“Stable-on-the-Table” Enzymes: Engineering the Enzyme–Graphene Oxide Interface for Unprecedented Kinetic Stability of the Biocatalyst

Abstract

A rational strategy is presented here to enhance enzyme stability by gaining control over the noncovalent interactions at the enzyme–graphene interface (EGI). The charge (n) on a model enzyme, glucose oxidase (GOx), was systematically varied from −67 to +78 via chemical modification of its COOH groups with polyamines, and chemically modified GOx­(n) has been adsorbed onto graphene oxide (GO). Control of the net charge on GOx­(n) provided an excellent handle to engineer the EGI to enhance the stability of the GOx­(n)/GO biocatalyst while retaining full activity. Enzyme loading (w/w, GOx­(n) to GO) increased with increased n, and a maximum enzyme loading of 420% (w/w) was noted when n = 0; a further increase in n decreased loading. Enzymatic activities of the GOx­(n)/GO hybrids increased steadily with n, and the maximum specific activity was about 1.6 times greater than that of GOx. There has been a good correlation between the retention of enzyme secondary structure and the corresponding enzymatic activities. These indicated an unprecedented increase in kinetic stability as a function of n, measured at 40 °C, with a maximum half-life of 38 days (n = 0, +35), which is about 150 times higher than that of GOx, under the same conditions. The kinetic barrier to denaturation increased steadily from 58.7 kcal/mol for GOx to 294 kcal mol–1 for GOx­(+35)/GO and then decreased slightly with increased n. Chemical denaturation studies showed that the improved stability is not due to changes in thermodynamic stability and that the increased kinetic stability is due to an increased barrier to denaturation. Adsorption onto GO played a key role in enhancing enzyme stability, and without GO, the stability enhancements were only marginal. Thus, engineering the EGI provided exciting opportunities to generate highly stable biocatalysts that can be stored under ambient conditions, while retaining high activities, with very long lifetimes, much needed for practical applications.

Green synthesis of enzyme/metal-organic framework composites with high stability in protein denaturing solvents

“…One of the important aims of enzyme immobilization is to achieve high enzyme stability at harsh conditions such as high temperature and organic solvents which often existe in industrial biocatalytic processes. Although immobilization may compromise the activity of enzyme, the greatly enhanced stability would enable the long-term and repeated use of enzyme and therefore a significantly reduced cost of enzyme (Liu et al 2015 ; Liang et al 2016a ; Ji et al 2016 ; Novak et al 2015 ). Metal-organic frameworks (MOFs) have emerged as a new type of nanomaterials suitable for immobilization of enzyme (Feng et al 2015 ; Lykourinou et al 2011 ; Chen et al 2012 ; Wen et al 2016 ) and other biomolecules (Zhang et al 2016 ; Li et al 2016a , b ).…”

2D Materials‐based Platforms for Electroanalysis Applications

“…In the case of enzymatic biosensors, the key to a good device is the immobilization of the enzyme onto the electrode surface. It has been reported that with the usage of graphene the denaturation of the enzyme is avoided, and the thermodynamic stability is improved generating in this way an excellent biocatalysis . For this reason graphene has been used as immobilization platform of various enzymes (ex.…”

Stimuli-responsive, protein hydrogels for potential applications in enzymology and drug delivery$$^{\S }$$

“…Oxidase activity of GOx was measured using D-glucose as a substrate by a known colorimetric method. 18 2.2h Self-degrading gels: BSA/ARG (75 mg/mL/ 100 mM) was prepared, as mentioned above, with the exception that trypsin (0.33 and 1 mg/mL) was dissolved in the BSA solution before gelation. Degradation of the gel by proteases with respect to temperature was measured by incubating the gel matrix in the buffer at 37 • C, in a water bath.…”