Improvement thermal stability of glucose isomerase from Anoxybacillus gonensis g2t by site directed mutagenesis K111R-L313I-V101R-A353G/F/W and T428E/S

Glucose isomerase is an intracellular enzyme that can be produced by thermophilic and mesophilic bacteria and various fungus. Even though the enzyme is named as xylose isomerase it is commonly known as glucose isomerase since it can catalyze the isomerization of glucose to fructose and it is mainly used in the production of fructose syrup or HFCS. This enzyme has great industrial importance for both ethanol production from hemicellulose and for being used in the production of fructose syrup or HFCS (high fructose corn syrup). In this study, we aim to improve the thermal stability of A.gonensis G2T glucose isomerase with previously improved kinetic properties by genetic manipulations. To this end, we tried to improve the thermal stability of the wild-type enzyme by using V101R, K111R, L313I, A353G, A353F, A353W, T428S and T428E site-specific mutations. V101R, K111R mutations increased the half-life of the wild-type to 31 and 37 days respectively from 19 days. These mutations were used together to create V101R – K111R double mutation. The double mutation decreased thermal stability of the enzyme to 11,5 days from 19 days. Moreover, V101R, K111R mutations were repeated in W137F –V184S mutant enzyme that has 9 times more catalytic efficiency. These trials resulted in an enzyme with W137F – V184S – K111R triple mutation which have low Km, high Vmax and catalytic efficiency.