Purification, characterization and biotechnological applications of chitinase enzyme from Stenotrophomonas maltophilia

Within the scope of this thesis, the chitinase activities of the bacterial isolates in our culture collection were investigated and the isolate with the best chitinase activity was determined and identified by classical (morphological, physiological, biochemical) and molecular (16S rRNA sequencing) methods. Chitinase enzyme was purified from the isolate identified as Stenotrophomonas maltophilia (Genbank No: MW600524) by ammonium sulphate precipitation and ion exchange chromatography. Optimum temperature and pH, Vmax and Km values, and temperature and pH stability of the purified enzyme were determined. The effects of various metal ions (Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Mg2+, Mn2+, Zn2+), reagents (%1-5 EDTA, H2O2, SDS, Tween 20 andTween 80) andsolvents (%5-15 DMSO, acheton, glycerol, ethanol, isopropanol) on enzyme activity were investigated. The effect of purified chitinase enzyme against major agricultural diseases and pests was investigated in order to determine its usability as antifungal and bioinsecticide. According to the results of the studies, the chitinase enzyme was purified 1,4 times at 30% ammonium sulphate concentration with a yield of 40,7%. Following partial purification, the enzyme was purified by ion exchange chromatography technique using HiPrep Q XL 16/10 column and HiPrep ™ 26/10 desalting column with 25,34% and 18,12% yields, respectively. The molecular weight of the purified enzyme was calculated as 52 kDa by SDS-PAGE method. The optimum temperature of the enzyme was determined to be 50 °C and the optimum pH value as 7,0. While Fe2+, Cd2+ and Mn2+ ions at 1 mM concentration and Mg2+ and Fe2+ ions at 5 mM concentration increased enzyme activity, all the tested reagents and solvents decreased the enzyme activity. Vmax and Km values of the enzyme for colloidal chitin were calculated as 1.07 µmol.ml-1.dk-1 and 0,6 mg/ml, respectively. Purified enzyme negatively affected the germination and hyphal development of Fusarium oxysporium, and caused deterioration in the chitin structure of the Leptinotarsa decemlineata insect. e Keywords: Stenotrophomanas maltophilia, chitinase, enzyme purification, antifungalactivity, bioinsecticide