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Journals / Türk Biyokimya Dergisi / 2015 / Cilt: 40 - Sayı: 4

Aspergillus niger C71 den geniş pH aralığında dengeli olan GH11 xylanaz klonlaması ve ekspresyonu

Cloning and expression of a broad pH stable GH11 xylanase from Aspergillus niger C71

Jian-Xin ZHANG
Jin-Feng SHAN
Hong MİNG
Dong-Ying SONG
Yan-Yan DUAN
Guo-Xing NİE
Pages
323–333
Publication Date
2022-11-05
DOI
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AI Summary & Key Takeaways

AI Insight
⚡ TL;DR(Too Long; Didn't Read)

The XYNB was purified by MonoQ an ion exchange chromatography and analyzed by SDS-PAGE.Results: The results showed that xynB is 678 bp in length, and encodes a 18 amino acid signal peptide as well as a 22 amino acid mature peptide with a calculated molecular weight of 24.127 kDa.

📌 3 Key Takeaways

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Objective & Hypothesis:Objective: To clone a full-length cDNA sequence of xynB, encoding endo-1,4-?-xylanase of Aspergillus niger C71 and express in Escherichia coli BL21(DE3).Methods: The xynB was cloned using rapid amplification of cDNA ends (RACE) methods.
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Key Findings & Data:The sequenced DNA was compared with the available sequences from GenBank using the BLASTX program, and the phylogenetic tree of the xylanases was then constructed using MEGA version 5.0.
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Conclusion & Impact:The XYNB was purified by MonoQ an ion exchange chromatography and analyzed by SDS-PAGE.Results: The results showed that xynB is 678 bp in length, and encodes a 18 amino acid signal peptide as well as a 22 amino acid mature peptide with a calculated molecular weight of 24.127 kDa.
🔬Design:Modeling & Simulation
📍Scope:Comprehensive Sample
⚙️Variables:Domain Parameters

Abstract

Objective: To clone a full-length cDNA sequence of xynB, encoding endo-1,4-?-xylanase of Aspergillus niger C71 and express in Escherichia coli BL21(DE3).Methods: The xynB was cloned using rapid amplification of cDNA ends (RACE) methods. The sequenced DNA was compared with the available sequences from GenBank using the BLASTX program, and the phylogenetic tree of the xylanases was then constructed using MEGA version 5.0. The amino acid sequences of XYNB were submitted to the ESyPred3D Web Server 1.0 for Homology modeling. The XYNB was purified by MonoQ an ion exchange chromatography and analyzed by SDS-PAGE.Results: The results showed that xynB is 678 bp in length, and encodes a 18 amino acid signal peptide as well as a 22 amino acid mature peptide with a calculated molecular weight of 24.127 kDa. Phylogenetic analysis of xynB, three-dimensional structure and overlap analysis of XYNB demonstrated that XYNB has a ?-jelly-roll architecture, which is more conversation in the catalytic domain of GH11 xylanases, belonging to the family 11 of glycosyl hydrolases. A maximum enzyme activity of 62,242.33 Uoml-1 was obtained from XYNB. The XYNB with MW of 41 kDa demonstrated a broad pH stability from pH 3.0 to pH 12. 0.5 mM of Fe2+ could greatly enhance activity of XYNB to 567.02%. The mainly hydrolysis products of beechwood xylan were xylobiose. The hydrolysis products of XYNB effecting on beechwood xylan were analysed by HPLC. Conclusion: The xynB had been successfully expressed in Escherichia coli BL21, with high enzyme activity and a broad pH stability, and it would be a very good application prospect as the feed enzyme.

Özet

Amaç: Aspergillus niger C71 in endo 1.4. - ? xylanaz xynB geninin tam uzunlukta cDNA sekansının klonlanması ve Escherichia. coli BL21(DE3) de ekspresyonu. Metod: xynB, cDNA uçlarının hızlı amplifikasyon (RACE) yöntemi kullanılarak klonlanmıştır. Elde edilen sekans DNA BLASTX programı kullanılarak GenBank içinde karşılaştırılmıştır. Xylanaz sekanslarının karşılaştırılmaları için filogenetik ağaç oluşturma MEGA 5.0 programı ile yapılmıştır. Türdeşlik araştırması, XYNB'nin amino asit sekansları ESyPred3D Web Server 1.0 ile yapılmıştır. XYNB iyon değiştirici kolon MonoQ ile saflaştırılmış ve SDS PAGE kromotografisi ile analiz edilmiştir.Bulgular: Elde edilen sonuçlara göre xynB 678 bp uzunluğundadır. Yapısal olarak 18 amino asitten oluşan sinyal peptiti ile 22 amino asitten oluşan olgun peptitten meydana gelmekte ve molekül ağırlığı 24.127kDa olarak ölçülmüştür. Enzimin (xynB) filiogenetik analizi , üç boyutlu yapısı ve örtüşme analizleri xynB'nin ?-jel rulo (jelly-roll) mimarisinde olduğunu, GH11 xylanaz ların katalitik bölgeleri ile ilişkili olduğunu dolayısı ile 11 glikosil hidrolazlar ailesinden olduğunu ortaya koymuştur. XYNB enziminden en fazla 62,242.33 Uoml-1 aktivite elde edilmiştir. Molekül ağırlığı 41kDa olan XYNB enziminin geniş bir pH aralığında (3.0 - 12.0) stabil olduğu saptanmıştır. 0.5 mM Fe2+ nin de XYNB enzim aktivitesini %567.02 arttırdığı bulunmuştur. Önemli hidroliz ürünü olan kayın ksilan ise ağırlıklı olarak ksilobiyolozdur. XYNB'nin hidroliz ürünlerinden kayın ksilazda HPLC ile analiz edilmiştir.Sonuç: xynB enzimi Escherichia. coli BL21 içinde, yüksek enzim aktivitesi ve geniş pH aralığında stabil olarak başarılı bir şekilde eksprese edilmiştir. Dolayısı ile besleme enzim olarak uygulamalarda kullanılabilecektir.

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Cite This Article

ZHANG, J., SHAN, J., MİNG, H., SONG, D., DUAN, Y., NİE, G. (2022). Aspergillus niger C71 den geniş pH aralığında dengeli olan GH11 xylanaz klonlaması ve ekspresyonu. Türk Biyokimya Dergisi, Cilt: 40 - Sayı: 4, 323–333. https://ajindex.com/articles/621091

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