Nanoarmoring: strategies for preparation of multi-catalytic enzyme polymer conjugates and enhancement of high temperature biocatalysis

Abstract

We report a general and modular approach for the synthesis of multi enzyme–polymer conjugates (MECs) consisting of five different enzymes of diverse isoelectric points and distinct catalytic properties conjugated within a single universal polymer scaffold. The five model enzymes chosen include glucose oxidase (GOx), acid phosphatase (AP), lactate dehydrogenase (LDH), horseradish peroxidase (HRP) and lipase (Lip). Poly(acrylic acid) (PAA) is used as the model synthetic polymer scaffold that will covalently conjugate and stabilize multiple enzymes concurrently. Parallel and sequential synthetic protocols are used to synthesise MECs, 5-P and 5-S, respectively. Also, five different single enzyme–PAA conjugates (SECs) including GOx–PAA, AP–PAA, LDH–PAA, HRP–PAA and Lip–PAA are synthesized. The composition, structure and morphology of MECs and SECs are confirmed by agarose gel electrophoresis, dynamic light scattering, circular dichroism spectroscopy and transmission electron microscopy. The bioreactor comprising MEC functions as a single biocatalyst can carry out at least five different or orthogonal catalytic reactions by virtue of the five stabilized enzymes, which has never been achieved to-date. Using activity assays relevant for each of the enzymes, for example AP, the specific activity of AP at room temperature and 7.4 pH in PB is determined and set at 100%. Interestingly, MECs 5-P and 5-S show specific activities of 1800% and 600%, respectively, compared to 100% specific activity of AP at room temperature (RT). The catalytic efficiencies of 5-P and 5-S are 1.55 × 10⁻³ and 1.68 × 10⁻³, respectively, compared to 9.11 × 10⁻⁵ for AP under similar RT conditions. Similarly, AP relevant catalytic activities of 5-P and 5-S at 65 °C show 100 and 300%, respectively, relative to native AP activity at RT as the native AP is catalytically inactive at 65 °C. The catalytic activity trends suggest: (1) MECs show enhanced catalytic activities compared to native enzymes under similar assay conditions and (2) 5-S is better suited for high temperature biocatalysis, while both 5-S and 5-P are suitable for room temperature biocatalysis. Initial cytotoxicity results show that these MECs are non-lethal to human cells including human embryonic kidney [HEK] cells when treated with doses of 0.01 mg mL⁻¹ for 72 h. This cytotoxicity data is relevant for future biological applications.