More Than One Enzyme: Exploring Alternative FMN-Dependent L-Lactate Oxidases for Biosensor Development.

The α-hydroxy acid oxidoreductase (HAOx) family contains a diverse group of enzymes that can be applied in biosensors for L-lactate detection, most prominently lactate oxidase (LOx). The limited availability and a lack of diversity of L-lactate-oxidizing enzymes have currently hindered advancements in L-lactate biosensor development. Until now, the field has mostly relied on a single, commercially available enzyme, namely Aerococcus viridans L-lactate oxidase (AvLOx). In this study, we present newly discovered alternative L-lactate oxidases that exhibit a narrow substrate specificity and varied kinetic efficiencies toward L-lactate, making them suitable for integration into existing biosensor configurations. Some of these FMN-dependent L-lactate oxidases could be obtained in substantial amounts from routine E. coli expression, potentially facilitating commercial production. Using electrochemical characterization with a mediated biosensor setup, we present 7 enzymes that perform comparable or even better than commercial AvLOx. Finally, we show that their electrochemical performance is not directly correlating with their biochemical performance, making predictions of the suitability of enzymes for biosensor applications extremely difficult. Our research emphasizes the significance of expanding the enzyme toolbox of L-lactate oxidases for the development of improved L-lactate biosensors.

Recent Advances in Electrochemical Enzyme-Based Biosensors for Food and Beverage Analysis.

Food analysis and control are crucial aspects in food research and production in order to ensure quality and safety of food products. Electrochemical biosensors based on enzymes as the bioreceptors are emerging as promising tools for food analysis because of their high selectivity and sensitivity, short analysis time, and high-cost effectiveness in comparison to conventional methods. This review provides the readers with an overview of various electrochemical enzyme-based biosensors in food analysis, focusing on enzymes used for different applications in the analysis of sugars, alcohols, amino acids and amines, and organic acids, as well as mycotoxins and chemical contaminants. In addition, strategies to improve the performance of enzyme-based biosensors that have been reported over the last five years will be discussed. The challenges and future outlooks for the food sector are also presented.

Flavin Mononucleotide-Dependent l-Lactate Dehydrogenases: Expanding the Toolbox of Enzymes for l-Lactate Biosensors.

The development of L-lactate biosensors has been hampered in recent years by the lack of availability and knowledge about a wider range and diversity of L-lactate-oxidizing enzymes that can be used as bioelements in these sensors. For decades, L-lactate oxidase of Aerococcus viridans (AvLOx) has been used almost exclusively in the field of L-lactate biosensor development and has achieved somewhat like a monopoly status as a biocatalyst for these applications. Studies on other L-lactate-oxidizing enzymes are sparse and are often missing biochemical data. In this work, we made use of the vast amount of sequence information that is currently available on protein databases to investigate the naturally occurring diversity of L-lactate-utilizing enzymes of the flavin mononucleotide (FMN)-dependent α-hydroxy acid oxidoreductase (HAOx) family. We identified the HAOx sequence space specific for L-lactate oxidation and additionally discovered a not-yet described class of soluble and FMN-dependent L-lactate dehydrogenases, which are promising for the construction of second-generation biosensors or other biotechnological applications. Our work paves the way for new studies on α-hydroxy acid biosensors and proves that there is more to the HAOx family than AvLOx.

Rapid Measurement of Lactate in the Exhaled Breath Condensate: Biosensor Optimization and In-Human Proof of Concept.

Lactate concentration is of increasing interest as a diagnostic for sepsis, septic shock, and trauma. Compared with the traditional blood sample media, the exhaled breath condensate (EBC) has the advantages of non-invasiveness and higher user acceptance. An amperometric biosensor was developed and its application in EBC lactate detection was investigated in this paper. The sensor was modified with PEDOT:PSS-PB, and two different lactate oxidases (LODs). A rotating disk electrode and Koutecky-Levich analysis were applied for the kinetics analysis and gel optimization. The optimized gel formulation was then tested on disposable screen-printed sensors. The disposable sensors exhibited good performance and presented a high stability for both LOD modifications. Finally, human EBC analysis was conducted from a healthy subject at rest and after 30 min of intense aerobic cycling exercise. The sensor coulometric measurements showed good agreement with fluorometric and triple quadrupole liquid chromatography mass spectrometry reference methods. The EBC lactate concentration increased from 22.5 µM (at rest) to 28.0 µM (after 30 min of cycling) and dropped back to 5.3 µM after 60 min of rest.