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Evolving stability and pH-dependent activity of the high redox potential Botrytis aclada laccase for enzymatic fuel cells.

ABSTRACT: Fungal high redox potential laccases are proposed as cathodic biocatalysts in implantable enzymatic fuel cells to generate high cell voltages. Their application is limited mainly through their acidic pH optimum and chloride inhibition. This work investigates evolutionary and engineering strategies to increase the pH optimum of a chloride-tolerant, high redox potential laccase from the ascomycete Botrytis aclada. The laccase was subjected to two rounds of directed evolution and the clones screened for increased stability and activity at pH 6.5. Beneficial mutation sites were investigated by semi-rational and combinatorial mutagenesis. Fourteen variants were characterised in detail to evaluate changes of the kinetic constants. Mutations increasing thermostability were distributed over the entire structure. Among them, T383I showed a 2.6-fold increased half-life by preventing the loss of the T2 copper through unfolding of a loop. Mutations affecting the pH-dependence cluster around the T1 copper and categorise in three types of altered pH profiles: pH-type I changes the monotonic decreasing pH profile into a bell-shaped profile, pH-type II describes increased specific activity below pH 6.5, and pH-type III increased specific activity above pH 6.5. Specific activities of the best variants were up to 5-fold higher (13 U mg-1) than BaL WT at pH 7.5.

SUBMITTER: Scheiblbrandner S

PROVIDER: S-EPMC5651918 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Effect of the L499M mutation of the ascomycetous Botrytis aclada laccase on redox potential and catalytic properties.

Project description:Laccases are members of a large family of multicopper oxidases that catalyze the oxidation of a wide range of organic and inorganic substrates accompanied by the reduction of dioxygen to water. These enzymes contain four Cu atoms per molecule organized into three sites: T1, T2 and T3. In all laccases, the T1 copper ion is coordinated by two histidines and one cysteine in the equatorial plane and is covered by the side chains of hydrophobic residues in the axial positions. The redox potential of the T1 copper ion influences the enzymatic reaction and is determined by the nature of the axial ligands and the structure of the second coordination sphere. In this work, the laccase from the ascomycete Botrytis aclada was studied, which contains conserved Ile491 and nonconserved Leu499 residues in the axial positions. The three-dimensional structures of the wild-type enzyme and the L499M mutant were determined by X-ray crystallography at 1.7 Å resolution. Crystals suitable for X-ray analysis could only be grown after deglycosylation. Both structures did not contain the T2 copper ion. The catalytic properties of the enzyme were characterized and the redox potentials of both enzyme forms were determined: E0 = 720 and 580 mV for the wild-type enzyme and the mutant, respectively. Since the structures of the wild-type and mutant forms are very similar, the change in the redox potential can be related to the L499M mutation in the T1 site of the enzyme.

| S-EPMC4220974 | biostudies-literature

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

Project description:A Myceliophthora thermophila laccase and a Rhizoctonia solani laccase were mutated on a pentapeptide segment believed to be near the type-1 Cu site. The mutation L513F in Myceliophthora laccase and the mutation L470F in Rhizoctonia laccase took place at a position corresponding to the type-1 Cu axial methionine (M517) ligand in Zucchini ascorbate oxidase. The triple mutations V509L,S510E,G511A in Myceliophthora laccase and L466V,E467S,A468G in Rhizoctonia laccase involved a sequence segment whose homologue in ascorbate oxidase is flanked by the M517 and a type-1 Cu-ligating histidine (H512). The single mutation did not yield significant changes in the enzymic properties (including any significant increase in the redox potential of the type-1 Cu). In contrast, the triple mutation resulted in several significant changes. In comparison with the wild type, the Rhizoctonia and Myceliophthora laccase triple mutants had a phenol-oxidase activity whose pH optimum shifted 1 unit lower and higher, respectively. Although the redox potentials were not significantly altered, the Km, kcat and fluoride inhibition of the laccases were greatly changed by the mutations. The observed effects are interpreted as possible mutation-induced structural perturbations on the molecular recognition between the reducing substrate and laccase and on the electron transfer from the substrate to the type-1 Cu centre.

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Anode-Driven Controlled Release of Cathodic Fuel via pH Response for Smart Enzymatic Biofuel Cell.

Project description:Enzymatic biofuel cells (EBFCs) with or without a membrane to separate the anodic and cathodic compartments generally suffered from high internal resistance or interactive interference, both of which restricted the improvement of their performance. Herein, a smart membrane-less EBFC was engineered based on anode-driven controlled release of cathodic acceptor via pH-responsive metal-organic framework ([Fe(CN)6]3-@ZIF-8) nanocarriers. The glucose anodic oxidation would produce gluconic acid accompanied by the change in pH value from neutral to the acidic case, which could drive the degradation of [Fe(CN)6]3-@ZIF-8 nanocarriers and further realize the controlled release of cathodic acceptor [Fe(CN)6]3-. More importantly, compared with controlled EBFC with or without membrane, the power output of the as-proposed EBFC enhanced at least 700 times due to the seamless electronic communication. Therefore, the ingenious strategy not only realized the successful engineering of the membrane-less EBFC but also provided an appealing idea for constructing smart devices.

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2018-11-30 | GSE115226 | GEO

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