Influence of Three Commercial Graphene Derivatives on the Catalytic Properties of a Lactobacillus plantarum α-l-Rhamnosidase When Used as Immobilization Matrices

Abstract

The modification of carbon nanomaterials with biological molecules paves the way toward their use in biomedical and biotechnological applications, such as next-generation biocatalytic processes, development of biosensors, implantable electronic devices, or drug delivery. In this study, different commercial graphene derivatives, namely, monolayer graphene oxide (GO), graphene oxide nanocolloids (GOCs), and polycarboxylate-functionalized graphene nanoplatelets (GNs), were compared as biomolecule carrier matrices. Detailed spectroscopic analyses showed that GO and GOC were similar in composition and functional group content and very different from GN, whereas divergent morphological characteristics were observed for each nanomaterial through microscopy analyses. The commercial α-l-rhamnosidase RhaB1 from the probiotic bacterium Lactobacillus plantarum, selected as a model biomolecule for its relevant role in the pharma and food industries, was directly immobilized on the different materials. The binding efficiency and biochemical properties of RhaB1-GO, RhaB1-GOC, and RhaB1-GN composites were analyzed. RhaB1-GO and RhaB1-GOC showed high binding efficiency, whereas the enzyme loading on GN, not tested in previous enzyme immobilization studies, was low. The enzyme showed contrasting changes when immobilized on the different material supports. The effect of pH on the activity of the three RhaB1-immobilized versions was similar to that observed for the free enzyme, whereas the activity-temperature profiles and the response to the presence of inhibitors varied significantly between the RhaB1 versions. In addition, the apparent K for the immobilized and soluble enzymes did not change. Finally, the free RhaB1 and the immobilized enzyme in GOC showed the best storage and reutilization stability, keeping most of their initial activity after 8 weeks of storage at 4 °C and 10 reutilization cycles, respectively. This study shows, for the first time, that distinct commercial graphene derivatives can influence differently the catalytic properties of an enzyme during its immobilization.

Interaction Analysis of Commercial Graphene Oxide Nanoparticles with Unicellular Systems and Biomolecules

“…The physical-chemical properties of the graphene oxide derivatives selected for this study were recently determined [4]. Microscopy analyses using AFM and TEM instruments showed that GO and GOC flakes were mostly in monolayer state and had a different size, while the analysis of their composition revealed a high similarity between both nanomaterials.…”

“…The protein binding capacity of nanomaterials determines possible biological applications and their toxicological potential too [60,61]. In case of commercial GO and GOC, both nanomaterial suspensions showed a high protein loading capacity and a good potential as enzyme immobilization supports [4]. However, their maximum protein binding capacity was not determined, and their polypeptide binding properties were determined using a single enzyme.…”

“…The interest in the immobilization of microorganisms and microbial enzymes for biotechnological applications has been continuously rising during the last decades because of several factors, including the increased availability of microbial strains and biocatalysts tailored to new applications, the development of new immobilization supports with improved properties, and the need of a shift toward the use of more sustainable processes in different industrial fields [1][2][3][4][5]. The immobilization of microorganisms and enzymes on solid carriers leads to a number of benefits.…”

“…Regarding the use of nanoparticles, an extensive number of studies have described the properties of different nanomaterials such as magnetic nanoparticles, including iron oxide (Fe 3 O 4 and γ-Fe 2 O 3 ), alloy-based (CoPt 3 and FePt), pure metal (Fe and Co), and spinel-type ferromagnets (MgFe 2 O 4 , MnFe 2 O 4 , and CoFe 2 O 4 ) [12], or carbon derived nanoparticles, namely single and multiwall carbon nanotubes, graphene, graphene oxide, fullerene, etc. [4,[13][14][15], as suitable carriers for enzymes of industrial interest. Similarly, applications for the use of these types of nanomaterials for the immobilization of prokaryotic and eukaryotic microorganisms have been investigated [11,[16][17][18].…”

“…The use of distinct commercial graphene oxide nanoparticles can influence dramatically the biocatalyst loading, biochemical properties, and stability. For this reason, the selection of an optimal biocatalyst-carrier combination makes advisable a thorough screening of the available options [4]. Also, in regard to the suitability of graphene oxide derivatives as support for microbial immobilization, conflicting results relating biocompatibility and cytotoxicity induced by these nanomaterials have been reported in the literature [21], which could be in part due to their heterogeneity in functional groups composition, the presence of different amounts of trace elements, their size and morphology, etc.…”