Galactose Oxidase: Applications of the covalently immobilized enzyme in a packed Bed configuration

Abstract

Galactose oxidase (E.C. 1.1.3.9) was covalently immobilized to chemically modified porous silica particles by reaction of the native enzyme with pendant benzoyl azide groups on the carrier. The enzyme loading on the carrier was 100-150 units per milliliter. The immobilized enzyme was incorporated into a hardware assembly suitable for the determination of galactose or lactose concentrations in complex biological fluids. The prototype instrument as described is suitable for continuous, on-line monitoring or discrete sample analysis. Reaction conditions can be readily provided which maintain global first order kinetics within the reactor and strict linearity of the procedure over a wide range of sample concentrations. Auto-inactivation of the immobilized enzyme can be prevented by K3Fe(CN)6 and long-term reactor stability can be achieved by the periodic application of the reagent to the enzyme reactor in situ.

Simultaneous determination of glucose, fructose, and sucrose in mixtures by amperometric flow injection analysis with immobilized enzyme reactors

“…While a number of chemically modified electrodes (CMEs) (1) have proved suitable as sensors in voltammetric determinations of trace metals (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16), their utility in practical analysis is largely unexplored. In our laboratories we are currently investigating the potentialities of CME-based procedures in such situations and have recently constructed CMEs with specific chemical reactivity toward nickel(II) (14) and copper(I) (15). In this paper we report on the results obtained by improvements of a crude but promising CME technique for quantitation of nickel(II).…”

“…For the determination of sugars, a large 1 Present address: Meiji Milk Products Co., Ltd., Kyobashi, Chuo-ku, Tokyo 104, Japan. number of sensing systems have been developed that use various kinds of membranes and sensor arrangements, such as glucose (3)(4)(5)(6)(7)(8)(9)(10), fructose (11,12), galactose (6,13-15), sucrose (5, 7, 9,14,16-21), lactose (14)(15)(16)22), and maltose (5,14,16,23).…”

Selective Oxidation of 5‐Hydroxymethylfurfural to 2,5‐Diformylfuran in Biphasic Media using Immobilized Galactose Oxidase: Proof of Concept and Limitations

“…[24] Immobilization of GalOx on chemically-modified porous silica beads enabled its use in a packed bed reactor. [25] Concerning biorefinery-oriented approaches, GalOx was encapsulated using Cu (II) and Zr (IV) to form nanoflowers, which were then used in the oxidation of HMF. [26] Nanoflower matrices based on Cu 3 (PO 4 ) 2 were also used to co-immobilize galactose oxidase, horseradish peroxidase, and catalase, to obtain a more active and inhibition-resistant biocatalyst for oxidation of HMF to DFF.…”

“…GalOx immobilized on mesoporous silica particles displayed increased thermal resistance to 60 °C [24] . Immobilization of GalOx on chemically‐modified porous silica beads enabled its use in a packed bed reactor [25] . Concerning biorefinery‐oriented approaches, GalOx was encapsulated using Cu (II) and Zr (IV) to form nanoflowers, which were then used in the oxidation of HMF [26] .…”

Coulometric flow analyzer for use with immobilized enzyme reactors

“…Potentiometric (17) and amperometric (18,19) electrodes have many virtues as detectors for immobilized enzyme reactors. These devices generally do not require the use of auxiliary color development reactions, thereby permitting simpler flow systems and improved throughput due to decreased sample dispersion.…”