Saleh A. Mohamed

Characterization of a cysteine protease from wheat Triticum aestivum (cv. Giza 164)

Enzymes, especially proteases, have become an important and indispensable part of the processes used by the modern food and feed industry to produce a large and diversified range of products for human and animal consumption. A cysteine protease, used extensively in the food industry, was purified from germinated wheat Triticum aestivum (cv. Giza 164) grains through a simple reproducible method consisting of extraction, ion exchange chromatography and gel filtration. The molecular weight of the enzyme was estimated to be 61000+/-1200-62000+/-1500 by SDS-PAGE and gel filtration. The cysteine protease had an isoelectric point and pH optimum at 4.4 and 4.0, respectively. The enzyme exhibited more activity toward azocasein than the other examined substrates with K(m) 2.8+/-0.15 mg azocasein/ml. In addition, it had a temperature optimum of 50 degrees C and based on a heat stability study 55% of its initial activity remained after preincubation of the enzyme at 50 degrees C for 30 min prior to substrate addition. All the examined metal cations inhibited the enzyme except Co(2+), Mg(2+), Mn(2+) and Li(+). The proteolytic activity of the enzyme was inhibited by thiol-specific inhibitors, whereas iodoacetate and p-hydroxymercuribenzoate caused a competitive inhibition with Ki values 6+/-0.3 mM and 21+/-1.2 microM, respectively. Soybean trypsin inhibitor had no effect on the enzyme. The enzyme activity remained almost constant for 150 days of storage at -20 degrees C. The properties of this enzyme, temperature and pH optima, substrate specificity, stability and sensitivity to inhibitors or activators, meet the prerequisites needed for food industries.

Distribution of lipases in the Gramineae. Partial purification and characterization of esterase from Avena fatua

Abstract The activity levels of esterase, lipid acylhydrolase and lipase were quantitatively screened in 23 species and cultivars of Gramineae. Their activity levels expressed as units g −1 seeds, were found to range from 10 to 123 for esterase, 0.28 to 7.67 for lipid acylhydrolase and 13.1 to 93.9 for lipase. Avena fatua , one of the grass species, exhibited the highest levels of esterase and lipase and could be potentially a good starting material for preparation of lipases. A. fatua esterase has been partially purified and characterized. Four isoenzymes, EI, EII, EIII and EIV, were separated by ion exchange chromatography. Esterases EII and EIII had Km values of 0.52 and 0.38 mM and a pH optimum at 9.0 with half maximal activities at pHs 8.5, 10 and 8, 10.5, respectively. Esterases EII and EIII had optimum activities at temperatures of 75°C and 65°C with activation energies of 3.3 and 4.3 kcal mol −1 , respectively. The enzymes were thermally stable as esterases EII and EIII retaining 39% and 23% of their activities at 90°C, respectively. Esterases EII and EIII were stimulated by Ba 2+ and Ca 2+ but were inhibited by Mn 2+ and Zn 2+ . A. fatua esterases exhibited optimum storage stability and were stable at high temperatures and alkaline pH. They possessed high affinity toward substrate and were resistant to inhibition by most divalent cations that were examined. These are important properties when considering the industrial application of these enzymes.

Horseradish peroxidase and chitosan: Activation, immobilization and comparative results

Recently, horseradish peroxidase (HRP) was immobilized on activated wool and we envisioned that the use of chitosan would be interesting instead of wool owing to its simple chemical structure, abundant nature and biodegradability. In this work, HRP was immobilized on chitosan crosslinked with cyanuric chloride. FT-IR spectroscopy and scanning electron microscopy were used to characterize immobilized HRP. The number of ten reuses of immobilized HRP has been detected. The pH was shifted from 5.5 for soluble HRP to 5.0 for immobilized enzyme. The soluble HRP had an optimum temperature of 30 °C, which was shifted to 35 °C for immobilized enzyme. The soluble HRP and immobilized HRP were thermal stable up to 35 and 45 °C, respectively. The apparent kinetic constant values (K(m)) of soluble HRP and chitosan-HRP were 35 mM and 40 mM for guaiacol and 2.73 mM and 5.7 mM for H2O2, respectively. Immobilization of HRP partially protected them from metal ions compared to soluble enzyme. The chitosan-HRP was remarkably more stable against urea, Triton X-100 and organic solvents. Chitosan-HRP exhibited large number of reuses and more resistance to harmful compounds compared with wool-HRP. On the basis of results obtained in the present study, chitosan-HRP could be employed in bioremediation application.

Antioxidant capacity of chewing stick miswak Salvadora persica

BackgroundChewing stick (miswak Salvadora persica L.) is an effective tool for oral hygiene. It possessed various biological properties including significant antibacterial and anti-fungal effects. In the present study, we evaluated the antioxidant compounds in miswak.MethodMiswak root was extracted with 80% methanol. Methanol extract as antioxidant was evaluated by using DPPH, ABTS and phosphomolybdenum complex assays and analysis by GC-MS. Peroxidase, catalase and polyphenoloxidase assays were performed for crude extract of miswak root.ResultsThe methanol extract of miswak contained the highest amount of crude extract among the various solvent extracts. The methanol extract showed a concentration dependent scavenging of DPPH and ABTS radicals with IC50 values 4.8 and 1.6 μg crude extract, respectively. The total antioxidant activities, based on the reduction of molybdenum (VI) to molybdenum (V), increased with increasing crude extract content. The correlation coefficients (R2) between total crude extract and DPPH, ABTS scavenging activities and the formation of phosphomolybdenum complex were 0.97, 0.99 and 0.95, respectively. The GC-MS analysis showed that the methanol extract doesn’t contain phenolic and flavonoid compounds or under detected limit. After silylation of methanol extract, three compounds namely 2-furancarboxaldehyde-5-(hydroxymethyl), furan-2-carboxylic acid-3-methyl- trimethylsilyl ester and D-erythro-pentofuranose-2-deoxy-1,3,5-tris-O-(trimethylsilyl) were identified by GC-MS analysis. These furan derivatives as they contain hydroxyl groups could be possessed antioxidant activities. The antioxidant enzymes were also detected in the miswak extract with high level of peroxidase and low level of catalase and polyphenoloxidase.ConclusionsThe synergistic actions of antioxidant compounds and antioxidant enzymes make miswak is a good chewing stick for oral hygiene and food purposes.

Biochemical Changes in Fruit of an Early and a Late Date Palm Cultivar During Development and Ripening

The biochemical changes in fruit of an early ‘Lonet-Mesaed’ and a late ‘Helali’ date palm cultivar during development and ripening including the activities of various degradative enzymes were studied. During the 2009 and 2010 seasons, in both cultivars, fruit growth, based on fruit and flesh weight, followed a smooth sigmoidal curve. The fruit and flesh weight gradually increased during development reaching a maximum at the immature green (Bisir) stage, but slightly decreased thereafter during ripening. Moisture percentage was highest at early stages and then gradually decreased to lower levels during the Bisir and the mature firm full-colored (Rutab) stages in both cultivars, with a further sharp decrease at the raisin-like stage (Tamer) in ‘Helali’. The accumulation of both total and reducing sugars in fruit slightly increased during development with a vast increase during maturation and ripening of both cultivars. The concentration of total proteins was highest at early stages and then gradually decreased during development to lower concentrations during ripening. A steady decrease in the membrane stability index (MSI %), as measured by the leakage of ions, was observed upon the progression of fruit development, especially during the Bisir and the Rutab stages, indicates a gradual loss of the membranes stability due to changes occurring in the biochemical and biophysical properties of cell membranes. During the 2009 season, both cultivars possessed polygalacturonase, cellulase xylanase, and α-amylase activities. Within the same fruit, the slightly softened apical half had activity of about 15 and 2 times for polygalacturonase and cellulase, respectively, higher than that of the firm basal half of the same fruit. Moreover, the activity of both xylanase and α-amylase was only detected in the apical tissues. The activities of these enzymes and fruit ripening were closely associated, suggesting their involvement in the ripening process of dates. The differences between the two cultivars in developmental and ripening patterns in conjunction with enzyme activities are discussed.

Solid state production of polygalacturonase and xylanase by Trichoderma species using cantaloupe and watermelon rinds

Different solid state fermentation (SSF) sources were tested such as cantaloupe and watermelon rinds, orange and banana peels, for the production of polygalacturonase (PG) and xylanase (Xyl) by Trichoderma harzianum and Trichoderma virens. The maximum production of both PG and Xyl were obtained by T. harzianum and T. virnes grown on cantaloupe and watermelon rinds, respectively. Time course, moisture content, temperature, pH, supplementation with carbon and nitrogen sources were optimized to achieve the maximum production of both PG and Xyl of T. harzianum and T. virens using cantaloupe and watermelon rinds, respectively. The maximum production of PG and Xyl of T. harzianum and T. virens was recorded at 4–5 days of incubation, 50–66% moisture, temperature 28–35°C and pH 6–7. The influence of supplementary carbon and nitrogen sources was studied. For T. harzianum, lactose enhanced PG activity from 87 to 120 units/g solid, where starch and maltose enhanced Xyl activity from 40 to 55–60 units/g solid for T. virnes. Among the nitrogen sources, ammonium sulphate, ammonium nitrate, yeast extract and urea increased PG activity from 90 to 110–113 units/g solid for T. harzianum. Similarly, ammonium chloride, ammonium sulphate and yeast extract increased Xyl activity from 45 to 55–70 units/g solid for T. virens.

Production, purification and characterization of α-amylase from Trichoderma harzianum grown on mandarin peel

The production, purification and characterization of α-amylase from Trichoderma harzianum grown on mandarin peel were investigated. The effect of incubation time and mandarin peel concentration on the production of α-amylase by T. harzianum was studied. α-Amylase A3 was purified from T. harzianum to electrophoretic homogeneity by using DEAE-Sepharose and Sephacryl S-200 columns. The enzyme had molecular weight of 70 kDa using gel filtration and SDS-PAGE. The affinity of the substrates toward A3 was in the order of amylopectin > glycogen > starch > β-cyclodextrin > dextrin > α-cyclodextrin. These findings tend to suggest that the enzyme has high affinity toward high-molecular mass substrates. The Km and Vmax values of the enzyme for hydrolyzing potato soluble starch and glycogen were 6.53, 4.5 mg/ml and 2 and 2.2 μmol reducing sugar/ml, respectively. The maximum activity of enzyme against soluble starch was determined at pH 4.5 and 40°C. α-Amylase A3 was stable up to 40°C for 30 min of incubation and retained 70 and 50% of its activity at 50 and 60°C, respectively. While all the examined metal cations were effective in inhibiting the enzyme, Ca2+ considerably enhanced the activity. The metal chelators, EDTA, sodium citrate and sodium oxalate had inhibitory effects on A3. The rate of breakdown of starch was higher than the rate of formation of reduced sugar indicating A3 is endo-acting enzyme. These properties of A3 with its remarkable activity meet the prerequisites needed for liquefaction and saccharification of starch industry.

Immobilization of Trichoderma harzianum α-Amylase on Treated Wool: Optimization and Characterization

α-Amylase from Trichoderma harzianum was covalently immobilized on activated wool by cyanuric chloride. Immobilized α-amylase exhibited 75% of its initial activity after 10 runs. The soluble and immobilized α-amylases exhibited maximum activity at pH values 6.0 and 6.5, respectively. The immobilized enzyme was more thermally stable than the soluble one. Various substrates were hydrolyzed by immobilized α-amylase with high efficiencies compared to those of soluble α-amylase. The inhibition of the immobilized α-amylase by metal ions was low as compared with soluble enzyme. On the basis of the results obtained, immobilized α-amylase could be employed in the saccharification of starch processing.

Characterization of polygalacturonases from fruit spoilage Fusarium oxysporum and Aspergillus tubingensis

We reported the partial purification and characterization of polygalacturonases from fruit spoilage Fusarium oxysporum and Aspergillus tubingensis isolated from banana and peach, respectively. By using diethylaminoethyl (DEAE)-Sepharose column, one and two forms of polygalacturonases were separated from F. oxysporum (PGase) and A. tubingensis (PGaseI and PGaseII), respectively. The polygalacturonases examined had higher affinity toward various polygalacturonic acids and pectins. The apparent Km and Vmax values were reported for the enzymes. Acidic pH optima (4.0 to 6.0) was also reported for the enzymes. Optimal temperature and thermal stability of the enzymes showed a range from 40 to 60°C. The effect of metal cations on the enzymes was studied. The most chemical compounds caused moderate inhibitory effect except benzoic and citric acids which had strong inhibitory effect on the polygalacturonases. The benzoic and citric acids were used as antifungal compounds for F. oxysporum and A. tubingensis. The citric acid was found to be more effective against fungal growth than benzoic acid.

Properties of peroxidase from chewing stick miswak

Miswak is a chewing stick prepared from the roots, twigs or stems of Salvadora persica L. and widely used in Middle Eastern and Estern African cultures. Currently, its chemical components had antimicrobial and antioxidant activities. In the present study, peroxidase, as antioxidant and antibacterial enzyme, was screened in 4 parts of miswak and the level of peroxidase activity was recorded in the order of peel of stem > root without peel > peel of root > stem without peel. Generally, the people used the root without peel. By chromatography of miswak root without peel on DEAESpharose 3 peroxidases POI, POII and POIII were separated. Peroxidase POII with highest activity was reached to homogeneity by chromatography on Sephacryl S-200. The molecular weight of POII was found to be 70 kDa. o-Phenylenediamine was found to be the best substrate for the enzyme followed by guaiacol, o-dianisidine, pyrogallol and p-aminoantipyrine. The apparent Km for catalysis of H2O2 and guaiacol were 0.9 and 17.33 mM respectively. The enzyme had an optimum pH and temperature at 5.5 and 40°C respectively. POII was stable at 10 to 40°C and unstable above 50°C. Most of the examined metal ions had partially inhibitory effects on POII, while Co