title: Combined cross-linked enzyme aggregates of horseradish peroxidase and glucose oxidase for catalyzing cascade chemical reactions.
abstract: 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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576, Singapore.
authors.forename: Le Truc
authors.initials: LT
authors.lastname: Nguyen
authors.affiliation: Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576, Singapore. Electronic address: cheyk@nus.edu.sg.
authors.forename: Kun-Lin
authors.initials: KL
authors.lastname: Yang
mesh_terms: D052160:Benzothiazoles / Q000378:metabolism; D055162:Biocatalysis; D002374:Catalase / Q000378:metabolism; D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D005949:Glucose Oxidase / Q000737:chemistry / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D010316:Particle Size; D066329:Protein Aggregates; D013451:Sulfonic Acids / Q000378:metabolism
chemical_list: D052160:Benzothiazoles; D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D066329:Protein Aggregates; D013451:Sulfonic Acids; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D006861:Hydrogen Peroxide; D005949:Glucose Oxidase; D006735:Horseradish Peroxidase; D002374:Catalase; D005947:Glucose
keywords: Cascade chemical reactions; Combi-CLEA; Glucose oxidase; Hollow sphere; Horseradish peroxidase; Millifluidic reactor