Immunoaffinity layering of enzymes - Stabilization and use in flow injection analysis of glucose and hydrogen peroxide

A general procedure for the high yield immobilization of enzymes with the h elp of specific anti-enzyme antibodies is described. Polyclonal antibodies were raised against Aspergillus niger glucose oxidase and horseradish perox idase in rabbits and the gamma globulin (IgG) fraction from the immune sera isolated by ammonium sulphate fractionation followed by ion-exchange chrom atography. Immobilization of glucose oxidase and horseradish peroxidase was achieved by initially binding the enzymes to a Sepharose matrix coupled wi th IgG isolated from anti-(glucose oxidase) and anti-(horseradish peroxidas e) sera, respectively. This was followed by alternate incubation with the I gG and the enzyme to assemble layers of enzyme and antibody on the support. The immunoaffinity-layered preparations obtained thus were highly active a nd, after six binding cycles, the amount of enzyme immobilized could be rai sed about 25 times over that bound initially. It was also possible to assem ble layers of glucose oxidase using unfractionated antiserum in place of th e IgG. The bioaffinity-layered preparations of glucose oxidase and horserad ish peroxidase exhibited good enzyme activities and improved resistance to heat-induced inactivation. The sensitivity of a flow injection analysis sys tem for measuring glucose and hydrogen peroxide could be remarkably improve d using immunoaffinity-layered glucose oxidase and horseradish peroxidase. For the detection of glucose, a Clark-type oxygen electrode, constructed as a small flow-through cell integrated with a cartridge bearing immunoaffini ty-layered glucose oxidase was employed. The hydrogen peroxide concentratio n was analysed spectrophotometrically using a flow-through cell and the lay ered horseradish peroxidase packed into a cartridge. The immunoaffinity-lay ered enzymes could be conveniently solubilized at acid pH and fresh enzyme loaded onto the support. Immunoaffinity-layered glucose oxidase was success fully used for the on-line monitoring of the glucose concentration during t he cultivation of Streptomyces cerevisiae.