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

AGUSTINA GODINO; AMARANTO MARILLA

Congreso; 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 theoxidation of L-lactate into pyruvate and generates hydrogen peroxide. LOX is widely used in thepharmaceutical, food and beverage industries. In medicine, the enzyme is used to measure lactateconcentration in blood and other body fluids. The association between higher levels of lactate andseveral health conditions (shocks, metabolic disorders, respiratory insufficiency and heart failure)implies that detecting lactate concentration in blood is essential as a diagnostics parameter. In thefood and beverage industries, the amount of lactate indicates the presence of bacterial fermentationand is related to the freshness and quality of several products. LOX can be obtained from severalbacterial sources and is used as the catalyst to develop enzyme-based biosensors and colorimetricin vitro tests for lactate detection. In enzyme-based biosensors, the enzyme immobilization on theelectrode surface is a crucially important process, which often determines the performancecharacteristics of the sensor. Among the techniques of enzyme immobilization, the avidin-biotinsystem allows uniform and efficient immobilization. The pre-requisite to exploit this specificimmobilization strategy is the production of biotin-tagged enzymes. In this context, the aim of thisstudy was to develop an expression system to produce, in Escherichia coli, a biotinylatedrecombinant LOX enzyme using an in vivo biotinylation approach. To address this objective, wedesigned two E. coli expression vectors: 1) To express a biotin ligase (BirA) enzyme that catalyzesthe covalent attachment of a biotin to the lysine within a short 15–23 amino acid peptide termed theAviTag. 2) To overexpress the Aerococcus viridans LOX fused with an N-terminal AviTag (forbiotinylation) and HisTag (for purification). For the first vector, the E. coli BirA coding sequence wascommercially synthesized and cloned into the pET11a (AmpR) plasmid. For the second one, theAviTag-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 bytransformation to co-express the BirA biotin ligase and the LOX fused to the AviTag. E. coli cells weregrown 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 andthe 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-PAGEand Western blot. The LOX protein was successfully overexpressed and biotinylated. Thebiotinylated LOX was observed in SDS-PAGE as a major protein band of approximately 45-50 kDa (41kDa LOX monomer, 4.1 kDa AviTag-linker-HisTag and biotin) in total protein fraction and biotinylationwas verified by Western blot assay using a streptavidin-conjugated secondary antibody. Thebiotinylated recombinant LOX was purified by Immobilized Metal Affinity Chromatography (IMAC)from soluble protein fraction and subsequently desalted. A high-purity biotinylated LOX wasefficiently 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 incomparison with a non-biotinalized LOX (without AviTag and biotinylation). Preliminary resultsAbstractsV Meeting & II Workshop of the Argentine Network of Enzymatic Technology (TEz Network) 68Book of Abstractsshowed 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 withinnormal values compared to specific activities of LOX enzymes reported in the literature and bycommercial manufacturers. Moreover, biotinylated LOX produced in our laboratory was successfullyimmobilized at glassy carbon electrodes modified with multi-walled carbon nanotubes non-covalently functionalized with avidin, making possible the amperometric quantification of lactatefrom the oxidation/reduction of the generated hydrogen peroxide. In conclusion, we demonstratedthe successful production of an active recombinant biotinylated LOX by using the in vivo biotinylationstrategy that allows obtaining the biotinylated protein of interest without further processing after thepurification 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 enzymefor building avidin-functionalized lactate biosensors.