İntraselüler Anoxybacillus amyloliticus ksilanaz’ının klonlanması, saflaştırılması ve karakterizasyonu

Hakan Karaoğlu; Züleyha Akpınar

doi:10.35229/jaes.1315724

EN TR

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

Abstract

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.

Keywords

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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Ayrıntılar

Birincil Dil

Türkçe

Konular

Gen İfadesi

Bölüm

Araştırma Makalesi

Yazarlar

Hakan Karaoğlu ^*
0000-0003-4615-1157
Türkiye

Züleyha Akpınar
0000-0003-0102-6651
Türkiye

Erken Görünüm Tarihi

15 Eylül 2023

Yayımlanma Tarihi

30 Eylül 2023

Gönderilme Tarihi

16 Haziran 2023

Kabul Tarihi

26 Ağustos 2023

Yayımlandığı Sayı

Yıl 2023 Cilt: 8 Sayı: 3

DOI

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

IZ

https://izlik.org/JA86WJ89BY

APA

Karaoğlu, H., & Akpınar, Z. (2023). İntraselüler Anoxybacillus amyloliticus ksilanaz’ının klonlanması, saflaştırılması ve karakterizasyonu. Journal of Anatolian Environmental and Animal Sciences, 8(3), 367-373. https://doi.org/10.35229/jaes.1315724

JAES, 2016- 11.yıl

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

Abstract

Keywords

İntraselüler Anoxybacillus amyloliticus ksilanaz’ının klonlanması, saflaştırılması ve karakterizasyonu

Öz

Anahtar Kelimeler

Kaynakça

Ayrıntılar

Birincil Dil

Konular

Bölüm

Yazarlar

Erken Görünüm Tarihi

Yayımlanma Tarihi

Gönderilme Tarihi

Kabul Tarihi

Yayımlandığı Sayı

DOI

IZ

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