Xylose/Glucose isomerase is an important industrial enzyme, which is being used for the conversion of glucose into fructose for decades. This research focuses on the production of glucose isomerase from Escherichia coli DH5¿. Fermentation optimization studies showed that higher amount of yeast extract and tryptone increased the cell growth and the optimum temperature for growth was 37°C. Partial purification of xylose isomerase was carried out using heat treatment and ammonium sulphate precipitation. 80% of the salt cut off value produced maximum activity for the enzyme concentrate. Xylose isomerase showed maximal activities at 75 °C temperature and pH 8. Enzyme activity at optimum conditions was 5.0 U/min/ml for E. coli DH5¿. Addition of cobalt ions and xylose enhanced the activity from 10-20 folds. The specific activity for E. coli DH5¿ was 60.5 nmol/min/mg of protein. Only 8% of the maximum D-fructose syrup was produced using 100mM of D-glucose as substrate after 14 days of incubation. Thereby, E. coli DH5¿ is not a better candidate for the production of High Fructose Syrup.