Combined cross-linked enzyme aggregates of horseradish peroxidase and glucose oxidase for catalyzing cascade chemical reactions

• Published in: Enzyme and microbial technology Vol. 100; pp. 52 - 59
• Main Authors: Nguyen, Le Truc, Yang, Kun-Lin
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2017
• Subjects: Benzothiazoles - metabolism; Biocatalysis; Cascade chemical reactions; Catalase - metabolism; Combi-CLEA; Cross-Linking Reagents; Enzymes, Immobilized - chemistry; Enzymes, Immobilized - metabolism; Glucose - metabolism; Glucose oxidase; Glucose Oxidase - chemistry; Glucose Oxidase - metabolism; Hollow sphere; Horseradish peroxidase; Horseradish Peroxidase - chemistry; Horseradish Peroxidase - metabolism; Hydrogen Peroxide - metabolism; Kinetics; Millifluidic reactor; Oxidation-Reduction; Particle Size; Protein Aggregates; Sulfonic Acids - metabolism; GOx; Glucose oxidase; Hollow sphere; Combi-CLEA; Millifluidic reactor; PTFE; HRP; Horseradish peroxidase; SEM; Cascade chemical reactions; CLEAs; ABTS
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2017.02.007

[Display omitted] •Two enzymes can be cross-linked as Combi-CLEA particles in millifluidic reactors.•Combi-CLEA particles are hollow spheres with a uniform sizeCombi-CLEA particles are hollow spheres with a uniform size.•Combi-CLEA exhibited higher catalytic efficiency than free enzymes.•Detection of glucose by using Combi-CLEA was demonstrated. Cascade reactions involved unstable intermediates are often encountered in biological systems. In this study, we developed combined cross-linked enzyme aggregates (combi-CLEA) to catalyze a cascade reaction which involves unstable hydrogen peroxide as an intermediate. The combi-CLEA contains two enzymes̶ glucose oxidase (GOx) and horseradish peroxidase (HRP) which are cross-linked together as solid aggregates. The first enzyme GOx catalyzes the oxidation of glucose and produces hydrogen peroxide, which is used by the second enzyme HRP to oxidize 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). The apparent reaction rate of the cascade reaction reaches 10.5±0.5μM/min when the enzyme ratio is 150:1 (GOx:HRP). Interestingly, even in the presence of catalase, an enzyme that quickly decomposes hydrogen peroxide, the reaction rate only decreases by 18.7% to 8.3±0.3μM/min. This result suggests that the intermediate hydrogen peroxide is not decomposed by catalase due to a short diffusion distance between GOx and HRP in the combi-CLEA. Scanning electron microscopy images suggest that combi-CLEA particles are hollow spheres and have an average diameter around 250nm. Because of their size, combi-CLEA particles can be entrapped inside a nylon membrane for detecting glucose by using the cascade reaction.