Investigation of the effect of D398S, I323V, I372Q/V and V380F mutations on the kinetic parameters and thermal stability of glucose isomerase from Anoxybacillus gonensis G2T

Glucose isomerase, known as xylose isomerase (E.C 5.3.1.5), catalyzes isomerization of glucose to fructose and found in many microorganisms. Until today, many GI' s from different thermophilic bacteria have been characterized and most of them have been used in industrial applications. In addition, a great deal of studies has been performed to improve kinetic and biochemical parameters of GI by bioengineering applications such as mutagenesis for using in industrial areas. GI of Anoxybacillus gonensis G2T was isolated as recombinant. According to the results, the G2T GI of Anoxybacillus gonensis was found to possess superior properties than the other GI' s being used today in terms of kinetic parameters. In this study, we aimed to improve biochemical and kinetic parameters of AgoGI with improved kinetic properties by genetic manipulations. To this end, I323V, I372V, I372Q, V380F, D398S site-specific mutations were created on AgoGI. In addition, successful I373V and V380F mutations were applied on GI with W137F/ V184S mutation with improved kinetic parameters. Kinetic parameters for W137F/ V184S/I372V were calculated as 64,37 ± 1,3 mM for Km, 99,05 ± 0,44 for Vmax, 82,62 1/sn for kcat and 1,283 for kcat/ Km. Kinetic parameters for W137F/ V184S/V380F were calculated as 116,57 ± 4,37 mM for Km, 37,22 ± 0,39 µmol/dk/mg protein for Vmax, 31,05 1/sn for kcat ve 0,266 for kcat/ Km.