Lytic Polysaccharide Monooxygenase from Talaromyces amestolkiae with an Enigmatic Linker-like Region: The Role of This Enzyme on Cellulose Saccharification.

The first lytic polysaccharide monooxygenase (LPMO) detected in the genome of the widespread ascomycete Talaromyces amestolkiae (TamAA9A) has been successfully expressed in Pichia pastoris and characterized. Molecular modeling of TamAA9A showed a structure similar to those from other AA9 LPMOs. Although fungal LPMOs belonging to the genera Penicillium or Talaromyces have not been analyzed in terms of regioselectivity, phylogenetic analyses suggested C1/C4 oxidation which was confirmed by HPAEC. To ascertain the function of a C-terminal linker-like region present in the wild-type sequence of the LPMO, two variants of the wild-type enzyme, one without this sequence and one with an additional C-terminal carbohydrate binding domain (CBM), were designed. The three enzymes (native, without linker and chimeric variant with a CBM) were purified in two chromatographic steps and were thermostable and active in the presence of H2O2. The transition midpoint temperature of the wild-type LPMO (Tm = 67.7 °C) and its variant with only the catalytic domain (Tm = 67.6 °C) showed the highest thermostability, whereas the presence of a CBM reduced it (Tm = 57.8 °C) and indicates an adverse effect on the enzyme structure. Besides, the potential of the different T. amestolkiae LPMO variants for their application in the saccharification of cellulosic and lignocellulosic materials was corroborated.

Scanning accuracy of nondental structured light extraoral scanners compared with that of a dental-specific scanner.

STATEMENT OF PROBLEM: Diagnostic stone casts can be digitized by using dental optical scanners based on structured light scanning technology. Nondental structured light scanning scanners could also be used; however, the accuracy of these nondental scanners remains unclear. PURPOSE: The purpose of this in vitro study was to measure the scanning accuracy (trueness and precision) of 3 nondental extraoral structured light scanners. MATERIAL AND METHODS: A representative maxillary diagnostic cast was obtained and digitized by using an extraoral dental scanner (Advaa Lab Scan; GC Europe), and a reference or control standard tessellation language file was obtained. Three nondental extraoral scanners were evaluated: groups ND-1 (Space Spider; Artec), ND-2 (Capture Mini; Geomagic), and ND-3 (DAVID SLS3; David). Ten digital scans per group were recorded at a constant room temperature (23 °C) by an experienced geodetic engineer following the manufacturer's recommendations. The control or reference file was used as a reference to measure the discrepancy between the digitized diagnostic cast and 3 different nondental scans by using an open-source software (CloudCompare v.2.6.1; CloudCompare) and the iterative closest point technique. The Shapiro-Wilk test revealed that the data were normally distributed. The data were analyzed by using 1-way ANOVA, followed by post hoc Bonferroni tests (α=.05). RESULTS: Significant differences between the 3 experimental nondental scanners and the control or reference scan (P<.001) were found. The ND-2 group had the lowest absolute mean error (trueness) and standard deviation (precision) (39 ±139 µm), followed by the ND-3 group (125 ±113 µm) and the ND1 group (-397 ±25 µm). No statistically significant differences were found in the mean error between the ND-2 and ND-3 groups (P=.228). CONCLUSIONS: Only 1 nondental extraoral scanner tested obtained trueness mean values similar to those of the reference dental scanner. In all groups, the precision mean values were higher than their trueness values, indicating low relative precision.

Lignin-degrading peroxidases from genome of selective ligninolytic fungus Ceriporiopsis subvermispora.

The white-rot fungus Ceriporiopsis subvermispora delignifies lignocellulose with high selectivity, but until now it has appeared to lack the specialized peroxidases, termed lignin peroxidases (LiPs) and versatile peroxidases (VPs), that are generally thought important for ligninolysis. We screened the recently sequenced C. subvermispora genome for genes that encode peroxidases with a potential ligninolytic role. A total of 26 peroxidase genes was apparent after a structural-functional classification based on homology modeling and a search for diagnostic catalytic amino acid residues. In addition to revealing the presence of nine heme-thiolate peroxidase superfamily members and the unexpected absence of the dye-decolorizing peroxidase superfamily, the search showed that the C. subvermispora genome encodes 16 class II enzymes in the plant-fungal-bacterial peroxidase superfamily, where LiPs and VPs are classified. The 16 encoded enzymes include 13 putative manganese peroxidases and one generic peroxidase but most notably two peroxidases containing the catalytic tryptophan characteristic of LiPs and VPs. We expressed these two enzymes in Escherichia coli and determined their substrate specificities on typical LiP/VP substrates, including nonphenolic lignin model monomers and dimers, as well as synthetic lignin. The results show that the two newly discovered C. subvermispora peroxidases are functionally competent LiPs and also suggest that they are phylogenetically and catalytically intermediate between classical LiPs and VPs. These results offer new insight into selective lignin degradation by C. subvermispora.

Kinetic and chemical characterization of aldehyde oxidation by fungal aryl-alcohol oxidase.

Fungal AAO (aryl-alcohol oxidase) provides H2O2 for lignin biodegradation. AAO is active on benzyl alcohols that are oxidized to aldehydes. However, during oxidation of some alcohols, AAO forms more than a stoichiometric number of H2O2 molecules with respect to the amount of aldehyde detected due to a double reaction that involves aryl-aldehyde oxidase activity. The latter reaction was investigated using different benzylic aldehydes, whose oxidation to acids was demonstrated by GC-MS. The steady- and presteady state kinetic constants, together with the chromatographic results, revealed that the presence of substrate electron-withdrawing or electron-donating substituents had a strong influence on activity; the highest activity was with p-nitrobenzaldehyde and halogenated aldehydes and the lowest with methoxylated aldehydes. Moreover, activity was correlated to the aldehyde hydration rates estimated by 1H-NMR. These findings, together with the absence in the AAO active site of a residue able to drive oxidation via an aldehyde thiohemiacetal, suggested that oxidation mainly proceeds via the gem-diol species. The reaction mechanism (with a solvent isotope effect, 2H2Okred, of approx. 1.5) would be analogous to that described for alcohols, the reductive half-reaction involving concerted hydride transfer from the alpha-carbon and proton abstraction from one of the gem-diol hydroxy groups by a base. The existence of two steps of opposite polar requirements (hydration and hydride transfer) explains some aspects of aldehyde oxidation by AAO. Site-directed mutagenesis identified two histidine residues strongly involved in gem-diol oxidation and, unexpectedly, suggested that an active-site tyrosine residue could facilitate the oxidation of some aldehydes that show no detectable hydration. Double alcohol and aldehyde oxidase activities of AAO would contribute to H2O2 supply by the enzyme.

Hemicellulases from Penicillium and Talaromyces for lignocellulosic biomass valorization: A review.

The term hemicellulose groups different polysaccharides with heterogeneous structures, mannans, xyloglucans, mixed-linkage ß-glucans and xylans, which differ in their backbone and branches, and in the type and distribution of glycosidic linkages. The enzymatic degradation of these complex polymers requires the concerted action of multiple hemicellulases and auxiliary enzymes. Most commercial enzymes are produced by Trichoderma and Aspergillus species, but recent studies have disclosed Penicillium and Talaromyces as promising sources of hemicellulases. In this review, we summarize the current knowledge on the hemicellulolytic system of these genera, and the role of hemicellulases in the disruption and synthesis of glycosidic bonds. In both cases, the enzymes from Penicillium and Talaromyces represent an interesting alternative for valorization of lignocellulosic biomass in the current framework of circular economy.

Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases.

Versatile peroxidase (VP) is defined by its capabilities to oxidize the typical substrates of other basidiomycete peroxidases: (i) Mn(2+), the manganese peroxidase (MnP) substrate (Mn(3+) being able to oxidize phenols and initiate lipid peroxidation reactions); (ii) veratryl alcohol (VA), the typical lignin peroxidase (LiP) substrate; and (iii) simple phenols, which are the substrates of Coprinopsis cinerea peroxidase (CIP). Crystallographic, spectroscopic, directed mutagenesis, and kinetic studies showed that these 'hybrid' properties are due to the coexistence in a single protein of different catalytic sites reminiscent of those present in the other basidiomycete peroxidase families. Crystal structures of wild and recombinant VP, and kinetics of mutated variants, revealed certain differences in its Mn-oxidation site compared with MnP. These result in efficient Mn(2+) oxidation in the presence of only two of the three acidic residues forming its binding site. On the other hand, a solvent-exposed tryptophan is the catalytically-active residue in VA oxidation, initiating an electron transfer pathway to haem (two other putative pathways were discarded by mutagenesis). Formation of a tryptophanyl radical after VP activation by peroxide was detected using electron paramagnetic resonance. This was the first time that a protein radical was directly demonstrated in a ligninolytic peroxidase. In contrast with LiP, the VP catalytic tryptophan is not beta-hydroxylated under hydrogen peroxide excess. It was also shown that the tryptophan environment affected catalysis, its modification introducing some LiP properties in VP. Moreover, some phenols and dyes are oxidized by VP at the edge of the main haem access channel, as found in CIP. Finally, the biotechnological interest of VP is discussed.

An anamorph of the white-rot fungus Bjerkandera adusta capable of colonizing and degrading compact disc components.

A Geotrichum-like fungus isolated from a biodeteriorated compact disc (CD) was able to degrade in vitro the components of different CD types. The fungal hyphae inside the CD fragments grew through the aluminium layer and produced the solubilization of this metal. Furthermore, examination of CDs by scanning electron microscopy showed that the fungus was able to destroy the pits and lands structures grooved in the polycarbonate layer, confirming degradation of this aromatic polymer. The fungus secretes aryl-alcohol oxidase and Mn2+-oxidizing peroxidase, two kinds of oxidoreductases characteristic of ligninolytic basidiomycetes. Analysis of the ITS region of ribosomal DNA, as well as the morphological characteristics, the lack of sexual forms and the profile of enzymes secreted in liquid medium identified the fungus as a Geotrichum-like anamorph of Bjerkandera adusta (Willd.) P. Karst.

Lignin modification during Eucalyptus globulus kraft pulping followed by totally chlorine-free bleaching: a two-dimensional nuclear magnetic resonance, Fourier transform infrared, and pyrolysis-gas chromatography/mass spectrometry study.

Chemical modification of eucalypt lignin was investigated during kraft pulping and chlorine-free bleaching by comparing milled wood lignin, kraft lignin, and pulp enzymatic residual lignins. The syringyl-to-guaiacyl ratio (S/G) from analytical pyrolysis slightly changed during pulping and bleaching (S/G, 3-4) but was higher in the kraft lignin. Semiquantitative heteronuclear single quantum correlation (HSQC) nuclear magnetic resonance (NMR) showed that the relative amount of beta-O-4' (around 80% side chains) and resinol type substructures (15%) was slightly modified during pulping and oxygen delignification. However, a decrease of resinol substructures (to only 6%) was found after alkaline peroxide bleaching. The relative amount of surviving linkages in the highly phenolic kraft lignin was dramatically modified; resinols were predominant. Oxygen delignification did not change interunit linkages, but a relative increase of oxidized units was found in the HSQC aromatic region, in agreement with the small increase of pyrolysis markers with oxidized side chains. NMR heteronuclear multiple bond correlations showed that the oxidized units after oxygen delignification bore conjugated ketone groups.

Versatile peroxidase oxidation of high redox potential aromatic compounds: site-directed mutagenesis, spectroscopic and crystallographic investigation of three long-range electron transfer pathways.

Versatile peroxidases (VP), a recently described family of ligninolytic peroxidases, show a hybrid molecular architecture combining different oxidation sites connected to the heme cofactor. High-resolution crystal structures as well as homology models of VP isoenzymes from the fungus Pleurotus eryngii revealed three possibilities for long-range electron transfer for the oxidation of high redox potential aromatic compounds. The possible pathways would start either at Trp164 or His232 of isoenzyme VPL, and at His82 or Trp170 of isoenzyme VPS1. These residues are exposed, and less than 11 A apart from the heme. With the purpose of investigating their functionality, two single mutations (W164S and H232F) and one double mutation (W164S/P76H) were introduced in VPL that: (i) removed the two pathways in this isoenzyme; and (ii) incorporated the absent putative pathway. Analysis of the variants showed that Trp164 is required for oxidation of two high redox potential model substrates (veratryl alcohol and Reactive Black 5), whereas the two other pathways (starting at His232 and His82) are not involved in long-range electron transfer (LRET). None of the mutations affected Mn2+ oxidation, which would take place at the opposite side of the enzyme. Substitution of Trp164 by His also resulted in an inactive variant, indicating that an indole side-chain is required for activity. It is proposed that substrate oxidation occurs via a protein-based radical. For the first time in a ligninolytic peroxidase such an intermediate species could be detected by low-temperature electron paramagnetic resonance of H2O2-activated VP, and was found to exist at Trp164 as a neutral radical. The H2O2-activated VP was self-reduced in the absence of reducing substrates. Trp164 is also involved in this reaction, which in the W164S variant was blocked at the level of compound II. When analyzing VP crystal structures close to atomic resolution, no hydroxylation of the Trp164 Cbeta atom was observed (even after addition of several equivalents of H2O2). This is in contrast to lignin peroxidase Trp171. Analysis of the crystal structures of both peroxidases showed differences in the environment of the protein radical-forming residue that could affect its reactivity. These variations would also explain differences found for the oxidation of some high redox potential aromatic substrates.

Comparison of different fungal enzymes for bleaching high-quality paper pulps.

Wild and recombinant hydrolases and oxidoreductases with a potential interest for environmentally sound bleaching of high-quality paper pulp (from flax) were incorporated into a totally chlorine free (TCF) sequence that also included a peroxide stage. The ability of feruloyl esterase (from Aspergillus niger) and Mn2+-oxidizing peroxidases (from Phanerochaete chrysosporium and Pleurotus eryngii) to decrease the final lignin content of flax pulp was shown. Laccase from Pycnoporus cinnabarinus (without mediator) also caused a slight improvement of pulp brightness that was increased in the presence of aryl-alcohol oxidase. However, the best results were obtained when the laccase treatment was performed in the presence of a mediator, 1-hydroxybenzotriazol (HBT), enabling strong delignification of pulps. The enzymatic removal of lignin resulted in high-final brightness values that are difficult to attain by chemical bleaching of this type of pulp. A partial inactivation of laccase by HBT was observed but this negative effect was strongly reduced in the presence of pulp. The good results obtained with the same laccase expressed in A. niger at bioreactor scale, revealed the feasibility of using recombinant laccase for bleaching high-quality non-wood pulps in the presence of a mediator.

Pleurotus ostreatus heme peroxidases: an in silico analysis from the genome sequence to the enzyme molecular structure.

An exhaustive screening of the Pleurotus ostreatus genome was performed to search for nucleotide sequences of heme peroxidases in this white-rot fungus, which could be useful for different biotechnological applications. After sequence identification and manual curation of the corresponding genes and cDNAs, the deduced amino acid sequences were converted into structural homology models. A comparative study of these sequences and their structural models with those of known fungal peroxidases revealed the complete inventory of heme peroxidases of this fungus. This consists of cytochrome c peroxidase and ligninolytic peroxidases, including manganese peroxidase and versatile peroxidase but not lignin peroxidase, as representative of the "classical" superfamily of plant, fungal, and bacterial peroxidases; and members of two relatively "new" peroxidase superfamilies, namely heme-thiolate peroxidases, here described for the first time in a fungus from the genus Pleurotus, and dye-decolorizing peroxidases, already known in P. ostreatus but still to be thoroughly explored and characterized.

Enzymatic delignification of plant cell wall: from nature to mill.

Lignin removal is a central issue in paper pulp manufacture, and production of other renewable chemicals, materials, and biofuels in future lignocellulose biorefineries. Biotechnology can contribute to more efficient and environmentally sound deconstruction of plant cell wall by providing tailor-made biocatalysts based on the oxidative enzymes responsible for lignin attack in Nature. With this purpose, the already-known ligninolytic oxidoreductases are being improved using (rational and random-based) protein engineering, and still unknown enzymes will be identified by the application of the different 'omics' technologies. Enzymatic delignification will be soon at the pulp mill (combined with pitch removal) and our understanding of the reactions produced will increase by using modern techniques for lignin analysis.

Protein radicals in fungal versatile peroxidase: catalytic tryptophan radical in both compound I and compound II and studies on W164Y, W164H, and W164S variants.

Lignin-degrading peroxidases, a group of biotechnologically interesting enzymes, oxidize high redox potential aromatics via an exposed protein radical. Low temperature EPR of Pleurotus eryngii versatile peroxidase (VP) revealed, for the first time in a fungal peroxidase, the presence of a tryptophanyl radical in both the two-electron (VPI) and the one-electron (VPII) activated forms of the enzyme. Site-directed mutagenesis was used to substitute this tryptophan (Trp-164) by tyrosine and histidine residues. No changes in the crystal structure were observed, indicating that the modified behavior was due exclusively to the mutations introduced. EPR revealed the formation of tyrosyl radicals in both VPI and VPII of the W164Y variant. However, no protein radical was detected in the W164H variant, whose VPI spectrum indicated a porphyrin radical identical to that of the inactive W164S variant. Stopped-flow spectrophotometry showed that the W164Y mutation reduced 10-fold the apparent second-order rate constant for VPI reduction (k(2app)) by veratryl alcohol (VA), when compared with over 50-fold reduction in W164S, revealing some catalytic activity of the tyrosine radical. Its first-order rate constant (k(2)) was more affected than the dissociation constant (K(D)(2)). Moreover, VPII reduction by VA was impaired by the above mutations, revealing that the Trp-164 radical was involved in catalysis by both VPI and VPII. The low first-order rate constant (k(3)) values were similar for the W164Y, W164H, and W164S variants, indicating that the tyrosyl radical in VPII was not able to oxidize VA (in contrast with that observed for VPI). VPII self-reduction was also suppressed, revealing that Trp-164 is involved in this autocatalytic process.

Lignin-derived compounds as efficient laccase mediators for decolorization of different types of recalcitrant dyes.

Ten phenols were selected as natural laccase mediators after screening 44 different compounds with a recalcitrant dye (Reactive Black 5) as a substrate. Their performances were evaluated at different mediator/dye ratios and incubation times (up to 6 h) by the use of Pycnoporus cinnabarinus and Trametes villosa laccases and were compared with those of eight known synthetic mediators (including -NOH- compounds). Among the six types of dyes assayed, only Reactive Blue 38 (phthalocyanine) was resistant to laccase-mediator treatment under the conditions used. Acid Blue 74 (indigoid dye), Reactive Blue 19 (anthraquinoid dye), and Aniline Blue (triarylmethane-type dye) were partially decolorized by the laccases alone, although decolorization was much more efficient and rapid with mediators, whereas Reactive Black 5 (diazo dye) and Azure B (heterocyclic dye) could be decolorized only in the presence of mediators. The efficiency of each natural mediator depended on the type of dye to be treated but, with the only exception being Azure B (< 50% decolorization), nearly complete decolorization (80 to 100%) was attained in all cases. Similar rates were attained with the best synthetic mediators, but the reactions were significantly slower. Phenolic aldehydes, ketones, acids, and esters related to the three lignin units were among the best mediators, including p-coumaric acid, vanillin, acetovanillone, methyl vanillate, and above all, syringaldehyde and acetosyringone. The last two compounds are especially promising as ecofriendly (and potentially cheap) mediators for industrial applications since they provided the highest decolorization rates in only 5 to 30 min, depending on the type of dye to be treated.

Induction, isolation, and characterization of two laccases from the white rot basidiomycete Coriolopsis rigida.

Previous work has shown that the white rot fungus Coriolopsis rigida degraded wheat straw lignin and both the aliphatic and aromatic fractions of crude oil from contaminated soils. To better understand these processes, we studied the enzymatic composition of the ligninolytic system of this fungus. Since laccase was the sole ligninolytic enzyme found, we paid attention to the oxidative capabilities of this enzyme that would allow its participation in the mentioned degradative processes. We purified two laccase isoenzymes to electrophoretic homogeneity from copper-induced cultures. Both enzymes are monomeric proteins, with the same molecular mass (66 kDa), isoelectric point (3.9), N-linked carbohydrate content (9%), pH optima of 3.0 on 2,6-dimethoxyphenol (DMP) and 2.5 on 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), absorption spectrum, and N-terminal amino acid sequence. They oxidized 4-anisidine and numerous phenolic compounds, including methoxyphenols, hydroquinones, and lignin-derived aldehydes and acids. Phenol red, an unusual substrate of laccase due to its high redox potential, was also oxidized. The highest enzyme affinity and efficiency were obtained with ABTS and, among phenolic compounds, with 2,6-dimethoxyhydroquinone (DBQH(2)). The presence of ABTS in the laccase reaction expanded the substrate range of C. rigida laccases to nonphenolic compounds and that of MBQH(2) extended the reactions catalyzed by these enzymes to the production of H(2)O(2), the oxidation of Mn(2+), the reduction of Fe(3+), and the generation of hydroxyl radicals. These results confirm the participation of laccase in the production of oxygen free radicals, suggesting novel uses of this enzyme in degradative processes.