The 1001 project titled “Identification, Biochemical Characterization and Biotechnological Applications of New Xylanase Enzymes with Different Extremophilic Properties Originating from Anatolian Water Buffalo Rumen through Sequence Homology Based Metagenomics Approach” with the number KBAG 121Z943, which was carried out by Dr. Yusuf Sürmeli from Tekirdağ Namık Kemal University, where Dr. Halil Kurt, a faculty member working in our center, is a researcher, was decided to be scientifically supported in the 2nd semester of 2021 within the scope of the Scientific and Technological Research Projects Support Program.
Lignocellulose represents a valuable source of undiscovered sustainable carbon and is predominantly composed of cellulose, hemicellulose and lignin. Hemicellulose, the second largest fraction of this renewable energy source, is mainly composed of the xylan structure. Xylanases are key enzymes involved in the degradation of xylan and cleave β-1,4-glycosidic bonds between xylopyranosyl residues in the xylan backbone. These enzymes belong to 17 glycoside hydrolase (GH) families including GH3, GH5, GH6, GH8, GH9, GH10, GH11, GH16, GH18, GH26, GH30, GH43, GH44, GH51, GH62, GH98 and GH141.
Xylanases are produced by a wide variety of organisms. Among them, fungal and bacterial xylanases are important because of their superior properties that can potentially be applied in industrial processes. These enzymes have attracted great interest in applications in biofuel, pulp, food and feed industries and are expected to be resistant to extreme conditions such as high temperature, acidic pH and lignocellulosic-derived inhibitor.
Xylanases have been reported from various sources, the most interesting of which is the rumen of ruminant animals. The rumen represents a potentially important source for classes of hydrolytic enzymes including bacterial and fungal xylanases. Çeşitli ruminant hayvanlarda yapılan rumen kaynaklı enzim çalışmaları sonucunda, farklı ekstremofilik özelliklere sahip ksilanaz enzimleri tanımlanmış ve karakterize edilmiştir.
Ksilanaz gibi lignoselülolitik enzimlerin geleneksel tanımlanması, mikroorganizmaların saf kültivasyonuna dayanmaktadır. Bu tanımlama ile rumen mikroorganizmalarının %85’inden fazlası kültüre edilemediği için çok sayıda fonksiyonel gen keşfedilmemiş olarak kalır. Bu nedenle, son zamanlarda, rumen gibi farklı ortamlardan ksilanazların da içinde olduğu yeni biyokatalizörlerin taranması, kültivasyondan bağımsız olan metagenomik yaklaşım ile mümkün hale gelmiştir. The metagenomic approach has been used to identify and characterize new extremophilic xylanases from Hu sheep, cow, Tibetan yak, cattle, camel rumen environments. However, to the best of our knowledge, no xylanase enzyme from Anatolian buffalo rumen has been studied so far.
The aim of this study was to identify different extremophilic xylanases from Anatolian buffalo rumen with thermostable, acidic pH stability and/or lignocellulosic-derived inhibitor stability through a sequence-based metagenomics approach, to perform their detailed biochemical characterization and to test their potential for use in three biotechnological applications for biofuel, animal feed and fruit juice industrial fields. For this purpose, Anatolian buffalo rumen metagenomic DNA will be sequenced by next generation sequencing and xylanase enzyme gene candidates will be identified. Xylanase enzyme gene candidates with different sequences will be cloned into expression vector, produced in E. coli BL21 (DE3), activity screening on agar, thermostability screening at crude protein level will be performed. Purification and detailed biochemical characterization of thermostable xylanase enzymes will be performed. The selected extremophilic xylanase enzymes with thermostable, acidic pH stability and/or lignocellulosic-derived inhibitor (ethanol and furfural) stability will be applied for biofuel, animal feed and fruit juice industrial applications. This study is unique because the identification and characterization of different extremophilic xylanase enzymes will be carried out for the first time using Anatolian buffalo rumen metagenome.
As a result of this study, we believe that different extremophilic xylanase enzymes with thermostable, acidic pH stability and/or lignocellulosic-derived inhibitor (ethanol and furfural) stability properties can be obtained and potentially used in biofuel, animal feed and fruit juice industrial processes. The outputs of this study will be able to meet the needs of these industries for these biotechnological processes. Thus, this study will contribute to the improvement of process efficiency in these three industrial areas.