Role of calcium in enhancing the activity and thermal stability of a new cationic peroxidase purified from euphorbia tirucalli latex

A new cationic peroxidase from Euphorbi and firucalli [pencil cactus] latex was purified to homogeneity using benzene fractionation, gel filtration and cation-exchange chromatography. The purified enzyme was found to be monomeric with a molecular weight of 44 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]. The purified enzyme had a broad specificity towards some phenolic substrates in the order of 2,2'-azino-bis [3-ethylbenzothiazoline-6-sulfonicacid] [ABTS] > guaiacol > 0-phenylenediamine > 4-aminoantipyrene, whereas no affinity towards ascorbic acid and o-dianisidine was recorded. The enzyme had pH and temperature optima at 7.0 and 40°C, respectively. Study of kinetic parameters demonstrated that ABTS had the highest affinity towards ELP, where K[m], F[max] and V[ms]/K[m] values were 0.503 mM, 500 U/assay and 994.04 U/mM, respectively. ELP was stable from 10°C up to 60°C and lost about 70% of its activity at 70°C. The thermal inactivation profile of ELP in absence of Ca[2+] is biphasic and characterized by a rapid decline in activity on exposure to heat, followed by a more gradual decrease in activity on continued exposure. However, the purified enzyme exhibited increased thermal stability in the presence of calcium ions. Furthermore, the activity of purified enzyme was enhanced by 550% in the presence of 15 mM CaCl[2], suggesting a pivotal role for Ca[2+] in conferring structural stability to the heme environment and in retaining the active site of ELP. Most of the examined metal ions [except for Ca and Mg] and compounds had differential inhibitory effects on ELP activity. In conclusion, a locally available plant [Euphorbia tirucalli] could be a potential candidate source for peroxidase, the most widely used enzyme in industrial and biomedical applications. In addition, calcium was found to be essential for enhancing enzymatic activity and thermal stability of the purified Euphorbia tirucalli latex peroxidase

Optimizing conditions for production of polygalacturonase from aspergillus niger NRRL3

The effect of varying cultural conditions was assessed on the production of polygalacturonase [PG] in Aspergillus niger NRRL3. The effect of different types of media and growth techniques [stationary and agitation cultures] on production of PG from A. niger was examined. The optimum activity of PG was recorded for potato dextrose broth medium with 0.1% citrus pectin as carbon source in agitation culture. There were variations in enzyme levels produced in culture filtrate with age of culture, the highest levels being in 3-day-old culture. The temperature and pH also had marked effects on the production of PG with the best conditions being 30°C and pH 5.0, respectively. The effect of different carbon sources on pH production showed that citrus pectin was the best carbon source. The addition of the different examined nitrogen sources and mineral salts decreased the productivity of PF enzyme compared to the PDB medium in absence of them

Purification and characterization of two cysteine proteases from germinated barley seeds

Different stages during barley [Hordium vulgare cv. Giza 126] gennination showed endoproteolytic activity. The changes of endoproteolytic activity and protein were detected with different stages during germination. In presence of gibberellic acid [GA 3], no changes could be detected in the profile of protease activity except on day 4 where the pinteolytic activity was increased to a 1.25 fold over its value in absence of GA3. During purification of H. vulgare protease, ion exchange chromatography on DEAE-cellulose led to five separate forms [from P1 to P5]. Proteases P3 and PS with the highest specific activities were pure after chromatography on Sephacryl 5-200. The molecular weights of P3 and PS were 25,000 and 30,000, respectively. By SDS-PAGE, the P3 and PS were composed of a single band of molecular weights of 24,000 and 30,000, respectively, indicating that the two proteases are brobably monomers. Barley P3 and P5 exhibited pH optima at 3.5 and 4.0, respectively. Km values for barley P3 and P5 were estimated to be 5.4 and 4.2 mg azocaseine/ml, respectively. Varying protease activity was detected for P3 and P5 when supplied with various proteins as substrates. P3 and P5 were found to have temperature optima at 30 and 40°C, respectively. P3 and P5 were stable up to 40°C and retained 45% and 35% of their activities at 60°C, respectively. The stability of P5 toward metal ions inactivation was considerably higher than the stability of P3. Only inhibitors of cysteine proteases significantly inhibited P3 and P5, while DTT as a reducing agent enhanced P3 and P5 activities. These results indicated unequivocally that P3 and P5 are cysteine proteases. With the purification of barley P3 and PS, the physiological roles of these specific proteases in germinating barley and the regulation of their expression by GA 3 can be addressed

Comparison of isoenzyme and random amplified polymorphic DNA data for determining intraspecific variation in date palm

Variation in isoenzyme and random amplified polymorphic DNA [RAPD] for Egyptian tissue culture-derived date palm cultivars were used to determine the utility of these markers assessing genetic variation among these cultivars. lsoenzyme patterns of esterase, polyphenol oxidase, peroxidase and acid phosphatase showed that the highest similarity index values were detected between individuals of cv. Hyani 1, 2 and 3 indicating the strong relationships and high similarity between the individuals in the same cv. Hyani. Surprisingly, Hyani 4 had low similarity index with Hyani 1, 2 and 3. The moderate similarity index values were detected between individuals of cv. Hyani 1, 2 and 3 and the three other cultivars Zaghloul, Amhat and Samani. DNA fingerprints and the DNA polymorphism for date palm cultivars were obtained with 2 of the tested primers [OPA 15 and OPA 16]. A total of eleven amplification products [bands] were generated using these two primers for the characterization of four date palm cultivars. Among the eleven bands generated in this study, nine bands were useful as polymorphic markers. The highest similarity index values, using DNA fingerprint, were detected between cv. Hyani 3 and each of cvs. Amhat and Hyani 4. DNA fingerprint dendrogram divided the date palm cultivars into cluster A [cvs. Hyani 1, 2, 3, 4, Amhat and Samani] and cv. Zaghloul with very low similarity index 0.068. In conclusion, our results showed that the similarity indices for DNA fingerprint data between date palm cultivars Hyani 1, 2, 3, 4, Zaghloul, Amhat and Samani were higher than those for isoenzyme patterns. Therefore, RAPD markers are reliable method to distinguish among most of the available commercial date palm cultivars

serine protease from germinated wheat seeds

A simple method for the purification of a serine endoprotease from wheat Triticum aestivum [cv. Giza 164] has been developed. It consists of ion-exchange and gel filtration chromatography. The molecular mass of the enzyme was 58 kDa by SDS/PAGE under reducing conditions and 57 kDa by gel filtration on a Sepharose 6B column. The enzyme had isoelectric point and pH optimum at 4.2 and 4.5, respectively. The substrate specificity of the enzyme was studied by the use of synthesized and natural substrates, azocasein, azoalbumin, hemoglobin, casein, gelatin and egg albumin. The enzyme appears to prefer azocasein with a Km of 2 mg azocasein/ml. The enzyme had a temperature optimum at 50°C with heat stability up to 40°C. While Co[2+] and Mg[2+] accelerated the enzyme activity by 54% and 56%, respectively, Ca[2+] and Ni[2+] had very little effect. The enzyme was strongly inhibited by PMSF [phenylmethylsulphonyl fluoride], but not by the other protease inhibitors, suggesting that the enzyme is a serine protease. From these results it can be concluded that the T. aestivum serine protease may be suitable for food processing

Distribution of acidic proteases in Gramineae. Kinetics of protein degradation in wheat Triticum aestivum [cv. Giza 164] during germination

Acidic protease activity was quantitatively screened in 28 species and cultivars of Gramineae family during germination. Most cultivars of cereals and some grasses as Oryza sativa and Avena fatua, not economic plants, had high acidic protease activity and could be a potentially good starting material for preparation of proteases. Kinetics of protein degradation in Triticum aestivum [cv. Giza 164] seeds during germination was examined. The results revealed that protease activity was dramatically increased from the second day of germination and reached its maximum activity on day 10. This increase in protease activity was accompanied by a decrease in soluble protein content. Gibberellic acid [GA3] caused an obvious increase in protease activity during germination that reached a 1.8-fold increase on day 10 over the untreated seeds concomitant with high decrease of the protein content. The isoenzyme pattern of T. aestivum protease during germination showed the differences in number and relative mobility of isoenzyme bands in absence and presence of GA3. The number of isoenzymes was increased from 9 to 12 in presence of GA3. Only three isoenzymes were detected in absence and presence ofGA3. The electrophoretic pattern of protein showed that the number and intensity of bands were increased, specially on days 8 and 10 and number of low molecular weight bands also appeared from days 4 to 14 in presence of GA3

Molecular characterization of wheat Triticum aestivum cultivars using isoenzyme analysis and RAPD fingerprint

Molecular characterization of Egyptian Triticum aestivum wheat cultivars including protease isoenzyme pattern and randomly amplified polymorphic DNA fingerprint [RAPD] was studied. Protease isoenzyme patterns indicated that the enzyme could exist as fast, intermediate or slow migrating forms. The numbers and relative mobility of protease bands of these cultivars were different and ranged between 2 and 5 bands. The degree of similarity between the wheat cultivars was determined and the results obtained indicated that Beni-Swif 3 and Sakha 8 have 100% similarity and could not be differentiated by the protease isoenzyme patterns. DNA fingerprints of wheat cultivars were studied using 10 random primers, of which only one primer P-262 gave visible and reproducible polymorphic bands. RAPD analysis and dendrogram indicated the genetic diversity and intervarietal relationships between the cultivars and also can be used for cultivar identification. RAPD analysis can differentiate between the cultivars that can not be differentiated by protease isoeznymes such as Beni-Swif 3 and Sakha 8. This study constitutes a contribution to the identification of molecular markers for genetic diversity and intervarietal relationships among Egyptian wheat cultivars

Purification and characterization of bacterial CM-cellulase from camel rumen fluid [Camelus dromedarius]

Bacterial CM-cellulase [1,4-beta-D-glucanohydrolase, [EC 3.2.1.4]] from camelrumen fluid, an enzyme that causes a random secession of cellulose chainyielding glucose and cellotriose, was purified and characterized. Thepurification procedure included ammonium sulfate precipitation andchromatographies on DEAE-cellulose, and Sepharose 6B columns. By ion exchangeon DEAE-cellulose, five isoenzymes of CM-cellulase were obtained. CM-cellulase CII, CIV and CV were purified 11.2, 13.9 and 25.9 fold with 17.3%, 12.5% and 13.7% recovery in activities, respectively. The molecularweights of CM-cellulase CII, CIV and CV were estimated to be 12000, 13000 and 14600, respectively, for the native and 13000 for the denatured enzymes,respectively, suggesting that isoforms are monomeric. Amino acid compositionof the three CM-cellulases were detected. CM-cellulases CII, CIV and CV hadisoelectric points at 5.1, 6.6 and 5.1 and km values of 8.3, 6.25 and 5 mgCM-cellulase/ml, respectively, with more affinity toward CM-cellulase. CM-cellulases CII and CV had similar temperature optima at 40C, while themaximum activity for cellulase CIV was at 50C and had identical pH optimaat 5.5. The effect of divalent metal cations and different inhibitor on thethree isoenzyme activities was examined