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Bacillus pumilus Y7 Katalaz (katX2) Geninin Klonlanması ve Açıklatılması

Katalazlar antioksidan metalloenzimler sınıfına dahil olup başlıca işlevleri hidrojen peroksidin (H2O2) suve moleküler oksijene parçalanmasıdır. Temel fonksiyonlarının yanında düşük seviyede peroksidazve(ya) oksidaz aktivitesi göstererek izoniazid (antitüberküloz ilacı) sentezi gibi tıbbi öneme sahip bazıbileşiklerin sentezini destekledikleri bilinmektedir. Yakın bir geçmişte Bacillus pumilus tarafındanüretilen 'Fe/hem' grubu içeren bir enzimin katalaz, peroksidaz ve penisilin oksidaz aktiviteleri gösterdiğiyayınlanmıştır. Bunun üzerine bu enzimin hem diğer katalaz, peroksidaz ve katalaz-peroksidazlar ilekarşılaştırılması hem de ikincil peroksidaz/oksidaz özelliğinin ayrıntılı olarak irdelenebilmesi için enzimikodlayan katX2 geninin klonlanması ve Escherichia coli’de üretimi gerçekleştirilmiştir. Restriksiyonenzimlerinden bağımsız gerçekleştirilen klonlama tekniğinde ilk olarak B. pumilus Y7’den elde edilenkatX2 geni kimerik primerler (5'katX2; 3'katX2) ile bir araya getirilerek megaprimer oluşturulmuş vesonrasında bu megaprimer ekspresyon vektörüne başarılı bir şekilde aktarılmıştır. 510 amino asittenoluşan olgun proteini kodlayan katX2 genini taşıyan pET28aTEV vektörü E. coli BL21 (DE3 star)hücrelerine transforme edilmiştir. Transformantlarda katalaz aktivitesi için en yüksek değer (8000 µmolmg-1 dak-1) 30°C’de, IPTG (0.1 mM) varlığında, 120 rpm çalkalama hızında 24 saat büyütüldüğüekspresyon koşullarında ulaşılmıştır.

Cloning and Expression of Catalase Gene (katX2) from Bacillus pumilus Y7

Catalases belonging to a class of antioxidant metalloenzymes function to decompose hydrogen peroxide (H2O2) to dioxygen and water. Besides their major activity, they are known to support the synthesis of certain compounds of medicinal important such as isoniazid (antituberculosis agent) with their peroxidase and/or oxidase activity at low level. Recently, it has been published that a Fe/heme containing enzyme produced by Bacillus pumilus presents catalase, peroxidase and penicillin oxidase activities. This report led us to investigate the enzyme for comparison with other catalase, peroxidase and catalase peroxidases and for elaboration its secondary peroxidase/oxidase activity. For this purpose, catalase encoding gene (katX2) obtained from B. pumilus Y7 was cloned by restriction free cloning technique and expressed in Escherichia coli. In this technique, the megaprimer was produced by assembling the katX2 with the chimeric primers (5’katX2; 3’katX2) and followed by its integration into the expression vector. Then, the pET28aTEV vector carrying the katX2 gene that encodes mature protein consisting of 510 amino acids was transformed into E. coli BL21 (DE3 Star) cells. Catalase activity of the transformants reached at their highest level (8000 μmole mg-1 min-1 ) when cells were expressed at 30°C and 120 rpm for 24 hours in the presence of IPTG (0.1 mM).

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