Abstract: A high activity immobilized enzyme composite is prepared by covalently bonding a second enzyme layer to a first enzyme layer that is immobilized to a carrier. The carrier is preferably silica gel which has been activated by treatment with a strong base followed by treatment with a strong acid. The first enzyme layer is covalently bonded to the activated silica gel with an aminosilane and a polyfunctional reactant, and the second enzyme layer is covalently bonded to the first layer with a polyfunctional reactant. Third, fourth and more successive enzyme layers may be covalently bonded. The composite has high activity per unit volume, superior stability and good half-life.

Abstract: An improved immobilized enzyme composite is prepared by subjecting a siliceous carrier to an initial treatment with alkali, then acid, then reacting the carrier with an organosilane. The organosilane may then be coupled through a covalent coupling agent with an enzyme. The alkali-acid treatment provides an exceptionally high number of hydroxyl groups per unit volume, so that the treated carrier is especially useful in the preparation of high performance immobilized enzyme compositions, particularly by multi-layering immobilization, providing a very high amount of enzyme activity per unit volume. Multi-layering is accomplished by successive steps of bonding a difunctional covalent coupling agent to a previously immobilized enzyme layer, then bonding an additional layer of enzyme to the coupling agent. This may be repeated to apply as many layers as desired.