The aim of study To purify GPCR from a local strain of S. cerevisiae using Ion exchange and gel filtration chromatography techniques , by packing materials for columns which will be chosen of low cost comparing to the already used in published researches, which depend on the costly affinity chromatography and other expensive methods of purification. Local strain of S. cerevisiae chosen for extraction and purification of G-protein coupled receptor (GPCR) .The strains were obtained from biology department in Al- Mosul University, Iraq. The isolated colony was activated on Yeast Extract Pepton Dextrose Broth (YEPDB) and incubated at 30 C˚ for 24 h .Loop fully of the yeast culture was transferred to (10ml) of yeast extract peptone glucose agar (YEPGA) slant , then incubated at 30C˚for 24h , after that it was stored at 4C˚ ,the yeast cultures were reactivated and persevered after each two weeks period. S.cerevisiae was identified by morphological, microscopic characterization and biochemical test . The GPCR that extract from whole cell of S.cerevisiae was purified by ion exchange chromatography using DEAE-Sepharose ,the bound proteins (negatively charged) were then eluted using gradient concentration of NaCl ranged between( 0.1 -0.5M). Gel filtration chromatography using Sepharose 6B was applied as a second step of purification. The optical density for each fraction was measured at 280 nm by UV-VS spectrophotometer then the GPCR concentration was determined by using ELISA Kit . The fractions which gave the highest absorbance and concentration of GPCR were collected .The molecular weight of GPCR was determined by gel filtration chromatography using blue dextrin solution. Standard curve was plotted between log of molecular weight for standard protein and the ratio of Ve/Vo of GPCR . The purity of the GPCR that extracted and purified from whole cell of S, cerevisiae were carried out by using SDS-PAGE electrophoresis . In ion exchange chromatography the fraction were collected with 5 ml tube at a flow rate 0.5 ml/ min and eluted with gradient (0.1-0.5M) of sodium chloride solution. Two proteins peaks appeared after eluted by the gradient concentration of sodium chloride, while no protein peaks appeared in the washing fractions. The GPCR concentration was measured in the fractions of these two protein peaks, data indicated that GPCR located in the first protein peak (eluted at 0.1M of NaCl) at fraction numbers between 3 and 9, the maximum concentration of GPCR was 9.281 with specific activity 71.58(ng/mg)protein , 3.125 purification folds and72.9(%) yield while the second peaks (eluted at 0.4 M of NaCl) don't give any concentration for GPCR, thus its neglected. Gel filtration chromatography was used as second step of purification which applied by using sepharose 6B. Results show single active protein peaks appeared that identical with the peak of GPCR at fractions numbers(29-35). The maximum concentration of GPCR was 9.082 (ng/ml)was observed in these fractions. The specific activity for these fractions was 151.37 (ng/mg) protein with 6.608 purification folds and 39.64 (%) yield. The present study a chive a relatively high purification of GPCR from whole cell of a local strain S. cerevisiae with fold purification 6.608 and a yield of 39.64 % and molecular weight about~33KD.
Twenty isolates of Serratia marcescens were isolated from inflammation of the urinary tract (UTI)., These isolates were found to produce hemolysin as indicated by blood agar plates in which the hemolysis of red blood cell indicate a positive result. Isolates were selected according to their hemolysis activity by measuring absorbance of hemoglobin at 405 nm that released from red blood cell. Hemolysin was completely purified using 50-75% saturation of ammonium sulphate followed by ion exchange chromatography with DEAE-cellulose then gel filtration chromatography by sepharose 4B. Accordingly molecular weight for the purified toxin was estimated as 45 KD.
Endoglucanase produced from Aspergillus flavus was purified by several steps including precipitation with 25 % ammonium sulphate followed by Ion –exchange chromatography, the obtained specific activity was 377.35 U/ mg protein, with a yield of 51.32 % .This step was followed by gel filtration chromatography (Sepharose -6B), when a value of specific activity was 400 U/ mg protein, with a yield of 48 %. Certain properties of this purified enzyme were investigated, the optimum pH of activity was 7 and the pH of its stability was 4.5, while the temperature stability was 40 °C for 60 min. The enzyme retained 100% of its original activity after incubation at 40 °C for 60 min; the optimum temperature for enzyme activity was 40 °C.
Soil samples from fields cultivated with barley and wheat in addition to samples
from spoiled orange and apple fruits and carrot roots were collected with the aim to
isolate cellulase producing bacterial strains. Bacterial isolates obtained from these
samples were grown on a selective medium containing carboxymethyl cellulose
(CMC) as a sole source for carbon and energy. Results showed that nine isolates out
of fifty were able to produce cellulase.The specific activity of cellulase in culture
filtrate of the most efficient isolate was 1.601 u/mg protein.This isolate was
identified according to its morphological characteristics and biochemical tests, and
then by using Api 20-E and VITEK-II identification systems an
Abstract:
Eclipta alba is a weed growing in damp, moist puddles distributed in the tropical and subtropical regions, so the most weight of the plant is water, which reached in to 90%. The extraction method by using different solvents (Methanol, Ethanol, Hexane and Aqueous) showed, the best yield with methanol reached to 76%, and the yield decreased with ethanol and hexane reached 55% and 53% respectively, while the minimum yield observed with aqueous hot and cold extraction reached 11% and 5% respectively. The phytochemical compound characterization showed the compounds (Coumarines , Flavones ,Volatile Oil ,Tannins , Saponines , Glycosides ,Carbohydrates ,Alkaloids , Resins) with different percentage. The thin layer chromatography det
Forty one isolates of genus Proteus were collected from 140 clinical specimens such as urine, stool, wound, burn, and ear swabs from patients of both sex. These isolates were identified to three Proteus spp. P. mirabilis, P. vulgaris and P. penneri .The ability of these bacteria to produce L-asparaginase II by using semi quantitative and quantitative methods was determined. P. vulgaris Pv.U.92 was distinguished for high level of L-asparaginase II production with specific activity 1.97 U/mg. Optimum conditions for enzyme production were determined; D medium with 0.3% of L-asparagine at pH 7.5 with temperature degree 35°C for incubation. Ultrasonication was used to destroy the P. vulgaris Pv.U.92 cells then ASNase II was extracted and pu
... Show More
In this study, the effect of Nd: YAG laser on the activity of superoxide dismutase (SOD) and alcoholdehydrogenase (ADH) was investigated. The Saccharomyces cells were irradiated using 532nm Q-Switched Nd: YAG laser with (1Hz) frequency. Different fluences 11.3, 22.6 and 33.9mJ/cm2 and different number of pulses 15, 30 and 60 pulse were used. The irradiated cells were incubated in a liquid nutritive medium for 24 hours. After incubation, the cells were harvested and disrupted to extract the intracellular enzymes and their activities were assessed. In comparison with the control, the irradiated cells showed a significant increase in the activity and the specific activity of SOD at energy densities of 11.3 and 22.6mJ/cm2 at 30 and 60 pulses
... Show MoreBeta-lactamase was purified from local isolate Klebsiella pneumonia by several steps included precipitation with ammonium sulphate at 20-40% saturation, DEAE- ion exchange chromatography and gel filtration on Sephacryl S-200 column. The obtained purification fold and recovery were 32.66; 47.04% respectively. The characterization of the purified beta-lactamase showed that the molecular weight was about 4000 daltons as determined by gel filtration.Purified enzyme had an optimal pH of 7 for activity and an optimal stability between pH 6.5-7.5, results shows that the optimal temperature appear to be 35 ? C .During storage the enzyme retained 72% at -20 ? C and retained 25% of the activity at the same period at 4 ? C.
The optimum conditions for the production of neutral protease from local strain Aspergillus niger var carbonarius by solid – state fermentation system (Wheat bran) moisted with 0.2 M phosphate buffer (PH7.0) . the hydration ratio was 1:5 (V:W) . the concentration of inoculum was 1×106 spores per 10 gram of solid materials , initial P H 6.5 and 96 hours of incubation period at 30? C .the enzyme activity was 1300 unit / ml and specific activity was 1550 unit / mg protein .
(2)