The preparation of an immobilised glucose isomerase. I. The production and selected properties of the partially-purified enzyme from Lactobacillus brevis

A strain of Lactobacillus brevis producing glucose isomerase was grown in a xylose-containing medium. Production of intracellular glucose isomerase was four times that previously reported. Methods for the release of enzyme were compared, and a process for the production of an enzyme concentrate proposed involving (i) heat autolysis of the cells at 40 °C for 8 h; (ii) precipitation of nucleic acids with manganese chloride; (iii) precipitation of enzyme with ammonium sulphate, collecting the fraction precipitated between 60 and 85% saturation, and (iv) heat treatment of the dissolved enzyme at 55 °C in the presence of Co²⁺ for 10 min. Enzyme concentrates prepared using steps (i) to (iii) only showed considerable competing activity, and produced sigmoid kinetics. The use of enzymes prepared using the full method showed that substrate inhibition was apparent at glucose concentrations above approximately 30% w/v. The apparent K_M value was calculated as 10 to 12% w/v (glucose), based on the definition of K_M as K_M= S_o at V_o - V_max(observed)/2. The equilibrium concentrations of fructose and glucose were 62 and 38% respectively, and were independent of temperature between 35 and 60 °C.