Several members of the glycoside hydrolase 61 (GH61) family of proteins have recently been shown to dramatically increase the breakdown of lignocellulosic biomass by microbial hydrolytic cellulases. However, purified GH61 proteins have neither demonstrable direct hydrolase activity on various polysaccharide or lignacious components of biomass nor an apparent hydrolase active site. Cellobiose dehydrogenase (CDH) is a secreted flavocytochrome produced by many cellulose-degrading fungi with no well-understood biological function. Here we demonstrate that the binary combination of Thermoascus aurantiacus GH61A (TaGH61A) and Humicola insolens CDH (HiCDH) cleaves cellulose into soluble, oxidized oligosaccharides. TaGH61A-HiCDH activity on cellulose is shown to be nonredundant with the activities of canonical endocellulase and exocellulase enzymes in microcrystalline cellulose cleavage, and while the combination of TaGH61A and HiCDH cleaves highly crystalline bacterial cellulose, it does not cleave soluble cellodextrins. GH61 and CDH proteins are coexpressed and secreted by the thermophilic ascomycete Thielavia terrestris in response to environmental cellulose, and the combined activities of T. terrestris GH61 and T. terrestris CDH are shown to synergize with T. terrestris cellulose hydrolases in the breakdown of cellulose. The action of GH61 and CDH on cellulose may constitute an important, but overlooked, biological oxidoreductive system that functions in microbial lignocellulose degradation and has applications in industrial biomass utilization.
1-Hydroxybenzotriazole, violuric acid, and N-hydroxyacetanilide are three N-OH compounds capable of mediating a range of laccase-catalyzed biotransformations, such as paper pulp delignification and degradation of polycyclic hydrocarbons. The mechanism of their enzymatic oxidation was studied with seven fungal laccases. The oxidation had a bell-shaped pH-activity profile with an optimal pH ranging from 4 to 7. The oxidation rate was found to be dependent on the redox potential difference between the N-OH substrate and laccase. A laccase with a higher redox potential or an N-OH compound with a lower redox potential tended to have a higher oxidation rate. Similar to the enzymatic oxidation of phenols, phenoxazines, phenothiazines, and other redox-active compounds, an "outer-sphere" type of single-electron transfer from the substrate to laccase and proton release are speculated to be involved in the rate-limiting step for N-OH oxidation.Laccases (EC 1.10.3.2) are multi-Cu oxidases that can catalyze the oxidation of a range of reducing substances with the concomitant reduction of O 2 (for recent reviews, see reference 24 and references therein). Because of their capability of catalyzing the oxidation of aromatic compounds, laccases are receiving increasing attention as potential industrial enzymes in various applications, such as pulp delignification, wood fiber modification, dye or stain bleaching, chemical or medicinal synthesis, and contaminated water or soil remediation (15, 37).Laccases contain one type 1 (T1) Cu center, one type 2 (T2) Cu center, and one type 3 (T3) Cu center. The T2 and T3 sites form a trinuclear Cu cluster onto which O 2 is reduced. The T1 Cu oxidizes the reducing substrate and transfers electrons to the T2 and T3 Cu. Laccase is able to oxidize certain phenols with E 0 values higher than its own (0.5 to 0.8 V versus the normal hydrogen electrode [NHE]) (36). However, many inorganic and organic compounds with comparable E 0 values (such as 1,2,3,5-tetramethoxybenzene [18]) are not laccase substrates due to unfavorable kinetics. Under certain conditions, however, these compounds can be indirectly oxidized by laccase through the mediation of small, redox-active laccase substrates. 2,2Ј-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was the first compound found capable of efficiently mediating the laccase oxidation of high-E 0 , nonsubstrate lignin model compounds (such as veratryl alcohol and nonphenolic lignin model dimers) (8). Based on product structure analysis, it has been proposed that laccase-oxidized ABTS can abstract an H atom from the lignin model compounds, leading to indirect laccase catalysis upon the oxidation of the compounds (25). To date, other types of mediators, particularly phenoxazines and N-OH compounds, also have been recognized for their mediation function in laccase catalysis (1, 6,17,29).Mediated laccase catalysis has been applied to a wide range of applications, such as pulp delignification (9, 10, 12, 22, 32), textile dye bleaching (31), polycyclic aromatic h...
Plant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of β-xylosidases and endo -β-1,4-xylanases in the secretomes of Aspergillus niger , by the use of chemical probes inspired by the β-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme–substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown.
Exploiting 1,3-dipolar cycloaddition and carbanilide cyclization transformations, novel isoxazolylmethylimidazolidinedione heterocycles have been prepared using both solution-and solid-phase methods.
Genome engineering methodologies are transforming biological research and discovery. Approaches based on CRISPR technology have been broadly adopted and there is growing interest in the generation of massively parallel edited cell libraries. Comparing the libraries generated by these varying approaches is challenging and researchers lack a common framework for defining and assessing the characteristics of these libraries. Here we describe a framework for evaluating massively parallel libraries of edited genomes based on established methods for sampling complex populations. We define specific attributes and metrics that are informative for describing a complex cell library and provide examples for estimating these values. We also connect this analysis to generic phenotyping approaches, using either pooled (typically via a selection assay) or isolate (often referred to as screening) phenotyping approaches. We approach this from the context of creating massively parallel, precisely edited libraries with one edit per cell, though the approach holds for other types of modifications, including libraries containing multiple edits per cell (combinatorial editing). This framework is a critical component for evaluating and comparing new technologies as well as understanding how a massively parallel edited cell library will perform in a given phenotyping approach.
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