Bir Bacillus clausii İzolatı Dört Farklı Pektinolitik Aktiviteye Sahip Görünmektedir

Bakteri kaynağı olarak Koyulhisar (Sivas, Türkiye) elma bahçesi toprak örnekleri kullanıldı. Elde edilen izolatlar tür düzeyinde kısmi protein- ve 16S rRNA gen dizisi homolojisi ile belirlendi. Hepsinin Bacillus clausii olduğu belirlendi. Bu izolatlar ile kültürler hazırlandı ve hücreler uzaklaştırıldıktan sonra üst sıvıdaki proteinler organik bir çözücü yardımı ile çöktürüldü. Pektinaz taraması yapılırken, mevcut bir proteazın diğer proteinleri sindirdiği fark edildi. Bu proteolitik aktivite, inkübasyon sıcaklığının 55oC'ye çıkarılmasıyla engellendi. Dört farklı pektinaz aktivitesi, poligalakturonaz, pektin liyaz, pektat liyaz ve pektin metilesteraz bundan sonra saptanabildi. SDS-PAGE'de görüntülenen ve bu aktivitelere karşılık gelen dört protein bandı, sırasıyla 80 kDa, 58 kDa, 46 kDa ve ~ 28 kDa idi. Enzimlerin tanımlanması için spektroskopik yöntemler kullanıldı. En küçük protein bandının (28 kDa) pektin metilesteraz olduğu bulundu. Bu enzimin Km ve Vmax değerleri sırasıyla 0.4 ve 217U idi.

Anahtar Kelimeler:

Bacillus clausii, pektat liyaz, pektin liyaz, pektin metilesteraz, poligalakturonaz

An Isolate of Bacillus clausii Appears to Possess Four Distinct Pectinolytic Activities

For the bacterial source, Koyulhisar (Sivas, Turkey) apple orchard soil samples were used. Obtained isolates were identified at the species level by partial protein- and 16S rRNA gene sequence homology All of them were determined to be Bacillus clausii. Cultures were prepared with these isolates and after removing the cells, the proteins in the upper fluid were precipitated with the help of an organic solvent. While performing pectinase screening, it was noticed that an existing protease digested other proteins. This proteolytic activity was eliminated by increasing the incubation temperature to 55oC. Four different pectinase activities, polygalacturonase, pectin lyase, pectate lyase, and pectin methylesterase, were then demonstrated. Four protein bands corresponding to these activities visualised by SDS-PAGE were 80 kDa, 58 kDa, 46 kDa and ~ 28 kDa, respectively. Spectroscopic methods were employed for the identification of the enzymes. The smallest protein band (28 kDa) was found to be pectin methylesterase. Km and Vmax values of this enzyme were 0.4 and 217U, respectively.

Keywords:

Bacillus clausii, pectate lyase, pectin lyase, pectin methylesterase, polygalacturonase,

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