Site-directed mutations in fungal laccase: effect on redox potential, activity and pH profile

Xu, Feng; Berka, Randy M.; Wahleithner, Jill A.; Nelson, Beth A.; Shuster, Jeffrey R.; Brown, Stephen H.M.; Palmer, Amy E.; Solomon, Edward I.

doi:10.1042/bj3340063

Cited by 249 publications

(192 citation statements)

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“…Although pH optima of laccases may depend on the substrate used, laccases from white rot fungi typically have pH optima in the range of pH 3-5, which corresponds well with the result for Tv laccase [19]. The higher pH of the Mt laccase is also in accordance with result previously determined on phenolic substrates [20]. The temperature optimum of 33°C for both laccases is lower than what is commonly reported for both enzymes (50 °C) [18][19][20], but could be due to the initial rate in other studies being determined during only a few minutes of reaction on model substrates.…”

Section: Real Time Assay Of Laccase Action On Lignin By Eprsupporting

confidence: 79%

“…A second purpose was to quantitatively compare the activities of two different fungal laccases directly on three types of lignin to assess any differences or similarities in kinetic rates; the three lignin substrates employed included two pure (organosolv) lignins and a lignin-rich residue from wheat straw obtained after extensive cellulase treatment. The laccases examined included a low redox potential (0.5 V vs NHE) laccase derived from the soft rot ascomycete Myceliophthora thermophila and a high redox potential (0.7 V vs NHE) laccase originating from the white rot basidiomycete Trametes versicolor [18][19][20].…”

Section: Introductionmentioning

confidence: 99%

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Direct rate assessment of laccase catalysed radical formation in lignin by electron paramagnetic resonance spectroscopy

Munk

Andersen

Meyer

2017

Enzyme and Microbial Technology

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Highlights Time course EPR measurement of direct radical formation by fungal laccases on genuine lignin samples  Assessment of enzyme kinetic rates of radical formation in lignin by two fungal laccases  Identification of different radical formation patterns by a low and high redox potential laccase  Demonstration of different laccase rates on different types of lignin substrates Abstract Laccases (EC 1.10.3.2) catalyse removal of an electron and a proton from phenolic hydroxyl groups, including phenolic hydroxyls in lignins, to form phenoxy radicals during reduction of O2. We employed electron paramagnetic resonance spectroscopy (EPR) for real time measurement of such catalytic radical 2 formation activity on three types of lignin (two types of organosolv lignin, and a lignin rich residue from wheat straw hydrolysis) brought about by two different fungal laccases, derived from Trametes versicolor (Tv) and Myceliophthora thermophila (Mt), respectively. Laccase addition to suspensions of the individual lignin samples produced immediate time and enzyme dose dependent increases in intensity in the EPR signal with g-values in the range 2.0047-2.0050 allowing a direct quantitative monitoring of the radical formation and thus allowed laccase enzyme kinetics assessment on lignin. The experimental data verified that the laccases acted upon the insoluble lignin substrates in the suspensions. When the action on the lignin substrates of the two laccases were compared on equal enzyme dosage levels (by activity units on syringaldazine) the Mt laccase exerted a significantly faster radical formation than the Tv laccase on all three types of lignin substrates. When comparing the equal laccase dose rates on the three lignin substrates the enzymatic radical formation rate on the wheat straw lignin residue was consistently higher than that of the organosolv lignins. The pH-temperature optimum for the radical formation rate in organosolv lignin was determined by response surface methodology to pH 4.8, 33°C and pH 5.8, 33°C for the Tv laccase and the Mt laccase, respectively. The results verify direct radical formation action of fungal laccases on lignin without addition of mediators and the EPR methodology provides a new type of enzyme assay of laccases on lignin.

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Section: Real Time Assay Of Laccase Action On Lignin By Eprsupporting

confidence: 79%

Section: Introductionmentioning

confidence: 99%

Direct rate assessment of laccase catalysed radical formation in lignin by electron paramagnetic resonance spectroscopy

Munk

Andersen

Meyer

2017

Enzyme and Microbial Technology

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“…Fluoride acts as a non-competitive inhibitor by binding to the T2/T3 copper cluster, blocking the ET pathway from the T1 site to the T2/T3 cluster [15][16][37][38]. This is confirmed by the complete removal of the biocatalytic activity of ThLc-modified nanoporous gold electrodes at a low concentration (2 mM) of F -( Fig.…”

Section: Resultssupporting

confidence: 52%

“…The non-competitive inhibitor F -does not bind to the T1 site, which accepts the electrons from the substrate, but to the T2/T3 trinuclear cluster. As a result, the electron transfer pathway from the T1 to the T2/T3 redox centres is blocked and the activity of the enzyme is inhibited at low concentrations of F - [15]. In the competitive inhibition mechanism, Cl -and I -compete with electron donors for access to the T1 redox centre of the enzyme, reducing the observed activity.…”

mentioning

confidence: 99%

Direct electron transfer of Trametes hirsuta laccase adsorbed at unmodified nanoporous gold electrodes

Salaj-Kośla

Pöller

Schuhmann

et al. 2013

Bioelectrochemistry

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The enzyme Trametes hirsuta laccase undergoes direct electron transfer at unmodified nanoporous gold electrodes, displaying a current density of 28 A/cm 2 . The response indicates that ThLc was immobilised at the surface of the nanopores in a manner which promoted direct electron transfer, in contrast to the absence of a response at unmodified polycrystalline gold electrodes. The bioelectrocatalytic activity of ThLc modified nanoporous gold electrodes was strongly dependent on the presence of halide ions. Fluoride completely inhibited the enzymatic response, whereas in the presence of 150 mM Cl -, the current was reduced to 50% of the response in the absence of Cl -. The current increased by 40% when the temperature was increased from 20°C to 37°C. The response is limited by enzymatic and/or enzyme electrode kinetics and is 30% of that observed for ThLc co-immobilised with an osmium redox polymer. Keywords: Laccase, direct electron transfer, nanoporous gold 2 IntroductionElectron transfer (ET) reactions are ubiquitous in nature. Controlling the rate of these reactions can be of significant benefit in developing biochemical systems that utilise redox proteins and enzymes and in particular, in applications that provide power for implanted or portable electronic devices. ET can be achieved directly (direct electron transfer, DET) or by the use of mediators (mediated electron transfer, MET) to shuttle electrons between the redox centre of the enzyme and the electrode. However, mediators are unselective and can also give rise to interfering effects [1][2][3]. DET-based devices offer high selectivity and sensitivity due to the absence of mediators. In addition, devices based on DET operate at potentials close to the redox potential of the enzyme, maximising the potential difference between the cathode and anode for biofuel cell applications [2]. Moreover, DET can be used to provide detailed information on the kinetics and thermodynamics of the ET process. However, the low stability of the enzyme layer together with the long distances over which ET occurs, represent major obstacles for DET based devices. In addition, the shielding mechanism of the enzymatic redox centre by the protein shell can disrupt DET [2,4]. One approach in optimizing and enabling rapid DET is to design the electrode surface with an architecture which promotes efficient rates of ET. In this way the electrode morphology ensures the most efficient orientation of the enzymatic redox centre and facilitates communication with the electroactive surface. Porous electrodes can be used to encapsulate the enzyme within a network of cavities, shortening the distance for electron transfer to the redox active site, which will promote more efficient and rapid rates of electron transfer [5]. However, the number of redox enzymes capable of interacting directly with the electrode while catalyzing the enzymatic reaction is limited, with estimates of approximately 5% of all enzymes exhibiting such a response [6].DET in enzymes was first described in 1978 fo...

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“…canonical ligand corresponds to a Leu residue (L506) instead of the Met or Phe residues observed in ascorbate oxidases and some laccases, respectively (Xu et al, 1998). Further analysis of this alignment can be found in the Discussion.…”

Section: Identification and Characterization Of Pc-fet3mentioning

confidence: 99%

Cloning and characterization of the genes encoding the high-affinity iron-uptake protein complex Fet3/Ftr1 in the basidiomycete Phanerochaete chrysosporium

et al. 2007

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Site-directed mutations in fungal laccase: effect on redox potential, activity and pH profile

Cited by 249 publications

References 32 publications

Direct rate assessment of laccase catalysed radical formation in lignin by electron paramagnetic resonance spectroscopy

Direct rate assessment of laccase catalysed radical formation in lignin by electron paramagnetic resonance spectroscopy

Direct electron transfer of Trametes hirsuta laccase adsorbed at unmodified nanoporous gold electrodes

Cloning and characterization of the genes encoding the high-affinity iron-uptake protein complex Fet3/Ftr1 in the basidiomycete Phanerochaete chrysosporium

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