Cloning of thermostable xylanase genes from anatolian water buffalo rumen metagenome

Xylanases are a group of enzymes that degrade xylan. These enzymes are examined according to their usage in different sectors such as feed and animal husbandry, pulp bleaching and bioconversion of agricultural wastes. No xylanase enzyme study has been conducted so far from Anatolian buffalo rumen. In this study, the cloning of thermostable xylanase genes from Anatolian buffalo rumen was performed. For this purpose, two different possible thermostable xylanase enzyme genes (M68 and E65) were selected using metagenomic DNA sequences from Anatolian buffalo rumen and these genes were first cloned into the cloning vector (pJET 1.2 blunt) and then into the expression vector (pET28a(+)). According to the BLASTp analysis result, the amino acid sequence of M68 xylanase enzyme was closest to the gene sequence of xylanase seen in bacteria in the Bacteroidales order, while E65 xylanase enzyme showed the highest level of similarity to the xylanase enzyme produced by Clostridia species. As a result of this study, it was suggested that M68 and E65 xylanase enzymes have potential for use in various industrial areas. PCR analyses showed that the annealing temperatures of the designed primers against the M68 and E65 genes were 60oC and 54oC, respectively. In addition, as a result of these analyses, it was determined that the M68 gene was slightly larger than 1500 bp, while the E65 gene was between 1000-1500 bp. Colony PCR and restriction enzyme digest analyses showed that both genes were successfully cloned. Agar activity results showed that clones carrying the recombinant E65 enzyme degraded xylan and had xylanase enzyme activity. Thermostability analysis showed that the E65 enzyme maintained its activity at 70oC for 120 minutes.