Chemical Modification of Glycoproteins’ Carbohydrate Moiety as a General Strategy for the Synthesis of Efficient Biocatalysts by Biomimetic Mineralization: The Case of Glucose Oxidase

Abstract

Zeolitic imidazolate framework-8 (ZIF-8) is widely used as a protective coating to encapsulate proteins via biomimetic mineralization. The formation of nucleation centers and further biocomposite crystal growth is entirely governed by the pure electrostatic interactions between the protein’s surface and the positively charged Zn(II) metal ions. It was previously shown that enhancing these electrostatic interactions by a chemical modification of surface amino acid residues can lead to a rapid biocomposite crystal formation. However, a chemical modification of carbohydrate components by periodate oxidation for glycoproteins can serve as an alternative strategy. In the present study, an industrially important enzyme glucose oxidase (GOx) was selected as a model system. Periodate oxidation of GOx by 2.5 mM sodium periodate increased negative charge on the enzyme molecule, from −10.2 to −36.9 mV, as shown by zeta potential measurements and native PAGE electrophoresis. Biomineralization experiments with oxidized GOx resulted in higher specific activity, effectiveness factor, and higher thermostability of the ZIF-8 biocomposites. Periodate oxidation of carbohydrate components for glycoproteins can serve as a facile and general method for facilitating the biomimetic mineralization of other industrially relevant glycoproteins.

The Influence of Isoenzyme Composition and Chemical Modification on Horseradish Peroxidase@ZIF-8 Biocomposite Performance

“…Periodate oxidation of purified HRP prior to biomineralization yields biocomposites with higher specific activities and thermostability. This is in accordance with our previous findings with GOx [14]. Also, one should pay attention to the buffer composition used for determination of performance parameters of enzyme@ZIF-8 biocomposites in order to avoid artefacts.…”

“…Oxidizing the carbohydrate component of commercial HRP with 2.5 mM NaIO 4 , followed by biomineralization, drastically increased the specific activity of the biocomposite, similar to the results that we obtained with glucose oxidase [14]. The results are in accordance with a higher protein balance and loading compared to the biocomposite with non-oxidized HRP.…”

“…As demonstrated in the results given in Table 4, the thermostability of purified and oxidized HRP-C was significantly increased after biomineralization. The biocomposite ox-HRP-C@ZIF-8 did not show a drop in activity even after washing with NaCl followed by heating for 1 h at 60 • C. This was in accordance with our previous findings that periodate oxidation of the carbohydrate moiety of enzyme molecules, when performed before biomineralization, leads to biocomposites with much higher activity and thermostability [14]. Table 4.…”

“…Thus, the electrostatic properties of a protein's surface, i.e., its pI and zeta potential, can be used to predict whether a protein will induce ZIF-8 growth from an aqueous solution [13]. Indeed, we recently demonstrated that periodate oxidation of carbohydrate components of another industrially relevant glycoprotein-glucose oxidase (GOx), where the negative charge on the enzyme molecule is increased, can facilitate biomimetic mineralization [14]. This process led to the rapid formation of biocomposites with higher thermal stability and activity compared to their native counterparts.…”

State of the Art Technologies for High Yield Heterologous Expression and Production of Oxidoreductase Enzymes: Glucose Oxidase, Cellobiose Dehydrogenase, Horseradish Peroxidase, and Laccases in Yeasts P. pastoris and S. cerevisiae

“…During the first phase cofactor of GOx, flavine adenine dinucleotide (FAD) is reduced to FADH 2 . In the second phase, the oxidative half-reaction, oxygen reoxidizes the reduced GOx to produce H 2 O 2 and FADH 2 oxidizes to FAD [5]. In 1999, the structure of glucose oxidase from Aspergillus was confirmed, which determined that GOx consists of two uniform subunits and that both subunits contain two separate domains: one is not covalently bound with FAD and the second attaches to the substrate.…”