Rapid Detection of Phenol Using a Membrane Containing Laccase Nanoflowers

Abstract

Dedicated to Professor Chunli Bai, President of the Chinese Academy of Sciences, in celebration of his 60th birthday.With the rapid development of nanoscience and nanotechnology, [1a] nanostructured biocatalysts that take the advantage of nanomaterials in terms of both functional and structural availability have offered new opportunities for improving biological functions of enzymes and expanding applications in areas such as biosensors, bioanalytical devices, and industrial biocatalysis. [1] Recently, we reported a method of preparing protein-inorganic hybrid nanostructures with flower-like shapes, [2] which have shown much greater activities than free enzymes and most of the reported immobilized enzymes. [3] To bring this appealing catalyst into practical use, however, an effective accommodation of these high-performance enzyme catalysts is required. One way is to weakly attach these enzyme nanoflowers to porous materials by physical adsorption. Recently, Krieg et al. [4] reported the fabrication of a supramolecular membrane by noncovalent modification of a commercial membrane, which suggests the possibility of fabricating functional filtration membranes by a simple post-modification procedure, thus enabling many new and interesting applications. It thus came to our mind to fabricate a membrane incorporating enzyme nanoflowers for the rapid detection of hazardous compounds through visualization of the catalyzed product. Owing to their high toxicity even at a low concentration, [5] phenols are listed as major toxic pollutants by the Environmental Protection Agency of the USA and other countries. [6] Sensitive detection of phenolic compounds has been well established using instrumental analysis such as liquid chromatography. [7] However, these methods usually require sophisticated instrumentation and a multistep procedure, making them less convenient for rapid and on-site detection.The present study started by the fabrication of an enzyme nanoflower incorporated into a membrane. As shown in Figure 1, a suspension of laccase-inorganic hybrid nanoflowers, which have a high activity (ca. 200 % that of free laccase) for phenol oxidization, as we observed previously, [2] was injected into a commercial disposable syringe filter equipped with a cellulose acetate membrane (pore size 0.2 mm). This procedure thus deposited enzyme nanoflowers with an average size of 4 mm onto the membrane. Then the aqueous sample containing phenol was mixed with an aqueous solution of 4-aminoantipyrine and was passed through the membrane with incorporated laccase nanoflowers, causing oxidative coupling of phenol with 4-aminoantipyrine to form an antipyrine dye [8] that has an absorption maximum at 495 nm. This procedure allowed rapid analysis by a UV/ Vis spectrophotometer or by the naked eye. Finally, pure water was injected into the filter to remove unreacted reagents and the reaction products, followed by drying the membrane in air for the next use.For the preparation of laccase-copper phosphate nanoflowers, typically, 0.8 mm aqueous Cu...

A new generation approach in enzyme immobilization: Organic-inorganic hybrid nanoflowers with enhanced catalytic activity and stability

“…Thus, sensitively, rapidly and on-site detection of these compounds are very important. In this regard, Zare and coworkers immobilized the laccase nanoflowers on a filter membrane for the rapid detection of hazardous compounds via visualization of catalyzed product [38]. To obtain reproducible measurements, this is a simple, accurate and successful approach.…”

Synthesis of copper ion incorporated horseradish peroxidase-based hybrid nanoflowers for enhanced catalytic activity and stability

“…35,36 Zare and co-workers also reported synthesis of a membrane containing laccase nanoflowers for detection of phenol. 37 In subsequent studies, Zeng and co-workers utilized the same synthetic route to produce CaHPO 4 -α-amylase-based nanoflowers 38 and Yang and co-workers used enzyme-inorganic nanoflowers as sensors for detection of hydrogen peroxide and phenol.…”

Synthesis of Lutein Esters by Using a Reusable Lipase-Pluronic Conjugate as the Catalyst

“…However, the lipase-catalyzed esterification needs to be carried out in organic solvents in which most enzymes display much lower activities than in aqueous solution mainly due to the formation of agglomeration of the insoluble enzyme molecules in organic solvents making enzyme less accessible to substrates. Nanostructured enzyme catalysts [6][7][8][9] such as enzyme nanoparticles [10,11], enzyme nanogels [12][13][14][15][16][17], flower-like enzyme-inorganic hybrid crystals [18][19][20][21], enzyme-metal-organic framework hybrid composites [22] and enzyme-polymer conjugates [23][24][25] have been demonstrated as effective chemical ways to re-engineer enzyme catalysts for improved activity and stability. For example, conjugation with an amphiphilic polymer such as Pluronic can enhance the solubilization of enzyme in organic solvents and therefore increase the apparent enzymatic activity by orders of magnitudes, as compared to its native counterpart.…”