized Rhizopus oryzae lipase (RoL) as a potential biocatalyst in the hydrolysis of oil-contaminated wastewater. Immobilization of the RoL occurred via the preparation of cross-linked enzyme aggregates of the RoL using ammonium sulphate (50% w/v) and glutaraldehyde (0.125% v/v) as the precipitant and cross-linker respectively. Characterization of the RoL-CLEA was carried out using scanning electron microscopy. Prior to the enzymatic treatment of the wastewater, the lipid concentration and the chemical oxygen demand levels were determined. The RoL-CLEA could be reused up to five cycles and its catalysis increased free fatty acid levels in the wastewater by 84.8% and 142.1% at 0.5% and 1% (w/v) CLEA respectively. There was an increase in chemical oxygen demand (COD) removal by 7.6% and 29.5% from the oil-contaminated wastewater when 0.5% (w/v) and 1% (w/v) of the immobilized biocatalyst was used. In conclusion, the RoL CLEA is a unique biocatalyst in the treatment of oil-contaminated wastewater. Hence, there is the possibility of utilizing this biocatalyst for the large-scale remediation of oil-contaminated water bodies.

Elucidation of lipase–substrate interactions will guide the proper industrial use and applicability of the enzyme. The aim of this study was to predict the 3 D structure of Rhizopus oryzae ZAC3 (RoZAC3) lipase, study its interactions with some natural substrates and evaluate the feasibility of fatty acid methyl esters (FAME) production by the immobilized lipase. Protein identification of RoZAC3 lipase was carried out using LC-MS/MS. The 3 D structure of the lipase was built using homology modelling and natural substrates such as tributyrin, tripalmitin and triolein were docked to the optimized 3 D model for investigation of enzyme-ligand interactions. RoZAC3 lipase, immobilized by adsorption on Lewatit VP OC 1600 was applied in the synthesis of fatty acid methyl esters (FAME). From the phylogenetic analysis, it was observed that RoZAC3 lipase was closely related (48%) to Rhizopus javanicus lipase (Q7M4U7). The predicted 3 D model was validated using the SWISS model validation server. Ramachandran and ERRAT plots were used to assess the amino acid environment and overall quality of the model. From the docking studies, the values of the binding energies obtained for tributyrin, tripalmitin and triolein were − 5.37, −5.27 and −5.77 respectively. At an enzyme:immobilization support ratio of 50 mg/g, transesterification reaction duration of 18 h and a temperature of 40 oC, the conversion reached above 80%. The molecular docking studies provided information on the interaction/modifications between the RoZAC3 lipase and triacylglycerols that can be exploited for numerous applications. The immobilized lipase could serve in hydro-esterification reactions adaptable for biodiesel production. Communicated by Ramaswamy H. Sarma

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.

This study was undertaken to isolate and identify a novel cellulase-producing strain from a waste site (7°28’11’’N 4°31’24’’E), optimise the growth conditions, partially purify and biochemically characterise the enzyme. The potentials of the purified cellulase to hydrolyse the lignocellulosic component of some agro-industrial wastes (e.g. orange peels etc.) was also investigated. The best cellulase-producing fungus was identified as Mucor ramanniacus and the optimum conditions for cellulase production were pH (4.5), inoculum size (12 mm), carbon and nitrogen sources were carboxymethyl cellulose and sodium nitrate respectively resulting in a specific activity of 1423 Units/mg protein. A purification fold of 1.56 and 45.37 % yield were obtained after purification. The optimum pH and temperature were at 9.0 and 40°C respectively. The kinetic parameters were 0.63 ± 0.495 mg/ml, 20.21 ± 11.28 U/ml, 1001.4s− 1 for Km and Vmax and kcat respectively. Na+, K+, Ca+, Cysteine, β-mercaptoethanol and SDS were activators while Tween 80, Triton X-100 EDTA, Hg2+ and Ba2+ inhibited the enzyme. M. ramanniacus cellulase hydrolysed all agro-industrial wastes used. The partially purified M. ramanniacus cellulase showed great potential in biodegradation of various lignocellulosic substrates and the biochemical characteristics exhibited makes it suitable in industrial applications.

Background Biochemical, hematological and histological changes are major observable clinical and pathological factors associated with Diabetes mellitus. Derangement in the levels of these parameters increases the risk of the development of complications. In another hand, gastrointestinal intolerance due to the development of lactic acidosis on the gastrointestinal tract and the intestinal microbiome is the toxic side effect of various synthetic antidiabetic agents. The use of Kigelia africana fruit extract for the treatment of diabetes has been scientifically validated. This study therefore aimed at investigating changes in the biochemical, hematological and histological parameters as well as the determination of the functional groups present in the hexane fraction of the fruit. Methods The fruits were extracted with ethanol and partitioned with n-hexane to obtain the hexane fraction. Diabetic rats induced with streptozotocin (STZ) were divided into 5 groups of 5 animals each and treated with 100, 200 and 400 mg/kg body weight (BW) hexane fraction alongside reference standard; glibenclamide. Fasting blood glucose levels and their body weights were monitored weekly. Animals were sacrificed at the end of 28-day treatment. Blood, liver, and kidney were collected for biochemical, hematological and histopathological analyses. Fourier transform infrared resonance (FTIR) spectroscopic analysis was carried out on the hexane fraction for functional group determination. Results The hexane fraction of K. africana fruit extract decreased fasting blood glucose (FBG) levels significantly with ameliorative effects on the hematological parameters such as packed cell volume (PCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and red blood cells (RBC) etc. There were significant regenerative differences in the biochemical activities as well as the renal cortex and midzone sections of the rat's kidney and liver when compared with untreated diabetic rats. The presence of polyphenolic functional groups via FTIR analysis suggested high antioxidant activities of the fruit extract. Conclusion The use of Kigelia africana fruit extracts protects against biochemical, hematological and histological changes that are injurious to diabetic patients. Therefore, Kigelia africana fruit is a good hepatic- and nephroprotective agent and has a hemato-protective ability., Diabetes mellitus; Kigelia africana; Streptozotocin; FTIR; Hyperglycemia; Metformin; Chemistry; Food Science; Agricultural Science; Biological Sciences; Health Sciences

In this study, the preparation, characterization, and evaluation of the cross-linked enzyme aggregates (CLEAs) of fructosyltransferase isolated from the mutant-type Aureobasidium pullulans NAC8 for the production of fructooligosaccharides (FOS) with prebiotic properties were investigated. The reaction conditions were optimized statistically by using response surface methodology. CLEAs were characterized by using both photo-microscopy and scanning electron microscopy energy-dispersive X-ray spectroscopy (SEM-EDX). The secondary structure of the protein was predicted by using Fourier transform infrared spectroscopy (FTIR). FOSs produced as a result of the biotransformation of sucrose was quantified by using a chemometric analysis of FTIR spectral data. The prepared FOSs were investigated for the prebiotic effects under anaerobic conditions using a standard strain of Lactobacillus sp. Following were the statistically determined, optimum conditions for the production of FOSs: 5% (v/v) glutaraldehyde, pH 5.5, and a temperature of 32°C. The values of R 2 , F value, adjusted precision, and coefficient of variation were found to be 0.94, 11.50, 9.34, and 35.34%, respectively. Validation of the model resulted in the reusability of the CLEAs in six reaction cycles as it retained 70% of the residual activity. The SEM-EDX and photomicrographic analysis showed that the sizes of CLEAs varied, which ranged between 10 and 40 μm. Aggregation leads to the formation of a higher number of beta sheets in the CLEAs than that in the free enzyme. The total FOSs produced by the CLEAs was around 57% (w/v). A. pullulans FOS supplemented to the medium had a stimulatory effect on the growth of bi dobacteria compared with the control medium. The growth curves of Lactobacillus sp. reached the plateau phase afteR 2 4 h of cultivation with FOS. The CLEA of fructosyltransferase catalyzed the biocatalysis of sucrose to FOSs, a process with a biotechnological importance as prebiotics.

Background β-Cyanoalanine synthase plays essential roles in germinating seeds, such as in cyanide homeostasis. Methods β-Cyanoalanine synthase was isolated from sorghum seeds, purified using chromatographic techniques and its biochemical and catalytic properties were determined. Results The purified enzyme had a yield of 61.74% and specific activity of 577.50 nmol H2S/min/mg of protein. The apparent and subunit molecular weight for purified β-cyanoalanine synthase were 58.26±2.41 kDa and 63.4 kDa, respectively. The kinetic parameters with sodium cyanide as substrate were 0.67±0.08 mM, 17.60±0.50 nmol H2S/mL/min, 2.97×10−1 s−1 and 4.43×102 M−1 s−1 for KM, Vmax, kcat and kcat/KM, respectively. With L-cysteine as substrate, the kinetic parameters were 2.64±0.37 mM, 63.41±4.04 nmol H2S/mL/min, 10.71×10−1 s−1 and 4.06×102 M−1 s−1 for KM, Vmax, kcat and kcat/KM, respectively. The optimum temperature and pH for activity were 35°C and 8.5, respectively. The enzyme retained more than half of its activity at 40°C. Inhibitors such as HgCl2, EDTA, glycine and iodoacetamide reduced enzyme activity. Conclusion The biochemical properties of β-cyanoalanine synthase in germinating sorghum seeds highlights its roles in maintaining cyanide homeostasis.

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.

Background. Many natural aquatic bodies have been contaminated with heavy metals released from domestic, industrial and other anthropogenic activities. Fish are an important bioindicator species and play an important role in the monitoring of water pollution.Objectives. This study shows the effect of heavy metals on the distribution of glutathione S-transferases (GST), catalase, rhodanese and 3-mercaptopyruvate sulphur transferase (3-MST) isolated from the liver, gills, fins and muscle of Clarias gariepinus.Methods . Glutathione S-transferase, catalase, rhodanese and 3-mercaptopyruvate S-transferase enzymes were isolated from the liver and gills of fish by homogenization of each tissue (with specific buffers for each enzyme) and centrifugation. Serial dilutions of the crude enzymes were then assayed for residual enzymatic activities using standard enzyme assay protocol.Results. The results showed heavy metals in the liver and muscle of the investigated fish. This study indicated significant accumulation of heavy metals in the tissues/organ of the fish from Ilesha, Osogbo and Yakoyo fish ponds. These are three main towns in Osun State where the major occupation is fish farming. The relationship between enzymatic activities and heavy metal content in C gariepinus tissue showed positive and significant (pConclusions. The study concluded that the pattern of response of GST, catalase, rhodanese and 3-MST activities in the various organs/tissues of C gariepinus to the heavy metals suggests that the excitation or inhibitions of their activities are organ specific. Further biochemical studies of fish tissues/organs are needed to characterize the enzymatic changes associated with heavy metal pollution.Competing Interests: The authors declare no competing financial interests

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.

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 o...

Background. Heavy metals in water systems are a human health concern as they can enter the food chain. Objectives. Heavy metal and particle size determinations were used to determine the sediment quality of three fishponds in Osun State, Nigeria and their feeder streams. Methods. Sediment samples were collected in triplicates from the streams and their associated fishponds and the accumulations of nine heavy metals (lead (Pb), cobalt (Co), cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), iron (Fe), manganese (Mg), zinc (Zn)) in the sediment were investigated seasonally. Particle size distribution and textural class of the samples were also determined. Results. Metal concentrations recorded in bottom sediment varied widely and exhibited fluctuations among the different ponds and streams, especially in the values of Fe, Cu, Mn, Cr, Zn and Pb. There was a significant difference (pCu>Ni>Zn>Pb>Cr>Mn>Co in Ilesha, Fe>Cu>Ni>Zn>Mn>Co>Pb>Cr in Osogbo and Zn>Fe>Ni>Mn>Cu>Co>Cr>Pb in Yakoyo. A closely related order of Fe>Mn>Ni>Zn>Cr>Cu>Se>Pb>Mo>Cd was observed in River Eku. The selected streams and their associated fishponds were fairly polluted, with the Yah fishpond (Ilesha) having the highest heavy metal pollution compared to Arula fishpond (Osogbo) and Ewuru/Rara fishpond (Yakoyo). The concentrations of accumulated heavy metals depended on the textural class of the sediment. Conclusion. Pollution has reached levels hazardous to human health compared to standard limits for aquaculture development. Stream and fishpond sediments should be analyzed at regular intervals as a quality assurance process to ensure that there are no toxic substances in the ponds, leading to possible bio-accumulation and magnification. This will help guarantee the health of the aquatic ecosystem, humans and the environment.

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 effect of sodium and chloride salts on the activity of some enzymes, namely, rhodanese and 3–mercaptopyruvate sulphur transferase, arginase, acid phosphatase, β-glucosidase and cellulase in the haemolymph of the giant African snail (Archachatina marginata) were investigated. This is an attempt to providing scientific reasoning(s) for the traditional belief that terrestrial snails cannot tolerate a saline environment. Only acid phosphatase, β-glucosidase and cellulase activities were affected by this treatment while rhodanese, 3-mercaptopyruvate sulphur transferase and arginase activities were slightly affected. The former enzymes are known to be involved in energy metabolism while the latter play secondary roles such as detoxification and urea metabolism. Keywords: Archachatina, heamolymph, rhodanese, 3-mercaptopyruvate sulphur transferase, arginase, β-glucosidase, cellulose, salt toxicityInternational Journal of Biological and Chemical Sciences Vol. 2 (1) 2008 pp. 66-71

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.