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انتاج وتنقية اللكتين من بكتريا Acinetobacter baumannii المعزولة من القشع وتاثيره في بعض الاحياء المجهرية خارج الجسم الحي
Author name:
رونق مهدي هاشم
Supervisor name:
ساهرة نصيف مسلم
General topic:
Biology
Specific topic:
Microbiology - Bacteria
Degree:
Master
University:
Mustansiriyah University - College Of Science
Language:
Arabic
University location:
Baghdad
First pages:
24T3264 - p.pdf
Abstract:
In order to isolate Acinetobacter baumannii , 104 samples of sputum were collected from patients infected with respiratory tract infections, from Teaching Baghdad Hospital / Medical City, General Teaching Al - Kindi hospital and Al - Imam Ali Hospital . Depending on microscopic , cultural and biochemical tests ,23 isolates belonge to it were obtained.The ability of A.baumannii isolates to produce lectin was detected by using glass slide and microtiteration plates methods and the results showed that all the isolates were able to produce lectin , A.baumannii S12 was the best isolate for produce it and the blood group O was the best for detection the titration of agglutination activity.The optimum conditions for lectin production were determined and the results showed that the cultivation of A.baumannii S12 in Colonization Factor Antigen medium with pH value of 7.2 at 37 ºC for 24 hours in shaker incubator at 100 rpm / min. were the best to produce it. The lectin was extracted from cilia of A.baumannii S12 isolate by using six different breaking methods at different times , the glass beads method for 50 minutes was the best to extract it, the agglutination activity reached to 512 AU / ml with specific activity 174.14 AU / mg protein in comparison with other breaking methods. Lectin was purified by three steps, included ; precipitation by 35% saturation ammonium sulfate , ion exchange chromatography by DEAE - cellulose, then gel filtration chromatography by Sephadex G - 150. The final lectin recovery was 36.92% with 20.63 folds of purification and 3592 U / mg protein of specific activity .The purified lectin was also partially characterized and the results showed that the optimal activity of the lectin was at 30 ºC and its thermostability was between (10 and 30) ºC ,the optimum pH for lectin activity was 7.2 while the pH value for its stability was between 7 and 7.2 . Lectin retained whole activity when treated with sucrose, raffinose, maltose, mannitol, fructose, lactose, arabinose, ribose and rhamminose, while it lost whole activity when treated with glucose , mannose and galactose. Also, lectin retained whole activity when treated with EDTA - NaN3 and trypsin , whereas its agglutination activity was reduced to the half when treated with pepsin , and to 12.5% and 0.39% when treated with 2 - mercaptoethanol and formalin, respectively. In addition, the effect of some substances on lectin stability was studied and the results showed that the mixture of 2% glycerol and physiological normal saline followed by 20% ammonium sulfate were the best for its stability for 20 and 10 days, respectively. while the solutions of the rest materials(Tween - 80, mannitol, sucrose, lactose and SDS) showed inhibitory effect on lectin activity.Also, the antimicrobial activity of purified lectin against 12 bacterial and fungal isolates was studied and the results showed that the lectin had antibacterial activity against Klebsiella pneumonia , Pseudomonas aeruginosa and Proteus mirabilis with (50 , 38.88 and 36.84 )% of inhibition, respectively, and antifungal activity against Penicillium spp. , Cladosporium spp. and Fusarium spp. with (33 , 18.75 and 17.39 )% of inhibition, respectively. whereas ,it had no antimicrobial activity against the bacteria; Escherichia coli , Enterobacter cloacae ,and Staphylococcus aureus ,and the fungi ; Aspergilus niger, Aspergilus terius , and Candida albicans.
