Studies on the combined action of amylases and glucose isomerase on starch and its hydrolyzate. Part I. Production, extraction, purification and kinetic behavior of glucose isomerase.

Abstract

Glucose isomerase was produced from Streptomyces phaeochromogenes by aerobic fermentation at 28 degrees C for 24 hrs. The crude enzyme was obtained by disintegrating the harvested cells. It was found that ammonium sulphate at a saturation of 0.3-0.5 gave the maximum enzyme recovery (88.8%) from the crude extract, while acetone gave 66.2% at a concentration of 3/1 (V/V). On this basis the crude enzyme extract was purified following several steps as concentration, dialysis, precipitation with (NH4)2 SO4, then passing through column of Amberlite CG-50, and the eluate was treated with acetone to precipitate the enzyme. The kinetics behavior was studied and it was found that: optimum D-glucose concentration was 0.8 M, Km was 0.25 M, optimum pH was 7.0 and temperature was 70 degrees C. Magnesium at concentration of 0.07 M gave the maximum activity and its Km was 0.024 M. Antagonistic effects of Na+, Ca++ and Fe+++ in presence of 0.07 M of Mg++ were studied. Km and Vmax at different levels of Mg++ concentration were determined and no change in Km value was observed, while Vmax was affected. These findings indicate that the Mg++ combined with enzyme independently of the substrate.