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Article
Purification and characterization of hemolysin produced by a local isolates of Staphylococcus aureus

Authors: Mohammed I. Nadir --- Hussain S. Al-Hassani --- Amer H. Al-Shammary
Journal: Karbala Journal of Medicine مجلة كربلاء الطبية ISSN: 19905483 Year: 2012 Volume: 5 no 2 Issue: 12 Pages: 1455-1463
Publisher: Kerbala University جامعة كربلاء

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Abstract

background: Staphylococcus aureus is a ubiquitous bacterium that is generating increasingly bad press coverage due to its propensity to adopt a pathogenic lifestyle in hospital and community settings. S. aureus colonies are found in approximately 30% of the general population. It colonizes the skin readily and can lead to a wide range of pathological conditions from skin lesions to osteomyelitis, endocarditis, and septicemia. Hemolysins are extracellular toxic proteins which are produced by many gram negative (e.g. Escherichia coli, Serratia spp., Proteus spp., Vibrio spp., Pasteurella spp., Pseudomonas aeruginosa) and gram positive bacteria (e.g. Streptococcus spp., Staphylococcus aureus, Listeria spp., Bacillus cerius, Clostridium tetani), all of which possess a certain pathogenic potential. Hemolysins have been therefore always considered as virulence factors. Most hemolysins cause lysis of erythrocytes by forming pores of varying diameters in the membrane and are designated as such because they have the ability to lyse red blood cells (RBCs). Objectives1-Purification of hemolysin from a local isolate of S. aureus.2-Characterization of hemolysin produced by a local isolate of S. aureus.Methods: Bacterial samples were identified by subjecting them to the standard laboratory procedures while semi quantitative screening on blood agar (containing 5% human blood) revealed that all isolates were hemolysin producer but in different efficiencies. Hemolysin was extracted by cooling centrifugation and purified by many steps including: precipitation by ammonium sulphate, dialysis, ionic exchange chromatography by using DEAE-Cellulose, and gel filtration chromatography by using Sephadex G-100. The molecular weight of hemolysin was determined by gel filtration chromatography on Sephadex G-100 while the optimum pH and temperature for hemolysin stability were also determined.Results: The results showed that forty isolates out of 100 were identified as Staphylococcus aureus. Hemolysin was extracted by cooling centrifugation and purified by many steps including: precipitation by ammonium sulphate with 50-75% saturation percentage, dialysis, ionic exchange chromatography by using DEAE-Cellulose, and gel filtration chromatography by using Sephadex G-100. The results showed that hemolysin was purified 135 fold with a yield of 1.16%.The molecular weight of hemolysin determined by gel filtration chromatography on Sephadex G-100 column was about 35000 daltons, while the optimum pH for enzyme stability was 7 and the optimum temperature for enzyme stability was between 25-35oC.Conclusions1.Conventional methods can be performed to extract hemolysins.2.Hemolysin was maximally produced when the pH was near neutrality and incubation temperature was 37oC and this conclusion indicates that hemolysin was produced when the conditions were similar to that of the host.


Article
EXTRACTION, PURIFICATION AND CHARACTERIZATION OF LIPASE PRODUCED BY A LOCAL ISOLATE OF STAPHYLOCOCCUS AUREUS

Authors: Amer H.R. Al-Shammary عامر هاني رزاق الشمري --- Asia F.R. Al-Husseiny اسيا فاضل رضا الحسيني
Journal: IRAQI JOURNAL OF MEDICAL SCIENCES المجلة العراقية للعلوم الطبية ISSN: P16816579,E22244719 Year: 2014 Volume: 12 Issue: 3 Pages: 254-260
Publisher: Al-Nahrain University جامعة النهرين

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Abstract

Background:Staphylococcus aureus is a ubiquitous bacterium that is generating increasingly bad press coverage due to its propensity to adopt a pathogenic lifestyle in hospital and community settings. Lipases catalyze both the hydrolysis and synthesis of triacylglycerols. Many of these enzymes are characterized by stability at high temperatures and in organic solvents.Objective:Purification of the enzyme by using the conventional methods and characterization of lipase.Methods:Purification included: extraction of the enzyme, the precipitation of the enzyme by ammonium sulphate, dialysis, ionic exchange chromatography by using DEAE-Cellulose (Diethylaminoethyl-Cellulose), and gel filtration by using Sephacryl S-200. Equal amounts of purified lipase solution were mixed with PBS (Phosphate buffer sodium) solutions of different pH (4,5,... until 10) and incubated in a water bath at 37 oC for 30 minutes, then the lipase activity was estimated. The purified lipase was incubated at different degrees of temperature (5, 15, ...until 85 oC) for 30 minutes. The molecular weight was determined by gel filtration chromatography.Results:The results revealed that the crude enzyme solution had a total protein concentration of 21.3 mg/ml and an enzyme activity of 257 µmole/ml. The lipase was precipitated by ammonium sulphate with 50-75%. Then the protein concentration was 4.7 mg/ml while the enzyme activity was 812 µmole/ml. Revealed that the protein concentration was 2.3 mg/ml and enzyme activity was 1020 µmole/ml. This revealed that the protein concentration was 0.9 mg/ml and the enzyme activity was 1669 µmole/ml.Conclusion:Lipase was purified to a considerable homogeneity and the characterization experiments revealed that the enzyme showed considerable heat stability and was optimally active at alkaline pH.Key words:Lipase, ion exchange chromatography, gel filtration chromatography, molecular weight.


Article
EXTRACTION AND PURIFICATION OF TWO OUTER MEMBRANE PROTEINS (PORINS) FROM KLEBSIELLA PNEUMONIAE LOCAL ISOLATE
استخلاص وتنقية بروتينات الغشاء الخارجي من عزله محليه لبكتريا الـ Klebsiella pneumoniae

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Abstract

Background: The porins are present in large amounts in the outer membrane of gram negative bacteria and form water-filled channels that permit the diffusion of small hydrophilic solutes across the outer membrane. Porins are generally divided into two classes: nonspecific porins (e.g., OmpC and OmpF), which permit the general diffusion of small polar molecules (600 Da), and specific porins (e.g., LamB), which facilitate the diffusion of specific substrates.Objective: To purify and characterize outer membrane proteins (porins) from a local isolate of Klebsiella pneumoniae. Materials and methods: An identified local isolate of Klebsiella pneumoniae was used as a primary source for the isolation and purification of porins. Outer membrane protein (porins) was purified and characterized and the contaminating lipopolysaccharides (LPS) were detected by thiobarbituric acid assay. Results: The final preparation contained porins in a concentration of 3.2 mg/ml. The results of electrophoretic separation revealed that porins appeared as two distinct bands with molecular weights of porins were estimated to be 35 and 36 kDa, respectively. Conclusions: Porins were expressed by the local isolate of Klebsiella pneumoniae with molecular weights highly similar to that of porins preparations produced by other gram negative bacteria and Klebsiella pneumoniae expressed two types of porins under standard laboratory conditions.Keywords: Porins, Thiobarbituric acid, Gel filtration chromatography, Ketodeoxyoctinate.

خلفيه الدراسة: توجد بروتينات الغشاء الخرجي ( البورينات) بكميات كبيره في الغشاء الخارجي للبكتريا السالبه لصبغة الكرام حيث تكون قنوات مملوءة بالماء تسمح بتنافذ الجزيئات المحبه للماء خلال الغشاء الخارجي للخلايا البكتيريه. تقسم البورينات بصوره عامه الى بورينات لا نوعيه (مثل OmpC , OmpF) التي تسمح بتنافذ الجزيئات القطبيه الصغيره (600 Da) وبورينات نوعيه (مثل LamB) والتي تسهل تنافذ مواد اساسيه لعمل الانزيمات.هدف الدراسه: تنقية وتوصيف بروتينات الغشاء الخارجي (البورينات) من عزله محليه من بكتريا ال Klebsiella pneumoniae .طريقة العمل: استعملت احدى عزلات بكتريا ال Klebsiella pneumoniae المحليه كمصدر لعزل وتنقية بروتينات الغشاء الخارجي. تم تنقية وتوصيف بروتينات الغشاء الخارجي وتم كذلك التحري عن وجود متعدد السكريد الشحمي في المستحضر النهائي باستخدام طريقة الكشف عن حامض الثايوباربجوريك.النتائــج: احتوى المستحضر النهائي على بورينات وبنسبة 3.2 mg/ml. واظهرت نتائج عملية الترحيل الكهربائي ظهور البورينات بشكل حزمتين ذواتي وزنين جزيئيين هما 35 kDa , 36 على التوالي.الاستنتاجات: تنتج العزله المحليه لبكتريا ال Klebsiella pneumoniae قيد الدراسه بورينات باوزان جزيئيه مشابهه لما تنتجه البكتريا الاخرى السالبه لصبغة الكرام وتنتج نفس العزله نوعين من البورينات تحت الضروف المختبريه القياسيه.مفتاح الكلمات: البورينات, حامض الثايوباربجوريك, كروموتوكرافيا الهلام, كيتودياوكستنيت.


Article
Purification and determination the molecular weight and study the kinetic properties of Glucose-6-phosphate dehydrogenase from Diabetic patients
دراسة الخصائص الحركية لإنزيم [G6PD] المستخلص والمنقى من مرضى السكري وتقدير الوزن الجزيئي له

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Abstract

This study was conducted to purifity Glucose 6-phosphate dehydrogenase from diabetic patients by gel filtration technique on Sephadex G100 and determine the molecular weight of enzyme, concentration and kinetic constant [Km, Vmax], study the effect of optimum temperature, substrate, pH. The study includes [60] patients with diabetes mellitus and [60] healthy individual as control. The activity of G6PD was measured after precipitated by [75%] Ammonium Sulfate concentration, using [Tris-HCl] buffer at pH 8.2. The specific activity was calculated [21.5UImg], total activity [706.8UI], number of purification [3.45] enzyme yield [23.188%] and enzyme activity [17.67UIml]. and the molecular weight were [57.82] k.D. The effect of substrate concentration was found to be increased with increasing substrate concentration. The optimum pH was found to be [8.4] and the optimum temperature was [38C]. Michaelis- Menten [Km] value for the enzyme was [3.33mM] and Vmax value was [0.263IUml ].

تنقية إنزيم [G6PD] من مرضى السكري باستخدام تقنية بسيطة هي الترشيح الهلامي بواسطة هلام السيفادكس G-100 وتقدير الوزن الجزيئي للأنزيم في المرضى ومقارنته مع الوزن الجزيئي ومقارنته مع النسبة الطبيعية للوزن الجزيئي وكذلك تقدير الثوابت الحركية للإنزيم وهي ثابت ميكاليس- منتن Kmوقيمة السرعة القصوى Vmax وتقدير قيم تركيز المادة الأساس ودرجة الحرارة والأس الهيدروجيني الأمثل للإنزيم. تتضمن الدراسة 60 عينة من مرضى السكري و60 عينة من الأصحاء حيث تم قياس الكلوكوز وفعالية الإنزيم في المصل وأظهرت النتائج ارتباط معنوي بين نقصان فعالية الإنزيم ومرض السكري وكذلك تم ترسيب الإنزيم باستخدام كبريتات الأمونيوم بنسبة إشباع [75%] ثم بعد ذلك تم تنقية الإنزيم بتقنية الترشيح الهلامي على هلام السيفادكس G-100 واعتمادا على الطريقة ذاتها تم تقدير الوزن الجزيئي للإنزيم. تم حساب الفعالية النوعية [21.5وحدة/ملغم والفعالية الكلية [706.8 وحدة] وعدد مرات تنقية بلغت 3.45مرة والحصيلة الإنزيمية [23.188%] والفعالية الإنزيمية [17.67وحدة/مل]. وتم قياس الوزن الجزيئي باستخدام نفس الطريقة وكان مقدار الوزن الجزيئي للإنزيم [57.82] كيلو دالتون وتم قياس تأثير زيادة تركيز المادة الأساس على فعالية الإنزيم فوجد إن فعالية الإنزيم تزداد بزيادة تركيز المادة الأساس إلى أن تبلغ مستوى ثابت تتوقف فيه الفعالية عن الزيادة مهما زاد تركيز المادة الأساس وأظهر الرسم البياني بينهما بشكل قطع زائد وتم دراسة تأثير الأس الهيدروجيني على الإنزيم فوجد إن الأس الهيدروجيني الأمثل هو [8.4] وتم دراسة تأثير درجة الحرارة على فعالية الإنزيم فوجد إن درجة الحرارة المثلى لعمل الإنزيم هي [38] وتم تقدير الثوابت الحركية فوجد إن قيمة ثابت ميكاليس – منتن هو [Km=3.33mM] وكانت قيمة السرعة القصوى [Vmax=0.263IUml] .

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