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.

All Wrapped up: Stabilization of Enzymes within Single Enzyme Nanoparticles

“…Despite the distribution of particle sizes, in some cases close to 100% activity could be retained after grafting, along with increased shelf life and good resistance against heat and trypsin degradation. Similar results have been observed with the use of this approach on a range of other enzymes including glucose oxidase, HRP, lactate dehydrogenase, acid phosphatase and lipase …”

“…Similar results have been observed with the use of this approach on a range of other enzymes including glucose oxidase, HRP, lactate dehydrogenase, acid phosphatase and lipase. 121 In situ polymerization and cross-linking. An alternative to grafting polymers to or from an enzyme is to polymerize a cross-linked network or gel around the enzyme.…”

Artificial Multienzyme Scaffolds: Pursuing in Vitro Substrate Channeling with an Overview of Current Progress

“…As with any immobilization strategy, careful selection of the chemistries used for immobilization is critical to ensure activity. Zore et al examined different approaches for enzyme assembly to a poly­(acrylic acid) abiotic scaffold to form multienzyme polymer conjugates (MECs) . In both of the strategies employed for MEC assembly, carbodiimide chemistry was used to form the abiotic scaffold.…”

Multimodal Enzyme‐Carrying Suprastructures for Rapid and Sensitive Biocatalytic Cascade Reactions

“…We investigated Michaelis‐Menten reaction kinetics, including catalytic efficiency of the suprastructure and the NPs dispersion in terms of GOX/HRP cascade reaction (Figure 3c,d ). [ 49 ] We monitored the initial velocity of the reactions ( V ) in different glucose concentrations, then calculated the affinity of substrates ( K m ), turnover number ( k cat ), and catalytic efficiency ( k cat / K m ) (Figure S10 , Supporting Information). It was calculated that the k cat / K m of the suprastructure (7.7 × 10 –4 m m –1 S –1 ) is higher than that of the NP dispersion (2.7 × 10 –4 m m –1 S –1 ) by a factor of 2.9.…”