▎ 摘　　要

An example of a highly stable and functional bienzyme-polymer conjugate triad assembled on a topologically orthogonal support, layered graphene oxide (GO), is reported here. Glucose oxidase (GOx) and horseradish peroxidase (HRP) catalytic dyad were used as the model system for cascade biocatalysis. Poly(acrylic acid) (PAA) was used to covalently conjugate these enzymes, and then the conjugate has been subsequently adsorbed onto GO. The resultant nanobiocatalysts are represented as GOx-HRP-PAA/GO. Their morphology and structural characteristics were examined by transmission electron microscopy (TEM), agarose gel electrophoresis, circular dichroism (CD), and zeta potential. These robust conjugates remarkably functioned as active catalysts under biologically challenging conditions such as extreme pHs, high temperature (65 degrees C), and in the presence of a chemical denaturant. In one example, GOx-HRP-PAA/GO presented doubling of the K-cat (TON) (68 x 10(-2) s(-1)) at pH 7.0 and room temperature, when compared to the corresponding physical mixture of GOx/HRP (32 X 10(-2) s(-1)) under similar conditions. In another case, at 65 degrees C, GOx-HRP-PAA/GO displayed similar to 120% specific activity, whereas GOx/HRP showed only 16% of its original activity. At pH 2.0 and in the presence of 4.0 mM SDS as the denaturant, GOx-HRP-PAA/GO presented greater than 100% specific activity, whereas GOx/HRP was completely deactivated under these conditions. Thus, we combined two concepts, enzyme-polymer conjugation followed by adsorption onto a 2D nanolayered material to obtain enhanced substrate channeling and excellent enzyme stability under challenging conditions. These features have never been attained by either traditional enzyme-polymer conjugates or enzyme-GO hybrids. This general, modular, and powerful approach may also be used to produce environmentally benign, biologically compatible (edible), and efficient cascade biocatalysts.