Abstract
Flavoenzyme dye-linked l-lactate dehydrogenase (Dye-LDH) is primarily involved in energy generation through electron transfer and exhibits potential utility in electrochemical devices. In this study, a gene encoding a Dye-LDH homolog was identified in a hyperthermophilic archaeon, Sulfurisphaera tokodaii. This gene was part of an operon that consisted of four genes that were tandemly arranged in the Sf. tokodaii genome in the following order: stk_16540, stk_16550 (dye-ldh homolog), stk_16560, and stk_16570. This gene cluster was expressed in an archaeal host, Sulfolobus acidocaldarius, and the produced enzyme was purified to homogeneity and characterized. The purified recombinant enzyme exhibited Dye-LDH activity and consisted of two different subunits (products of stk_16540 (α) and stk_16550 (β)), forming a heterohexameric structure (α3β3) with a molecular mass of approximately 253 kDa. Dye-LDH also exhibited excellent stability, retaining full activity upon incubation at 70 °C for 10 min and up to 80% activity after 30 min at 50 °C and pH 6.5–8.0. A quasi-direct electron transfer (DET)-type Dye-LDH was successfully developed by modification of the recombinant enzyme with an artificial redox mediator, phenazine ethosulfate, through amine groups on the enzyme’s surface. This study is the first report describing the development of a quasi-DET-type enzyme by using thermostable Dye-LDH.
Highlights
Dye-linked L-lactate dehydrogenase (Dye-LDH) is a flavoenzyme containing FMN as a cofactor, which catalyzes oxidation of L-lactate to pyruvate in the presence of natural electron acceptors such as cytochrome c
The constitutive promoter sso 10610 was selected for expression of the gene cluster in S. acidocaldarius SK1 cells based on a previous report [13]
S. acidocaldarius SK1-carrying pSAVex-STK16540-16570 was cultivated in an xylose and tryptone (XT) medium, and a crude extract prepared from these cells exhibited dye-linked L-lactate dehydrogenase (Dye-LDH) activity, indicating successful production of the enzyme
Summary
Dye-linked L-lactate dehydrogenase (Dye-LDH) is a flavoenzyme containing FMN as a cofactor, which catalyzes oxidation of L-lactate to pyruvate in the presence of natural electron acceptors such as cytochrome c. Dye-LDH can utilize artificial electron acceptors, such as 2,6-dichlorophenolindophenol (DCIP) and potassium ferricyanide, in place of its natural electron acceptor These artificial electron acceptors can efficiently transfer electrons derived from substrates to electrodes. Dye-LDH exhibits significant potential for utilization as a specific element in electrochemical devices, such as. Hyperthermophiles, which can grow at or near the boiling point of water, are excellent sources of microbial enzymes that are highly stable under a variety of conditions, such as high temperature, acidity, and alkalinity [10,11,12] It is, worth exploring hyperthermophiles as potential sources of stable Dye-LDH for electrochemical applications. We performed detailed biochemical characterizations of this enzyme and examined its utility as a specific element for L-lactate electrochemical devices
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