Flavin Mononucleotide-Dependent <scp>l</scp>-Lactate Dehydrogenases: Expanding the Toolbox of Enzymes for <scp>l</scp>-Lactate Biosensors

Tsvik, Lidiia; Steiner, Beate; Herzog, Peter L.; Haltrich, Dietmar; Sützl, Leander

doi:10.1021/acsomega.2c05257

Cited by 8 publications

(24 citation statements)

References 70 publications

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“…LOx Sequence Selection and Alignment. Nine bacterial protein sequences from a recently defined LOx clade and two additional sequences that showed good oxidase activity with Llactate in an initial screening 26 were selected for recombinant expression together with AvLOx as a reference. The full species names and abbreviations of the 12 selected sequences, as well as their phylogenetic relationship, are listed in Figure 2.…”

Section: ■ Resultsmentioning

confidence: 99%

“…Protein Purification. As previously reported, 26 cells were disrupted using multiple cycles in a French Press, and after centrifugation, crude extracts were filtered through a 0.22-μm membrane filter. A kta FPLC system (GE Healthcare, USA) was used to conduct IMAC purification on 5 mL HisTrap FF columns (Cytiva, USA).…”

Section: ■ Conclusionmentioning

confidence: 99%

“…24,25 This dynamic lid-loop is the least conserved region when comparing amino acid sequences within the HAOx family. 26 In its first half-reaction, LOx catalyzes the oxidation of Llactate to pyruvate while FMN is reduced to FMNH 2 . The ensuing half-reaction reoxidizes the cofactor by reducing molecular oxygen to H 2 O 2 (Figure 1); alternatively, LOx can also employ other electron acceptors such as 2,6-dichlor-ophenol indophenol (DCIP), albeit to a varying extent.…”

Section: ■ Introductionmentioning

confidence: 99%

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

Tsvik,

Zhang,

O’Hare

et al. 2024

ACS Omega

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Section: ■ Resultsmentioning

confidence: 99%

Section: ■ Conclusionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Tsvik,

Zhang,

O’Hare

et al. 2024

ACS Omega

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“…This exceptional pH tolerance enables robust and versatile biocatalytic processes by allowing the utilisation of a single enzyme across multiple applications with varying pH conditions (Table S7). The broad pH optima of these enzymes suggest their robustness in industrial settings, which often encounter pH fluctuations, thereby improving operational stability in large-scale and continuous processes [60][61][62][63] . Regarding their activity levels, all three groups (controls, zero-shot, fine-tuned) have comparable performance, with no significant difference observed between either of them (ANOVA f-statistic = 1.99, p = 0.17, n.s., Fig.…”

Section: Lactate Dehydrogenasesmentioning

confidence: 99%

Conditional language models enable the efficient design of proficient enzymes

Munsamy,

Illanes-Vicioso,

Funcillo

et al. 2024

Preprint

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The design of functional enzymes holds promise for transformative solutions across various domains but presents significant challenges. Inspired by the success of language models in generating nature-like proteins, we explored the potential of an enzyme-specific language model in designing catalytically active artificial enzymes. Here, we introduce ZymCTRL ('enzyme control'), a conditional language model trained on the enzyme sequence space, capable of generating enzymes based on user-defined specifications. Experimental validation at diverse data regimes and for different enzyme families demonstrated ZymCTRL's ability to generate active enzymes across various sequence identity ranges. Specifically, we describe the design of carbonic anhydrases and lactate dehydrogenases in zero-shot, without requiring further training of the model, and showcasing activity at sequence identities below 40% compared to natural proteins. Biophysical analysis confirmed the globularity and well-folded nature of the generated sequences. Furthermore, fine-tuning the model enabled the generation of lactate dehydrogenases more likely to pass in silico filters and with activity comparable to their natural counterparts. Two of the artificial lactate dehydrogenases were scaled up and successfully lyophilised, maintaining activity and demonstrating preliminary conversion in one-pot enzymatic cascades under extreme conditions. Our findings open a new door towards the rapid and cost-effective design of artificial proficient enzymes. The model and training data are freely available to the community.

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“…The first reported Fc b 2 -based ABSs were developed using cells or cell lysates, from the yeasts S. cerevisiae and Hansenula anomala as the parental cells of this enzyme [ 30 , 31 ]. Many Lact-sensitive ABSs based on the recombinant cells have been proposed, especially in the last several years [ 32 , 33 , 34 , 35 , 36 , 37 ].…”

Section: Introductionmentioning

confidence: 99%

Flavocytochrome b2-Mediated Electroactive Nanoparticles for Developing Amperometric L-Lactate Biosensors

et al. 2023

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L-Lactate is an indicator of food quality, so its monitoring is essential. Enzymes of L-Lactate metabolism are promising tools for this aim. We describe here some highly sensitive biosensors for L-Lactate determination which were developed using flavocytochrome b2 (Fcb2) as a bio-recognition element, and electroactive nanoparticles (NPs) for enzyme immobilization. The enzyme was isolated from cells of the thermotolerant yeast Ogataea polymorpha. The possibility of direct electron transfer from the reduced form of Fcb2 to graphite electrodes has been confirmed, and the amplification of the electrochemical communication between the immobilized Fcb2 and the electrode surface was demonstrated to be achieved using redox nanomediators, both bound and freely diffusing. The fabricated biosensors exhibited high sensitivity (up to 1436 A·M−1·m−2), fast responses, and low limits of detection. One of the most effective biosensors, which contained co-immobilized Fcb2 and the hexacyanoferrate of gold, having a sensitivity of 253 A·M−1·m−2 without freely diffusing redox mediators, was used for L-Lactate analysis in samples of yogurts. A high correlation was observed between the values of analyte content determined using the biosensor and referenced enzymatic-chemical photometric methods. The developed biosensors based on Fcb2-mediated electroactive nanoparticles can be promising for applications in laboratories of food control.

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

Cited by 8 publications

References 70 publications

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

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

Conditional language models enable the efficient design of proficient enzymes

Flavocytochrome b2-Mediated Electroactive Nanoparticles for Developing Amperometric L-Lactate Biosensors

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