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    Anadolu manda rumen metagenom kaynaklı termostabil ksilanaz genlerinin klonlanması
    (Tekirdağ Namık Kemal Üniversitesi, 2024) Albayrak, Ebru; Velioğlu, Hasan Murat; Sürmeli, Yusuf
    Ksilanazlar, ksilanı parçalayan enzim grubudur. Bu enzimler yem ve hayvancılık, küspenin ağartılması ve tarımsal atıkların biyodönüşümü gibi farklı sektörlerdeki kullanımlarına göre incelenir. Şimdiye kadar Anadolu manda rumen kaynaklı ksilanaz enzim çalışması yapılmamıştır. Bu çalışmada Anadolu manda rumen kaynaklı termostabil ksilanaz genlerinin klonlanmasını gerçekleştirilmiştir. Bu amaçla, Anadolu manda rumen kaynaklı metagenomik DNA dizileri kullanılarak iki ayrı muhtemel termostabil ksilanaz enzim geni (M68 ve E65) seçilmiştir ve bu genler, önce klonlama vektörüne (pJET 1.2 blunt), ardından ekspresyon vektörüne (pET28a(+)) klonlanmıştır. BLASTp analiz sonucuna göre, M68 ksilanaz enziminin amino asit dizisi, Bacteroidales takımında yer alan bakterilerde görülen ksilanazın gen dizisine en yakın, E65 ksilanaz enziminin ise, Clostridia türleri tarafından üretilen ksilanaz enzimine en yüksek düzeyde benzerlik gösterdiği görülmüştür. Bu çalışma sonucunda M68 ve E65 ksilanaz enzimlerinin, çeşitli endüstriyel alanlarda kullanım potansiyeline sahip oldukları önerilmiştir. PCR analizleri, tasarlanan primerlerin M68 ve E65 genlerine karşı bağlanma sıcaklıklarının, sırasıyla, 60oC ve 54oC olduğunu göstermiştir. Ayrıca, bu analizler sonucunda, M68 geninin 1500 bç'den biraz daha büyük olduğu, E65 geninin ise 1000-1500 bç arasında olduğu tespit edilmiştir. Koloni PCR ve restriksiyon enzim kesimi analizleriyle, her iki genin başarılı bir şekilde klonlandığını göstermiştir. Agar aktivite sonucu, rekombinant E65 enzimini taşıyan klonların ksilanı parçaladıklarını ve ksilanaz enzim aktivitesinin varlığını göstermiştir. Termostabilite analizi E65 enziminin 120 dakika boyunca 70oC'de aktivitesini koruduğunu göstermiştir.
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    Discovery and In Silico Characterization of Anatolian Water Buffalo Rumen-Derived Bacterial Thermostable Xylanases: A Sequence-Based Metagenomic Approach
    (American Chemical Society, 2025) Kurt, Halil; Sever Kaya, Dilek; Akçok, İsmail; Sarı, Ceyhun; Albayrak, Ebru; Velioğlu, Hasan Murat; Şamlı, Hasan Ersin
    This study involved shotgun sequencing of rumen metagenomes from three Anatolian water buffalos, an exploration of the relationship between microbial flora and xylanases, and in silico analyses of thermostable xylanases, focusing on their sequence, structure, and dynamic properties. For this purpose, the rumen metagenome of three Anatolian water buffalos was sequenced and bioinformatically analyzed to determine microbial diversity and full-length xylanases. Analyses of BLAST, biophysicochemical characteristics, phylogenetic tree, and multiple sequence alignment were performed with Blastp, ProtParam, MEGA11 software, and Clustal Omega, respectively. Three-dimensional homology models of three xylanases (AWBRMetXyn5, AWBRMetXyn10, and AWBRMetXyn19) were constructed by SWISS-MODEL and validated by ProSA, ProCheck, and Verify3D. Also, their 3D models were structurally analyzed by PyMOL, BANΔIT, thermostability predictor, What If, and Protein Interaction Calculator (PIC) software. Protein-ligand interactions were examined by docking and MD simulation. Shotgun sequence and Blastp analyses showed that Clostridium (Clostridiales bacterial order), Ruminococcus (Oscillospiraceae bacterial family), Prevotella (Bacteroidales bacterial order), and Butyrivibrio (Lachnospiraceae bacterial family) were found as dominant potential xylanase-producer genera in three rumen samples. Furthermore, the biophysicochemical analysis indicated that three xylanases exhibited an aliphatic index above 80, an instability index below 40, and melting temperatures (Tm) surpassing 65 °C. Phylogenetic analysis placed three xylanases within the GH10 family, clustering them with thermophilic xylanases, while homology modeling identified the optimal template as a xylanase from a thermophilic bacterium. The structural analysis indicated that three xylanases possessed the number of salt bridges, hydrophobic interactions, and Tm score higher than 50, 165, and 70 °C, respectively; however, the reference thermophilic XynAS9 had 43, 145, and 54.41 °C, respectively. BANΔIT analysis revealed that three xylanases exhibited lower B′-factor values in the β3-α1 loop/short-helix at the N-terminal site compared to the reference thermophilic XynAS9. In contrast, six residues (G79, M123, D150, T199, A329, and G377) possessed higher B′-factor values in AWBRMetXyn5 and their aligned positions in AWBRMetXyn10 and AWBRMetXyn19, relative to XynAS9 including Gln, Glu, Ile, Lys, Ser, and Val at these positions, respectively. MD simulation results showed that the β9-η5 loop including catalytic nucleophile glutamic acid in the RMSF plot of three xylanases had a higher fluctuation than the aligned region in XynAS9. The distance analysis from the MD simulation showed that the nucleophile residue in AWBRMetXyn5 and AWBRMetXyn10 remained closer to the ligand throughout the simulation compared with XynAS9 and AWBRMetXyn19. The most notable difference between AWBRMetXyn5 and AWBRMetXyn10 was the increased amino acid fluctuations in two specific regions, the η3 short-helix and the η3-α3 loop, despite a minimal sequence difference of only 1.24%, which included three key amino acid variations (N345, N396, and T397 in AWBRMetXyn5; D345, K396, and A397 in AWBRMetXyn10). Thus, this study provided computational insights into xylanase function and thermostability, which could inform future protein engineering efforts. Additionally, three xylanases, especially AWBRMetXyn5, are promising candidates for various high-temperature industrial applications. In a forthcoming study, three xylanases will be experimentally characterized and considered for potential industrial applications. In addition, the amino acid substitutions (G79Q, M123E, D150I, T199K, A329S, and G377V) and the residues in the β3-α1 loop will be targeted for thermostability improvement of AWBRMetXyn5. The amino acids (N345, N396, and T397) and the residues on the β9-η5 loop, η3 short-helix, and η3-α3 loop will also be focused on development of the catalytic efficiency. © 2025 The Authors. Published by American Chemical Society.