Production of an-in vivo biotinylated recombinant L-lactate oxidase for biosensing applications

AGUSTINA GODINO; MARILLA AMARANTO; LOPEZ MUJICA M ; TAMBORELLI A; DALMASSO PABLO ; RIVAS GUSTAVO; BARRA JOSÉ LUIS

Encuentro; V Encuentro y II Workshop de la Red Argentina de tecnología Enzimática; 2023

L-lactate oxidase (LOX) (EC 1.1.3.2) is a member of the family of flavoproteins and catalyzes the oxidation of L-lactate into pyruvate and generates hydrogen peroxide. LOX is widely used in the pharmaceutical, food and beverage industries. In medicine, the enzyme is used to measure lactate concentration in blood and other body fluids. The association between higher levels of lactate and several health conditions (shocks, metabolic disorders, respiratory insufficiency and heart failure) implies that detecting lactate concentration in blood is essential as a diagnostics parameter. In the food and beverage industries, the amount of lactate indicates the presence of bacterial fermentation and is related to the freshness and quality of several products. LOX can be obtained from several bacterial sources and is used as the catalyst to develop enzyme-based biosensors and colorimetric in vitro tests for lactate detection. In enzyme-based biosensors, the enzyme immobilization on the electrode surface is a crucially important process, which often determines the performance characteristics of the sensor. Among the techniques of enzyme immobilization, the avidin-biotin system allows uniform and efficient immobilization. The pre-requisite to exploit this specific immobilization strategy is the production of biotin-tagged enzymes. In this context, the aim of this study was to develop an expression system to produce, in Escherichia coli, a biotinylated recombinant LOX enzyme using an in vivo biotinylation approach. To address this objective, we designed two E. coli expression vectors: 1) To express a biotin ligase (BirA) enzyme that catalyzes the covalent attachment of a biotin to the lysine within a short 15–23 amino acid peptide termed the AviTag. 2) To overexpress the Aerococcus viridans LOX fused with an N-terminal AviTag (for biotinylation) and HisTag (for purification). For the first vector, the E. coli BirA coding sequence was commercially synthesized and cloned into the pET11a (AmpR) plasmid. For the second one, the AviTag-HisTag-LOX coding sequence was commercially synthesized and cloned into pET50b(+) (KmR) vector. The obtained plasmids were incorporated together into the E. coli BL21 (DE3) strain by transformation to co-express the BirA biotin ligase and the LOX fused to the AviTag. E. coli cells were grown at 37 ◦C with shaking in 500 mL LB medium supplemented with the appropriated antibiotics (Km and Amp) to an OD600 of 0.6–0.8. Protein expression was induced by adding 0.17% lactose and the medium supplemented with 50 μM biotin, followed by incubation overnight at 20°C with shaking. The cells were then collected and the expression of biotinylated LOX protein evaluated by SDS-PAGE and Western blot. The LOX protein was successfully overexpressed and biotinylated. The biotinylated LOX was observed in SDS-PAGE as a major protein band of approximately 45-50 kDa (41 kDa LOX monomer, 4.1 kDa AviTag-linker-HisTag and biotin) in total protein fraction and biotinylation was verified by Western blot assay using a streptavidin-conjugated secondary antibody. The biotinylated recombinant LOX was purified by Immobilized Metal Affinity Chromatography (IMAC) from soluble protein fraction and subsequently desalted. A high-purity biotinylated LOX was efficiently recovered after a single affinity purification step, obtaining 15-20 mg/L of bacterial culture. Finally, the purified biotinylated LOX was functionally evaluated. We analyzed its specific activity in comparison with a non-biotinalized LOX (without AviTag and biotinylation). Preliminary resultsshowed that the activity of biotinylated LOX (102±2 U/mg) was approximately half that of non-biotinylated LOX activity (216±4 U/mg). However, the biotinylated LOX activity obtained is within normal values compared to specific activities of LOX enzymes reported in the literature and by commercial manufacturers. Moreover, biotinylated LOX produced in our laboratory was successfully immobilized at glassy carbon electrodes modified with multi-walled carbon nanotubes non-covalently functionalized with avidin, making possible the amperometric quantification of lactate from the oxidation/reduction of the generated hydrogen peroxide. In conclusion, we demonstrated the successful production of an active recombinant biotinylated LOX by using the in vivo biotinylation strategy that allows obtaining the biotinylated protein of interest without further processing after the purification process, and that, at variance with chemical biotinylation, ensures homogeneous site-specific modification. In addition, this LOX offers the great advantage to be a ready-to-use enzyme for building avidin-functionalized lactate biosensors.