Cloning, purification, and characterization of intracellular Anoxybacillus amyloliticus’ xylanase

Yıl 2023, , 367 - 373, 30.09.2023

Hakan Karaoğlu , Züleyha Akpınar

https://doi.org/10.35229/jaes.1315724

Öz

Xylanases [endo-1,4-β-D-ksilanaz (E.C. 3.2.1.8)] are extremely important enzymes used in many industrial applications where xylan needs to be processed with an environmentally friendly method. In this study, the gene of the Anoxybacillus amyloliticus’ intracellular xylana was expressed by cloning into the pET28a+ vector. Then, the enzyme (AnaXYN329) was purified, and its biochemical and kinetic properties were revealed. According to the obtained results, AnaXYN329 exhibited the optimum activity at pH 6.5, and 60 and 65 °C. Thermal stability experiments of AnaXYN329 maintained approximately 120 minutes and the enzyme activity was halved at the end of the 40th minutes. In addition, values of Km, Vmax, kcat, kcat/Km were determined as 3.631 ± 0.162 µg/µL, 100.05 ± 0.00292 µmoL/min/mg protein, 128.978 1/sec, and 35.514, respectively. Consequently, AnaXYN329 has potential to be used in industrial applications.

Anahtar Kelimeler

Thermophilic, Anoxybacillus amyloliticus, xylanase, expression

İntraselüler Anoxybacillus amyloliticus ksilanaz’ının klonlanması, saflaştırılması ve karakterizasyonu

Öz

Ksilanazlar [endo-1,4-β-D-ksilanaz (E.C. 3.2.1.8)] ksilanın çevre dostu bir yöntemle işlenmesine ihtiyaç duyulan endüstriyel birçok uygulamada kullanılan son derece önemli enzimlerdir. Bu çalışmada Anoxybacillus amyloliticus bakterisine ait intraselüler ksilanaz geni pET28a+ vektörüne klonlanarak üretilmiştir. Daha sonra enzim (AnaXYN329) saflaştırılarak enzimin sahip olduğu biyokimyasal ve kinetik özellikler açığa çıkarılmıştır. Elde edilen sonuçlara göre, AnaXYN329 en yüksek aktiviteyi pH 6.5’da; 60 ve 65 °C’de, göstermiştir. AnaXYN329 enziminin ısıl kararlılık deneyleri yaklaşık 120 dakika sürmüştür ve enzim yarılanma ömrünü 40. dakikada tamamlamıştır. Ayrıca enzimin Km değeri 3.631 ± 0.162 µg/µL, Vmax değeri 100.05 ± 0.00292 µmoL/dk/mg protein, kcat değeri 128,978 1/sn, kcat/Km değeri ise 35.514 olarak hesaplandı. Sonuç olarak AnaXYN329, endüstriyel uygulamalarda kullanılma potansiyeline sahiptir.

Anahtar Kelimeler

Termofilik, Anoxybacillus amyloliticus, ksilanaz, ekspresyon

Kaynakça

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