Bilirubin Oxidase Activity of
            <i>Bacillus subtilis</i>
            CotA

Sakasegawa, Shin-ichi; Ishikawa, Hidehiko; Imamura, Shigeyuki; Sakuraba, Haruhiko; Goda, Shuichiro; Ohshima, Toshihisa

doi:10.1128/aem.72.1.972-975.2006

Cited by 39 publications

(46 citation statements)

References 21 publications

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“…Same may be said for other bacterial laccases. For example, the spore coat protein CotA from B. subtilis is a model bacterial laccase, yet it has been shown to be a billirubin oxidase (Sakasegawa et al 2006) and can also use N 2 O as the electron acceptor (Fernandes et al 2010), which is not typical for a laccase. Similarly, CueO from E. coli can oxidize many of the classical laccase substrates, but it has been shown to have a strong cuprous oxidase activity and is, in fact, involved in vivo in the copper homeostasis (Singh et al 2004).…”

Section: Discussionmentioning

confidence: 98%

Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity in Thioalkalivibrio sp

Ausec

Črnigoj

Šnajder

et al. 2015

Appl Microbiol Biotechnol

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Laccases are oxidoreductases mostly studied in fungi, while bacterial laccases remain poorly studied despite their high genetic diversity and potential for biotechnological application. Our previous bioinformatic analysis identified alkaliphilic bacterial strains Thioalkalivibrio sp. as potential sources of robust bacterial laccases that would be stable at high pH. In the present work, a gene for a laccase-like enzyme from Thioalkalivibrio sp. ALRh was cloned and expressed as a 6× His-tagged protein in Escherichia coli. The purified enzyme was a pH-tolerant laccase stable in the pH range between 2.1 and 9.9 at 20 °C as shown by intrinsic fluorescence emission spectrometry. It had optimal activities at pH 5.0 and pH 9.5 with the laccase substrates 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,6-dimethoxyphenol, respectively. In addition, it could oxidize several other monophenolic compounds and potassium hexacyanoferrate(II) but not tyrosine. It showed highest activity at 50 °C, making it suitable for prolonged incubations at this temperature. The present study shows that Thioalkalivibrio sp. encodes an active, alkaliphilic, and thermo-tolerant laccase and contributes to our understanding of the versatility of bacterial laccase-like multicopper oxidases in general.

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Section: Discussionmentioning

confidence: 98%

Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity in Thioalkalivibrio sp

Ausec

Črnigoj

Šnajder

et al. 2015

Appl Microbiol Biotechnol

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“…The affinity for O 2 of MCOs such as BOD or LAC is low because the in vivo function is not to reduce oxygen [K m is in the order of hundreds of micromoles (see Table 1)] [56,[71][72][318][319][320][321][322] . A direct Figure 13.…”

Section: Search For Enzymes With High O 2 Affinitymentioning

confidence: 99%

“…124/322 Rhus vernicifera [56] Plant LAC 0.22 [b] 103/560 [320] Trametes hirsute (Th) [56] Fungus LAC 0.57 [b] 220/210 Sreptomyces coelicolor (Sc) [57] Bacterium LAC 0.47 (pH 5.5) [c] 3600/5.8 [321] E. coli [329] Bacterium CcO aa3 0.2/n.d. Bradyrhizobium japonicum [330] Bacterium CcO cbb3 0.007/n.d.…”

Section: Enzymes For O 2 Reductionmentioning

confidence: 99%

Biohydrogen for a New Generation of H₂/O₂ Biofuel Cells: A Sustainable Energy Perspective

et al. 2014

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Among sustainable alternatives to fossil fuel, proton exchange membrane fuel cells are promising devices that deliver electricity from hydrogen and oxygen, producing only water. The transformation of the fuel and oxidant however relies on rare‐metal catalysts. H2/O2 enzymatic biofuel cells thus emerge as sustainable biotechnological devices in which chemical catalysts are replaced by hydrogenases at the anode and multicopper oxidases at the cathode. This review discusses the recent breakthroughs and the limitations in all the components of H2/O2 biofuel cells: 1) Catalytic mechanisms involved in multicopper oxidases and hydrogenases, in addition to the new potential of enzymes from the biodiversity pool, which exhibit outstanding properties. 2) The molecular basis for oriented enzyme immobilization on the electrochemical interfaces as a prerequisite for a fast direct electron‐transfer rate. 3) The requirement for 3D networks to enhance current densities. 4) The production of H2 from biomass. 5) The history of H2/O2 biofuel cells and recent devices, which also highlights the remaining issues that will allow the use of such devices in low‐power applications.

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“…The B. subtilis endospore coat protein, CotA, which is a prokaryotic laccase-like protein when expressed in E. coli was initially found to accumulate in IBs and initial refolding experiments were similarly unsuccessful (Martins et al 2002), but conditions were obtained to enable the production of a soluble and functional CotA protein. (Sakasegawa et al 2006) And, the fungal laccase from C. bulleri was successfully refolded and found to be active after subjecting it to several denaturating and metal chelating agents. (Salony et al 2008) Unlike TvL, this laccase does not contain cysteine residues which may have simplified the refolding process for this protein.…”

Section: Discussionmentioning

confidence: 99%

Pushing the limits of automatic computational protein design: design, expression, and characterization of a large synthetic protein based on a fungal laccase scaffold

et al. 2011

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The de novo engineering of new proteins will allow the design of complex systems in synthetic biology. But the design of large proteins is very challenging due to the large combinatorial sequence space to be explored and the lack of a suitable selection system to guide the evolution and optimization. One way to approach this challenge is to use computational design methods based on the current crystallographic data and on molecular mechanics. We have used a laccase protein fold as a scaffold to design a new protein sequence that would adopt a 3D conformation in solution similar to a wild-type protein, the Trametes versicolor (TvL) fungal laccase. Laccases are multi-copper oxidases that find utility in a variety of industrial applications. The laccases with highest activity and redox potential are generally secreted fungal glycoproteins. Prokaryotic laccases have been identified with some desirable features, but they often exhibit low redox potentials. The designed sequence (DLac) shares a 50% sequence identity to the original TvL protein.The new DLac gene was overexpressed in E. coli and the majority of the protein was found in inclusion bodies. Both soluble protein and refolded insoluble protein were purified, and their identity was verified by mass spectrometry. Neither protein exhibited the characteristic T1 copper absorbance, neither bound copper by atomic absorption, and neither was active using a variety of laccase substrates over a range of pH values. Circular dichroism spectroscopy studies suggest that the DLac protein adopts a molten globule structure that is similar to the denatured and refolded native fungal TvL protein, which is significantly different from the natively secreted fungal protein. Taken together, these results indicate that the computationally designed DLac expressed in E. coli is unable to utilize the same folding pathway that is used in the expression of the parent TvL protein or the prokaryotic laccases. This sequence can be used going forward to help elucidate the sequence requirements needed for prokaryotic multi-copper oxidase expression.

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Bilirubin Oxidase Activity of Bacillus subtilis CotA

Cited by 39 publications

References 21 publications

Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity in Thioalkalivibrio sp

Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity in Thioalkalivibrio sp

Biohydrogen for a New Generation of H₂/O₂ Biofuel Cells: A Sustainable Energy Perspective

Pushing the limits of automatic computational protein design: design, expression, and characterization of a large synthetic protein based on a fungal laccase scaffold

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Bilirubin Oxidase Activity of Bacillus subtilis CotA

Cited by 39 publications

References 21 publications

Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity in Thioalkalivibrio sp

Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity in Thioalkalivibrio sp

Biohydrogen for a New Generation of H2/O2 Biofuel Cells: A Sustainable Energy Perspective

Pushing the limits of automatic computational protein design: design, expression, and characterization of a large synthetic protein based on a fungal laccase scaffold

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Product

Resources

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Biohydrogen for a New Generation of H₂/O₂ Biofuel Cells: A Sustainable Energy Perspective