Cloning of cellulase genes from Thermobifida fusca EFTS 7A which is a thermophylic actinomyces and production of its recombinant protein

In this thesis, an isolate obtained from a mushroom farm compost situated in Izmir Eski Foca was used. The extracellular cellulase and xylanase production capacities of these isolates, were determined with methods such as Congo Red and Gram's Iodine in modified Thermoactinomyces medium. Identification of the microorganism was done by its 16S rRNA gene sequencing and it was named Thermobifida fusca EFTS 7A according to the GeneBank sequence comparison studies. Activities of extracellular cellulase, beta glucocidase and xylanase were measured spectrophotometrically. The optimum activity conditions of enzymes were designated by experimenting different carbon, organic, inorganic nitrogen sources and various pH areas. Under optimum conditions, the highest cellulase activity within the presence of avicel on 7th day measured as 230,6 U/L and the highest xylanase activity within the presence of xylan on the 7th day measured as 1350,6 U/L. The highest β-glucosidase activity has been determined as 203,3 U/L, in an environment that contains glycerol and cellobiose. The cellulolytic gene region to be cloned is designated based on the Thermobifida fusca YX actinomycete which its genome was sequenced. The depicted region tfu_0901(cel5A) is an endoglucanase. A primer couple is designed accordingly to be used in the kit which was used in cloning processes. This gene region was isolated via PCR and was purified by extracting from gel. This purified PCR product was sent to DNA sequence analysis and the sequences were compared to sequences in databases so that the region was proven the significant region of PCR. The gene region cel5A and pKLAC2 vector were cut off with restriction enzymes and both products were purified again. The PCR product was transferred to pKLAC2 vector, by performing ligation process. In order to form linear expression cassette, the pKLAC2 vector was cut by BstXI enzyme. The linear expression cassette was transformed to the compotent Kluyveromyces lactis GG79 cells. The recombinant cellulase activity measurements performed via DNS methodology were calculated as 50,4 U/L on 2nd day and 44,7 U/L on 3rd day. Keywords: Thermophilic actinomyces, cellulase, primers, PCR, restriction enzymes and cloning