Activity enhancement of multicopper oxidase from a hyperthermophile via directed evolution, and its application as the element of a high performance biocathode

Satomura, Takenori; Hirano, Tatsunari; Inagaki, Kohei; Horinaga, Kosaku; Takamura, Eiichiro; Sakamoto, Hiroaki; Ohshida, Tatsuya; Ohshima, Toshihisa; Sakuraba, Haruhiko; Suye, Shin‐ichiro

doi:10.1016/j.jbiotec.2020.10.019

Cited by 5 publications

(5 citation statements)

References 20 publications

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“…The 3F3 variant selected after DNA shuffling showed a ~ tenfold higher k cat than the 1B5 and a remarkable 2-order of magnitude higher k cat /K m compared to wild-type. Notably, the turnover number for ABTS of 3F3 is close to the one measured for the model bacterial CotA-laccase ( k cat = 144 s −1 ) [ 65 ]; these results significantly surpass the outcomes of a similar engineering approach, where a McoP variant featuring the four mutations G225S, L284H, F290I, and T341A demonstrated only a ninefold increase in enzymatic activity compared to wild-type [ 66 ]. The introduced mutations did not have any discernible impact on thermal stability.…”

Section: Resultsmentioning

confidence: 83%

“…For instance, the longer distance of M353 in chain B to segment 296–302 in chain A may induce additional destabilization in this region. Recently, it was reported that mutation F290I in McoP, located in the loop T288-E310, improved the turnover rate for ABTS by around sixfold (28 s −1 at 50 °C) [ 66 ]; the X-ray crystal structure analysis (PDB 6K3D) suggested that this mutation increased the flexibility of the loop, improving the ET between the enzyme and the substrate.…”

Section: Resultsmentioning

confidence: 99%

“…McoP was recently utilized to fabricate biocathodes employing various electrodes and immobilization strategies [ 66 – 69 ]. In this study, wild-type McoP and the 3F3 variant were adsorbed onto gold electrodes (AuEs).…”

Section: Resultsmentioning

confidence: 99%

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Flexible active-site loops fine-tune substrate specificity of hyperthermophilic metallo-oxidases

Brissos,

Borges,

Sancho

et al. 2024

J Biol Inorg Chem

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Hyperthermophilic (‘superheat-loving’) archaea found in high-temperature environments such as Pyrobaculum aerophilum contain multicopper oxidases (MCOs) with remarkable efficiency for oxidizing cuprous and ferrous ions. In this work, directed evolution was used to expand the substrate specificity of P. aerophilum McoP for organic substrates. Six rounds of error-prone PCR and DNA shuffling followed by high-throughput screening lead to the identification of a hit variant with a 220-fold increased efficiency (kcat/Km) than the wild-type for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) without compromising its intrinsic activity for metal ions. The analysis of the X-ray crystal structure reveals four proximal mutations close to the T1Cu active site. One of these mutations is within the 23-residues loop that occludes this site, a distinctive feature of prokaryotic MCOs. The increased flexibility of this loop results in an enlarged tunnel and one additional pocket that facilitates bulky substrate-enzyme interactions. These findings underscore the synergy between mutations that modulate the dynamics of the active-site loop enabling enhanced catalytic function. This study highlights the potential of targeting loops close to the T1Cu for engineering improvements suitable for biotechnological applications. Graphical Abstract

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Section: Resultsmentioning

confidence: 83%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Flexible active-site loops fine-tune substrate specificity of hyperthermophilic metallo-oxidases

Brissos,

Borges,

Sancho

et al. 2024

J Biol Inorg Chem

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“…The advancement of computer computing power and the emergence of advanced algorithms have led to the rapid development of computer‐aided enzyme design and modification, which has become a novel method for improving the industrial application characteristics of enzymes. Directed and in situ evolutions are the main engineering strategies to improve the catalytic efficiency and stability of multicopper oxidase enzymes (Satomura et al., 2021). The Ni 2+ dependence of urease poses potential safety concerns.…”

Section: Prevention Of Nmh Contamination In the Process Of Alcoholic ...mentioning

confidence: 99%

Mitigation of microbial nitrogen‐derived metabolic hazards as a driver for safer alcoholic beverage choices: An evidence‐based review and future perspectives

Liao,

Asif,

Huang

et al. 2023

Comp Rev Food Sci Food Safe

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Alcoholic beverages have been enjoyed worldwide as hedonistic commodities for thousands of years. The unique quality and flavor are attributed to the rich microbiota and nutritional materials involved in fermentation. However, the metabolism of these microbiota can also introduce toxic compounds into foods. Nitrogen‐derived metabolic hazards (NMH) are toxic metabolic hazards produced by microorganisms metabolizing nitrogen sources that can contaminate alcoholic beverages during fermentation and processing. NMH contamination poses a risk to dietary safety and human health without effective preventive strategies. Existing literature has primarily focused on investigating the causes of NMH formation, detection methods, and abatement techniques for NMH in fermentation end‐products. Devising effective process regulation strategies represents a major challenge for the alcoholic beverage industry considering our current lack of understanding regarding the processes whereby NMH are generated, real‐time and online detection, and the high degradation rate after NMH formation. This review summarizes the types and mechanisms of nitrogenous hazard contamination, the potential risk points, and the analytical techniques to detect NMH contamination. We discussed the changing patterns of NMH contamination and effective strategies to prevent contamination at different stages in the production of alcoholic beverages. Moreover, we also discussed the advanced technologies and methods to control NMH contamination in alcoholic beverages based on intelligent monitoring, synthetic ecology, and computational assistance. Overall, this review highlights the risks of NMH contamination during alcoholic beverage production and proposes promising strategies that could be adopted to eliminate the risk of NMH contamination.

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“…A directed evolution strategy allowed to isolate a McoP mutant exhibiting an enhanced activity both in solution or at the electrode, although the CuT1 redox potential was only slightly enhanced [61]. The amino acid F290, not involved in CuT1 coordination, was shown to be essential.…”

Section: Cueo Electrochemistry : How To Enhance Cut1 Redox Potentialmentioning

confidence: 99%

Electrochemistry of copper efflux oxidase-like multicopper oxidases involved in copper homeostasis

Mazurenko

Adachi

Ezraty

et al. 2022

Current Opinion in Electrochemistry

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Activity enhancement of multicopper oxidase from a hyperthermophile via directed evolution, and its application as the element of a high performance biocathode

Cited by 5 publications

References 20 publications

Flexible active-site loops fine-tune substrate specificity of hyperthermophilic metallo-oxidases

Flexible active-site loops fine-tune substrate specificity of hyperthermophilic metallo-oxidases

Mitigation of microbial nitrogen‐derived metabolic hazards as a driver for safer alcoholic beverage choices: An evidence‐based review and future perspectives

Electrochemistry of copper efflux oxidase-like multicopper oxidases involved in copper homeostasis

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