Functionalization of the NiTi Shape Memory Alloy Surface through Innovative Hydroxyapatite/Ag-TiO2 Hybrid Coatings.

The objective of this research was to develop a surface modification for the NiTi shape memory alloy, thereby enabling its long-term application in implant medicine. This was achieved through the creation of innovative multifunctional hybrid layers comprising a nanometric molecular system of silver-rutile (Ag-TiO2), known for its antibacterial properties, in conjunction with bioactive submicro- and nanosized hydroxyapatite (HAp). The multifunctional, continuous, crack-free coatings were produced using the electrophoretic deposition method (EPD) at 20 V/1 min. Structural and morphological analyses through Raman spectrometry and scanning electron microscopy (SEM) provided comprehensive insights into the obtained coating. The silver within the layer existed in the form of nanometric silver carbonates (Ag2CO3) and metallic nanosilver. Based on DTA/TG results, dilatometric measurements, and high-temperature microscopy, the heat treatment temperature for the deposited layers was set at 800 °C for 2 h. The procedures applied resulted in the creation of a new generation of materials with a distinct structure compared with the initial nanopowders. The resulting composite layer, measuring 2 µm in thickness, comprised hydroxyapatite (HAp), apatite carbonate (CHAp), metallic silver, silver oxides, Ag@C, and rutile exhibiting a defective structure. This structural characteristic contributes significantly to its heightened activity, influencing both bioactivity and biocompatibility properties.

Simple and efficient expression of Agaricus meleagris pyranose dehydrogenase in Pichia pastoris.

Pyranose dehydrogenase (PDH) is a fungal flavin-dependent sugar oxidoreductase that is highly interesting for applications in organic synthesis or electrochemistry. The low expression levels of the filamentous fungus Agaricus meleagris as well as the demand for engineered PDH make heterologous expression necessary. Recently, Aspergillus species were described to efficiently secrete recombinant PDH. Here, we evaluate recombinant protein production with expression hosts more suitable for genetic engineering. Expression in Escherichia coli resulted in no soluble or active PDH. Heterologous expression in the methylotrophic yeast Pichia pastoris was investigated using two different signal sequences as well as a codon-optimized sequence. A 96-well plate activity screening for transformants of all constructs was established and the best expressing clone was used for large-scale production in 50-L scale, which gave a volumetric yield of 223 mg L(-1) PDH or 1,330 U L(-1) d(-1) in space-time yield. Purification yielded 13.4 g of pure enzyme representing 95.8% of the initial activity. The hyperglycosylated recombinant enzyme had a 20% lower specific activity than the native enzyme; however, the kinetic properties were essentially identical. This study demonstrates the successful expression of PDH in the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, the feasibility of large-scale production of the enzyme with this expression system together with a simplified purification scheme for easy high-yield purification is shown.

Wiring of pyranose dehydrogenase with osmium polymers of different redox potentials.

In this study, five different flexible osmium based redox polymers were investigated for their ability to efficiently "wire" the oxidoreductase pyranose dehydrogenase (PDH, EC 1.1.99.29) from Agaricus meleagris, on graphite electrodes for possible applications in biofuel cells. A series of newly synthesised osmium based redox polymers covering the potential range between -270 and +160 mV vs. Ag|AgCl (0.1M KCl) was used. The performance of the redox polymers for enzyme wiring was investigated using glucose as substrate. The optimal operational conditions such as pH and potential were investigated.

Increasing the coulombic efficiency of glucose biofuel cell anodes by combination of redox enzymes.

A highly efficient anode for glucose biofuel cells has been developed by a combination of pyranose dehydrogenase from Agaricus meleagris (AmPDH) and cellobiose dehydrogenase from Myriococcum thermophilum (MtCDH). These two enzymes differ in how they oxidize glucose. AmPDH oxidizes glucose at the C(2) and C(3) carbon, whereas MtCDH at the C(1) carbon. Both enzymes oxidize efficiently a number of other mono- and disaccharides. They do not react directly with oxygen and produce no H(2)O(2). Electrodes were prepared by embedding (i) only AmPDH (in order to study this enzyme separately) and (ii) a mixture of AmPDH and MtCDH in an Os redox polymer hydrogel. Single-walled carbon nanotubes (SWCNTs) were added in order to enhance the current density. The electrodes were investigated with linear sweep and cyclic voltammetry in the presence of different substrates at physiological conditions. The electrochemical measurements revealed that the product of one enzyme can serve as a substrate for the other. In addition, a kinetic pathway analysis was performed by spectrophotometric measurements leading to the conclusion that up to six electrons can be gained from one glucose molecule through a combination of AmPDH and MtCDH. Hence, the combination of redox enzymes can lead to an enzymatic biofuel cell anode with an increased coulombic efficiency far beyond the usual yields of two electrons per substrate molecule.

Heterologous expression of an Agaricus meleagris pyranose dehydrogenase-encoding gene in Aspergillus spp. and characterization of the recombinant enzyme.

Pyranose dehydrogenase (PDH) is a flavin-dependant sugar oxidoreductase found in the family Agaricaceae, basidiomycetes that degrade lignocellulose-rich forest litter, and is catalytically related to the fungal enzymes pyranose 2-oxidase and cellobiose dehydrogenase. It has broad substrate specificity and displays similar activities with most sugar constituents of lignocellulose including disaccharides and oligosaccharides, a number of (substituted) quinones, and metal ions are suitable electron acceptors rather than molecular oxygen. In contrast to pyranose 2-oxidase and cellobiose dehydrogenase, which oxidize regioselectively at C-2 and C-1, respectively, PDH is capable of oxidation on C-1 to C-4 as well as double oxidations, depending on the nature of the substrate. This makes it a very interesting enzyme for biocatalytic applications, as many of the reaction products are otherwise unaccessible by chemical or enzymatic means. PDH was characterized in detail in a limited number of fungi, and the first encoding genes were isolated only recently. We report here, for the first time, the heterologous expression of one of these genes, encoding the major PDH protein in Agaricus meleagris, in the filamentous fungi Aspergillus nidulans, and Aspergillus niger.

Pyranose 2-oxidase from Phanerochaete chrysosporium--expression in E. coli and biochemical characterization.

The presented work reports the isolation and heterologous expression of the p2ox gene encoding the flavoprotein pyranose 2-oxidase (P2Ox) from the basidiomycete Phanerochaete chrysosporium. The p2ox cDNA was inserted into the bacterial expression vector pET21a(+) and successfully expressed in Escherichia coli. We obtained active, fully flavinylated recombinant P2Ox in yields of approximately 270 mg/l medium. The recombinant enzyme was provided with an N-terminal T7-tag and a C-terminal His(6)-tag to facilitate simple one-step purification. We obtained an apparently homogenous enzyme preparation with a specific activity of 16.5 U/mg. Recombinant P2Ox from P. chrysosporium was characterized in some detail with respect to its physical and catalytic properties, both for electron donor (sugar substrates) and - for the first time - alternative electron acceptors (1,4-benzoquinone, substituted quinones, 2,6-dichloroindophenol and ferricenium ion). As judged from the catalytic efficiencies k(cat)/K(m), some of these alternative electron acceptors are better substrates than oxygen, which might have implications for the proposed in vivo function of pyranose 2-oxidase.

Properties of pyranose dehydrogenase purified from the litter-degrading fungus Agaricus xanthoderma.

We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose-methanol-choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi-covalent flavinylation in PDH, 9-S-cysteinyl, 8-alpha-N3-histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are D-glucose, D-galactose, L-arabinose, and D-xylose. As shown by in situ NMR analysis, D-glucose and D-galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino-bis-(3-ethylbenzthiazolin-6-sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (k(cat)/K(m)). The enzyme may play a role in lignocellulose degradation.

Structural basis for substrate binding and regioselective oxidation of monosaccharides at C3 by pyranose 2-oxidase.

Pyranose 2-oxidase (P2Ox) participates in fungal lignin degradation by producing the H2O2 needed for lignin-degrading peroxidases. The enzyme oxidizes cellulose- and hemicellulose-derived aldopyranoses at C2 preferentially, but also on C3, to the corresponding ketoaldoses. To investigate the structural determinants of catalysis, covalent flavinylation, substrate binding, and regioselectivity, wild-type and mutant P2Ox enzymes were produced and characterized biochemically and structurally. Removal of the histidyl-FAD linkage resulted in a catalytically competent enzyme containing tightly, but noncovalently bound FAD. This mutant (H167A) is characterized by a 5-fold lower kcat, and a 35-mV lower redox potential, although no significant structural changes were seen in its crystal structure. In previous structures of P2Ox, the substrate loop (residues 452-457) covering the active site has been either disordered or in a conformation incompatible with carbohydrate binding. We present here the crystal structure of H167A in complex with a slow substrate, 2-fluoro-2-deoxy-D-glucose. Based on the details of 2-fluoro-2-deoxy-D-glucose binding in position for oxidation at C3, we also outline a probable binding mode for D-glucose positioned for regioselective oxidation at C2. The tentative determinant for discriminating between the two binding modes is the position of the O6 hydroxyl group, which in the C2-oxidation mode can make favorable interactions with Asp452 in the substrate loop and, possibly, a nearby arginine residue (Arg472). We also substantiate our hypothesis with steady-state kinetics data for the alanine replacements of Asp452 and Arg472 as well as the double alanine 452/472 mutant.