Cloning, expression, purification of the chitinase A gene region of serratia marcescens GBS19 isolation and investigation of insecticidal effect on agricultural pest Myzus persicae

It has many advantages such as the use of certain enzymes produced by microorganisms with the potential to be entomopathogenic for biological control and control purposes, and being safe for other living things apart from the targeted organism. This thesis study aimed to present the use of chitinase in biological control as a biological and environmentally friendly solution. The methods to be used in the thesis study carried out, the chiA gene region in the S. marcescens GBS19 genome was amplified by PCR using specific primers. The amplified gene region was cloned into the pBluescript II KS (+) of cloning vector and sequence analysis was performed. The chiA gene confirmed by sequence analysis, was cloned into the pET-22b (+) expression vector and the protein was expressed in Escherichia coli BL21(DE3). The expressed chiA protein was purified using his-taq affinity chromatography and visualized by SDS-PAGE. As expected, a protein band of 62.5 kDA was observed as a result of SDS-PAGE. Also, protein expression was confirmed using qPCR. The optimum temperature and pH levels for the chiA enzyme were examined. According to the results the temperature for the highest activity for the was found to be 40°C, and the pH was 5 level. After assessing the optimum conditions in which the enzyme works, its insecticidal activity on the agricultural pest Myzus persicae was investigated. The LD50 value of the pure chiA enzyme was found to be 15,804 ppm. By computional analysis using ligand- protein interaction, it was determined that the highest binding between the enzyme and chitin, which is the substrate, was -4.10 kcal/mol in addition to 8 different binding points. When the chiA enzyme was compared with the ref_seq chiA enzymes found in the NCBI database, it showed similarity to the closest chitinase with 98.93%.