Combining genetically engineered oxidase with hydrogen bonded organic framework (HOF) for highly efficient biocomposites.

Hydrogen bonded organic frameworks (HOFs) with enzymes incorporated during their bottom-up synthesis represent functional biocomposites with promising applications in catalysis and sensing. High enzyme loading while preserving high specific activity is fundamental for development, but to combine these biospecific features with a porous carrier is an unmet challenge. Here, we explored synthetic incorporation of D-amino acid oxidase (DAAO) with metal-free tetraamidine/tetracarboxylate-based BioHOF-1. Comparison of different DAAO forms in BioHOF-1 incorporation revealed that N-terminal enzyme fusion with the positively charged module Zbasic2 (Z-DAAO) promotes the loading (2.5-fold; ~500 mg g-1) and strongly boosts the activity (6.5-fold). To benchmark the HOF composite with metal-organic framework (MOF) composites, Z-DAAO was immobilized into the zeolitic imidazolate framework-8 (ZIF-8), the relatively more hydrophilic analogue metal azolate framework-7 (MAF-7). While sensitivity to the framework environment limited the activity of DAAO@MAF-7 (3.2 U mg-1) and DAAO@ZIF-8 (≤ 0.5 U mg-1), the activity of DAAO@BioHOF-1 was comparable (~45%) to that of soluble DAAO (50.1 U mg-1) and independent of the enzyme loading (100 – 500 mg g-1). The DAAO@BioHOF-1 composites showed superior activity with respect to every reported carrier for the same enzyme and excellent stability during solid catalyst recycling. Collectively, our results show that the fusion of the enzyme with a positively charged protein module enables the synthesis of highly active HOF biocomposites suggesting the use of genetic engineering for the preparation of biohybrid systems with unprecedented properties.