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1.
ACS Nano ; 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32644773

RESUMO

Colloidal metal halide perovskite nanocrystals (NCs) with chiral ligands are outstanding candidates as a circularly polarized luminescence (CPL) light source due to many advantages such as high photoluminescence quantum efficiency, large spin-orbit coupling, and extensive tunability via composition and choice of organic ligands. However, achieving pronounced and controllable polarized light emission remains challenging. Here, we develop strategies to achieve high CPL responses from colloidal formamidinium lead bromide (FAPbBr3) NCs at room temperature using chiral surface ligands. First, we show that replacing a portion of typical ligands (oleylamine) with short chiral ligands ((R)-2-octylamine) during FAPbBr3 NC synthesis results in small and monodisperse NCs that yield high CPL with average luminescence dissymmetry g-factor, glum = 6.8 × 10-2. To the best of our knowledge, this is the highest among reported perovskite materials at room temperature to date and represents around 10-fold improvement over the previously reported colloidal CsPbClxBryI3-x-y NCs. In order to incorporate NCs into any optoelectronic or spintronic application, the NCs necessitate purification, which removes a substantial amount of the chiral ligands and extinguishes the CPL signals. To circumvent this issue, we also developed a postsynthetic ligand treatment using a different chiral ligand, (R-/S-)methylbenzylammonium bromide, which also induces a CPL with an average glum = ±1.18 × 10-2. This postsynthetic method is also amenable for long-range charge transport since methylbenzylammonium is quite compact in relation to other surface ligands. Our demonstrations of high CPL and glum from both as-synthesized and purified perovskite NCs at room temperature suggest a route to demonstrate colloidal NC-based spintronics.

2.
J Am Chem Soc ; 142(30): 13030-13040, 2020 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-32602710

RESUMO

Incorporating chiral organic molecules into organic/inorganic hybrid 2D metal-halide perovskites results in a novel family of chiral hybrid semiconductors with unique spin-dependent properties. The embedded chiral organic moieties induce a chiroptical response from the inorganic metal-halide sublattice. However, the structural interplay between the chiral organic molecules and the inorganic sublattice, as well as their synergic effect on the resulting electronic band structure need to be explored in a broader material scope. Here we present three new layered tin iodide perovskites templated by chiral (R/S-)methylbenzylammonium (R/S-MBA), i.e., (R-/S-MBA)2SnI4, and their racemic phase (rac-MBA)2SnI4. These MBA2SnI4 compounds exhibit the largest level of octahedral bond distortion compared to any other reported layered tin iodide perovskite. The incorporation of chiral MBA cations leads to circularly polarized absorption from the inorganic Sn-I sublattice, displaying chiroptical activity in the 300-500 nm wavelength range. The bandgap and chiroptical activity are modulated by alloying Sn with Pb, in the series of (MBA)2Pb1-xSnxI4. Finally, we show that vertical charge transport through oriented (R-/S-MBA)2SnI4 thin films is highly spin-dependent, arising from a chiral-induced spin selectivity (CISS) effect. We demonstrate a spin-polarization in the current-voltage characteristics as high as 94%. Our work shows the tremendous potential of these chiral hybrid semiconductors for controlling both spin and charge degrees of freedom.

3.
Sci Adv ; 5(12): eaay0571, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31840072

RESUMO

Chiral-induced spin selectivity (CISS) occurs when the chirality of the transporting medium selects one of the two spin ½ states to transport through the media while blocking the other. Monolayers of chiral organic molecules demonstrate CISS but are limited in their efficiency and utility by the requirement of a monolayer to preserve the spin selectivity. We demonstrate CISS in a system that integrates an inorganic framework with a chiral organic sublattice inducing chirality to the hybrid system. Using magnetic conductive-probe atomic force microscopy, we find that oriented chiral 2D-layered Pb-iodide organic/inorganic hybrid perovskite systems exhibit CISS. Electron transport through the perovskite films depends on the magnetization of the probe tip and the handedness of the chiral molecule. The films achieve a highest spin-polarization transport of up to 86%. Magnetoresistance studies in modified spin-valve devices having only one ferromagnet electrode confirm the occurrence of spin-dependent charge transport through the organic/inorganic layers.

4.
World J Microbiol Biotechnol ; 34(3): 42, 2018 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-29480332

RESUMO

An increasing number of researchers working in biology, biochemistry, biotechnology, bioengineering, bioinformatics and other related fields of science are using biological molecules. As the scientific background of the members of different scientific communities is more diverse than ever before, the number of scientists not familiar with the rules for non-ambiguous designation of genetic elements is increasing. However, with biological molecules gaining importance through biotechnology, their functional and unambiguous designation is vital. Unfortunately, naming genes and proteins is not an easy task. In addition, the traditional concepts of bioinformatics are challenged with the appearance of proteins comprising different modules with a respective function in each module. This article highlights basic rules and novel solutions in designation recently used within the community of bacterial geneticists, and we discuss the present-day handling of gene and protein designations. As an example we will utilize a recent mischaracterization of gene nomenclature. We make suggestions for better handling of names in future literature as well as in databases and annotation projects. Our methodology emphasizes the hydrolytic function of multi-modular genes and extracellular proteins from bacteria.


Assuntos
Clostridium thermocellum/enzimologia , Clostridium thermocellum/genética , Biologia Computacional/métodos , Proteínas/genética , Bases de Dados Genéticas , Bases de Dados de Proteínas , Genoma Bacteriano , Genômica/métodos , Glucosidases/genética , Armazenamento e Recuperação da Informação/métodos , Anotação de Sequência Molecular
5.
Biotechnol Biofuels ; 11: 22, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29434665

RESUMO

Background: Thermophilic microorganisms and their enzymes offer several advantages for industrial application over their mesophilic counterparts. For example, a hyperthermophilic anaerobe, Caldicellulosiruptor bescii, was recently isolated from hot springs in Kamchatka, Siberia, and shown to have very high cellulolytic activity. Additionally, it is one of a few microorganisms being considered as viable candidates for consolidated bioprocessing applications. Moreover, C. bescii is capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This ability is accomplished by the production and secretion of free, multi-modular and multi-functional enzymes, one of which, CbCel9A/Cel48A also known as CelA, is able to outperform enzymes found in commercial enzyme preparations. Furthermore, the complete C. bescii exoproteome is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Therefore, understanding the functional diversity of enzymes in the C. bescii exoproteome and how inter-molecular synergy between them confers C. bescii with its high cellulolytic activity is an important endeavor to enable the production of more efficient biomass degrading enzyme formulations and in turn, better cellulolytic industrial microorganisms. Results: To advance the understanding of the C. bescii exoproteome we have expressed, purified, and tested four of the primary enzymes found in the exoproteome and we have found that the combination of three or four of the most highly expressed enzymes exhibit synergistic activity. We also demonstrated that discrete combinations of these enzymes mimic and even  improve upon the activity of the whole C. bescii exoproteome, even though some of the enzymes lack significant activity on their own. Conclusions: We have demonstrated that it is possible to replicate the cellulolytic activity of the native C. bescii exoproteome utilizing a minimal gene set, and that these minimal gene sets are more active than the whole exoproteome. In the future, this may lead to more simplified and efficient cellulolytic enzyme preparations or yield improvements when these enzymes are expressed in microorganisms engineered for consolidated bioprocessing.

6.
Biotechnol Biofuels ; 10: 274, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29213319

RESUMO

Glycoside hydrolase (GH) family 48 is an understudied and increasingly important exoglucanase family found in the majority of bacterial cellulase systems. Moreover, many thermophilic enzyme systems contain GH48 enzymes. Deletion of GH48 enzymes in these microorganisms results in drastic reduction in biomass deconstruction. Surprisingly, given their importance for these microorganisms, GH48s have intrinsically low cellulolytic activity but even in low ratios synergize greatly with GH9 endoglucanases. In this study, we explore the structural and enzymatic diversity of these enzymes across a wide range of temperature optima. We have crystallized one new GH48 module from Bacillus pumilus in a complex with cellobiose and cellohexaose (BpumGH48). We compare this structure to other known GH48 enzymes in an attempt to understand GH48 structure/function relationships and draw general rules correlating amino acid sequences and secondary structures to thermostability in this GH family.

7.
Biotechnol Biofuels ; 10: 283, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29209415

RESUMO

In the shadow of a burgeoning biomass-to-fuels industry, biological conversion of lignocellulose to fermentable sugars in a cost-effective manner is key to the success of second-generation and advanced biofuel production. For the effective comparison of one cellulase preparation to another, cellulase assays are typically carried out with one or more engineered cellulase formulations or natural exoproteomes of known performance serving as positive controls. When these formulations have unknown composition, as is the case with several widely used commercial products, it becomes impossible to compare or reproduce work done today to work done in the future, where, for example, such preparations may not be available. Therefore, being a critical tenet of science publishing, experimental reproducibility is endangered by the continued use of these undisclosed products. We propose the introduction of standard procedures and materials to produce specific and reproducible cellulase formulations. These formulations are to serve as yardsticks to measure improvements and performance of new cellulase formulations.

8.
Sci Rep ; 7(1): 9622, 2017 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-28851921

RESUMO

The crystalline nature of cellulose microfibrils is one of the key factors influencing biomass recalcitrance which is a key technical and economic barrier to overcome to make cellulosic biofuels a commercial reality. To date, all known fungal enzymes tested have great difficulty degrading highly crystalline cellulosic substrates. We have demonstrated that the CelA cellulase from Caldicellulosiruptor bescii degrades highly crystalline cellulose as well as low crystallinity substrates making it the only known cellulase to function well on highly crystalline cellulose. Unlike the secretomes of cellulolytic fungi, which typically comprise multiple, single catalytic domain enzymes for biomass degradation, some bacterial systems employ an alternative strategy that utilizes multi-catalytic domain cellulases. Additionally, CelA is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Furthermore we have determined that the factors negatively affecting digestion of lignocellulosic materials by C. bescii enzyme cocktails containing CelA appear to be significantly different from the performance barriers affecting fungal cellulases. Here, we explore the activity and degradation mechanism of CelA on a variety of pretreated substrates to better understand how the different bulk components of biomass, such as xylan and lignin, impact its performance.


Assuntos
Celulase/metabolismo , Celulose/metabolismo , Firmicutes/enzimologia , Celulase/química , Celulase/genética , Estabilidade Enzimática/efeitos da radiação , Temperatura Alta , Hidrólise , Domínios Proteicos
9.
Sci Rep ; 7(1): 4389, 2017 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-28663545

RESUMO

In planta expression of a thermophilic endoglucanase (AcCel5A) reduces recalcitrance by creating voids and other irregularities in cell walls of Arabidopsis thaliana that increase enzyme accessibility without negative impacts on plant growth or cell wall composition. Our results suggest that cellulose ß-1-4 linkages can be cut sparingly in the assembling wall and that these minimal changes, made at the proper time, have an impact on plant cell wall recalcitrance without negative effects on overall plant development.


Assuntos
Biomassa , Parede Celular/metabolismo , Celulase/genética , Plantas/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Celulase/química , Celulase/metabolismo , Celulose/metabolismo , Ordem dos Genes , Modelos Moleculares , Plantas/enzimologia , Plasmídeos/genética , Conformação Proteica , Relação Estrutura-Atividade
10.
Proteins ; 84(3): 295-304, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26572060

RESUMO

Family 48 cellobiohydrolases are some of the most abundant glycoside hydrolases in nature. They are able to degrade cellulosic biomass and therefore serve as good enzyme candidates for biofuel production. Family 48 cellulases hydrolyze cellulose chains via a processive mechanism, and produce end products composed primarily of cellobiose as well as other cellooligomers (dp ≤ 4). The challenge of utilizing cellulases in biofuel production lies in their extremely slow turnover rate. A factor contributing to the low enzyme activity is suggested to be product binding to enzyme and the resulting performance inhibition. In this study, we quantitatively evaluated the product inhibitory effect of four family 48 glycoside hydrolases using molecular dynamics simulations and product expulsion free-energy calculations. We also suggested a series of single mutants of the four family 48 glycoside hydrolases with theoretically reduced level of product inhibition. The theoretical calculations provide a guide for future experimental studies designed to produce mutant cellulases with enhanced activity.


Assuntos
Proteínas de Bactérias/química , Glicosídeo Hidrolases/química , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Domínio Catalítico , Inibidores Enzimáticos/química , Glicosídeo Hidrolases/genética , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Homologia Estrutural de Proteína , Termodinâmica
11.
Photosynth Res ; 128(1): 45-57, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26526668

RESUMO

The green alga Chlamydomonas reinhardtii contains six plastidic [2Fe2S]-cluster ferredoxins (FDXs), with FDX1 as the predominant isoform under photoautotrophic growth. FDX2 is highly similar to FDX1 and has been shown to interact with specific enzymes (such as nitrite reductase), as well as to share interactors with FDX1, such as the hydrogenases (HYDA), ferredoxin:NAD(P) reductase I (FNR1), and pyruvate:ferredoxin oxidoreductase (PFR1), albeit performing at low catalytic rates. Here we report the FDX2 crystal structure solved at 1.18 Å resolution. Based on differences between the Chlorella fusca FDX1 and C. reinhardtii FDX2 structures, we generated and purified point-mutated versions of the FDX2 protein and assayed them in vitro for their ability to catalyze hydrogen and NADPH photo-production. The data show that structural differences at two amino acid positions contribute to functional differences between FDX1 and FDX2, suggesting that FDX2 might have evolved from FDX1 toward a different physiological role in the cell. Moreover, we demonstrate that the mutations affect both the midpoint potentials of the FDX and kinetics of the FNR reaction, possibly due to altered binding between FDX and FNR. An effect on H2 photo-production rates was also observed, although the kinetics of the reaction were not further characterized.


Assuntos
Chlamydomonas reinhardtii/química , Ferredoxinas/química , Ferredoxinas/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/genética , Ferredoxina-NADP Redutase/metabolismo , Ferredoxinas/genética , Hidrogênio/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , NADP/metabolismo , Oxirredução , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína
12.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 9): 1946-54, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26327384

RESUMO

The unique active site of the Caldicellulosiruptor bescii family 3 pectate lyase (PL3) enzyme has been thoroughly characterized using a series of point mutations, X-ray crystallography, pK(a) calculations and biochemical assays. The X-ray structures of seven PL3 active-site mutants, five of them in complex with intact trigalacturonic acid, were solved and characterized structurally, biochemically and computationally. The results confirmed that Lys108 is the catalytic base, but there is no clear candidate for the catalytic acid. However, the reaction mechanism can also be explained by an antiperiplanar trans-elimination reaction, in which Lys108 abstracts a proton from the C5 atom without the help of simultaneous proton donation by an acidic residue. An acidified water molecule completes the anti ß-elimination reaction by protonating the O4 atom of the substrate. Both the C5 hydrogen and C4 hydroxyl groups of the substrate must be orientated in axial configurations, as for galacturonic acid, for this to be possible. The wild-type C. bescii PL3 displays a pH optimum that is lower than that of Bacillus subtilis PL1 according to activity measurements, indicating that C. bescii PL3 has acquired a lower pH optimum by utilizing lysine instead of arginine as the catalytic base, as well as by lowering the pK(a) of the catalytic base in a unique active-site environment.


Assuntos
Concentração de Íons de Hidrogênio , Polissacarídeo-Liase/química , Thermoanaerobacter/enzimologia , Catálise , Cristalografia por Raios X , Modelos Moleculares
13.
Biotechnol Biofuels ; 8: 113, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26269712

RESUMO

BACKGROUND: The Caldicellulosiruptor bescii genome encodes a potent set of carbohydrate-active enzymes (CAZymes), found primarily as multi-domain enzymes that exhibit high cellulolytic and hemicellulolytic activity on and allow utilization of a broad range of substrates, including plant biomass without conventional pretreatment. CelA, the most abundant cellulase in the C. bescii secretome, uniquely combines a GH9 endoglucanase and a GH48 exoglucanase in one protein. The most effective commercial enzyme cocktails used in vitro to pretreat biomass are derived from fungal cellulases (cellobiohydrolases, endoglucanases and a ß-d-glucosidases) that act synergistically to release sugars for microbial conversion. The C. bescii genome contains six GH5 domains in five different open reading frames. Four exist in multi-domain proteins and two as single catalytic domains. E1 is a GH5 endoglucanase reported to have high specific activity and simple architecture and is active at the growth temperature of C. bescii. E1 is an endo-1,4-ß-glucanase linked to a family 2 carbohydrate-binding module shown to bind primarily to cellulosic substrates. We tested if the addition of this protein to the C. bescii secretome would improve its cellulolytic activity. RESULTS: In vitro analysis of E1 and CelA shows synergistic interaction. The E1 gene from Acidothermus cellulolyticus was cloned and expressed in C. bescii under the transcriptional control of the C. bescii S-layer promoter, and secretion was directed by the addition of the C. bescii CelA signal peptide sequence. The vector was integrated into the C. bescii chromosome at a site previously showing no detectable detrimental consequence. Increased activity of the secretome of the strain containing E1 was observed on both carboxymethylcellulose (CMC) and Avicel. Activity against CMC increased on average 10.8 % at 65 °C and 12.6 % at 75 °C. Activity against Avicel increased on average 17.5 % at 65 °C and 16.4 % at 75 °C. CONCLUSIONS: Expression and secretion of E1 in C. bescii enhanced the cellulolytic ability of its secretome. These data agree with in vitro evidence that E1 acts synergistically with CelA to digest cellulose and offer the possibility of engineering additional enzymes for improved biomass deconstruction with the knowledge that C. bescii can express a gene from Acidothermus, and perhaps other heterologous genes, effectively.

14.
Front Plant Sci ; 6: 315, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26029221

RESUMO

Identifying the cell wall-ionically bound glycoside hydrolases (GHs) in Arabidopsis stems is important for understanding the regulation of cell wall integrity. For cell wall proteomics studies, the preparation of clean cell wall fractions is a challenge since cell walls constitute an open compartment, which is more likely to contain a mixture of intracellular and extracellular proteins due to cell leakage at the late growth stage. Here, we utilize a CaCl2-extraction procedure to isolate non-structural proteins from Arabidopsis whole stems, followed by the in-solution and in-gel digestion methods coupled with Nano-LC-MS/MS, bioinformatics and literature analyses. This has led to the identification of 75 proteins identified using the in-solution method and 236 proteins identified by the in-gel method, among which about 10% of proteins predicted to be secreted. Together, eight cell wall proteins, namely AT1G75040, AT5G26000, AT3G57260, AT4G21650, AT3G52960, AT3G49120, AT5G49360, and AT3G14067, were identified by the in-solution method; among them, three were the GHs (AT5G26000, myrosinase 1, GH1; AT3G57260, ß-1,3-glucanase 2, GH17; AT5G49360, bifunctional XYL 1/α-L-arabinofuranosidase, GH3). Moreover, four more GHs: AT4G30270 (xyloglucan endotransferase, GH16), AT1G68560 (bifunctional α-l-arabinofuranosidase/XYL, GH31), AT1G12240 (invertase, GH32) and AT2G28470 (ß-galactosidase 8, GH35), were identified by the in-gel solution method only. Notably, more than half of above identified GHs are xylan- or hemicellulose-modifying enzymes, and will likely have an impact on cellulose accessibility, which is a critical factor for downstream enzymatic hydrolysis of plant tissues for biofuels production. The implications of these cell wall proteins identified at the late growth stage for the genetic engineering of bioenergy crops are discussed.

15.
Biotechnol Biofuels ; 7(1): 170, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25489338

RESUMO

INTRODUCTION: The efficient conversion of lignocellulosic feedstocks remains a key step in the commercialization of biofuels. One of the barriers to cost-effective conversion of lignocellulosic biomass to sugars remains the enzymatic saccharification process step. Here, we describe a novel hybrid processing approach comprising enzymatic pre-digestion with newly characterized hyperthermophilic enzyme cocktails followed by conventional saccharification with commercial enzyme preparations. Dilute acid pretreated corn stover was subjected to this new procedure to test its efficacy. Thermal tolerant enzymes from Acidothermus cellulolyticus and Caldicellulosiruptor bescii were used to pre-digest pretreated biomass at elevated temperatures prior to saccharification by the commercial cellulase formulation. RESULTS: We report that pre-digestion of biomass with these enzymes at elevated temperatures prior to addition of the commercial cellulase formulation increased conversion rates and yields when compared to commercial cellulase formulation alone under low solids conditions. CONCLUSION: Our results demonstrating improvements in rates and yields of conversion point the way forward for hybrid biomass conversion schemes utilizing catalytic amounts of hyperthermophilic enzymes.

16.
Science ; 344(6184): 578, 2014 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-24812382

RESUMO

Gusakov critiques our methodology for comparing the cellulolytic activity of the bacterial cellulase CelA with the fungal cellulase Cel7A. We address his concerns by clarifying some misconceptions, carefully referencing the literature, and justifying our approach to point out that the results from our study still stand.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Celulase/química , Celulose/química
17.
Biotechnol Bioeng ; 111(8): 1541-9, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24522957

RESUMO

We report a novel approach to concurrently improve the tolerance to ionic liquids (ILs) as well as reduce lignin inhibition of Trichoderma reesei cellulase via engineering enzyme charge. Succinylation of the cellulase enzymes led to a nearly twofold enhancement in cellulose conversion in 15% (v/v) 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]). The improvement in activity upon succinylation correlated with the apparent preferential exclusion of the [Cl] anion in fluorescence quenching assays. Additionally, modeling analysis of progress curves of Avicel hydrolysis in buffer indicated that succinylation had a negligible impact on the apparent KM of cellulase. As evidence of reducing lignin inhibition of T. reesei cellulase, succinylation resulted in a greater than twofold increase in Avicel conversion after 170 h in buffer with 1 wt% lignin. The impact of succinylation on lignin inhibition of cellulase further led to the reduction in apparent KM of the enzyme cocktail for Avicel by 2.7-fold. These results provide evidence that naturally evolved cellulases with highly negative surface charge densities may similarly repel lignin, resulting in improved cellulase activity. Ultimately, these results underscore the potential of rational charge engineering as a means of enhancing cellulase function and thus conversion of whole biomass in ILs.


Assuntos
Celulases/genética , Celulases/metabolismo , Líquidos Iônicos/metabolismo , Lignina/metabolismo , Trichoderma/enzimologia , Celulases/química , Celulose/metabolismo , Hidrólise , Imidazóis/metabolismo , Engenharia de Proteínas , Eletricidade Estática , Trichoderma/genética , Trichoderma/metabolismo
18.
Biotechnol Bioeng ; 111(4): 664-73, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24264519

RESUMO

Lignocellulosic biomass is a potential source of sustainable transportation fuels, but efficient enzymatic saccharification of cellulose is a key challenge in its utilization. Cellulases from the glycoside hydrolase (GH) family 48 constitute an important component of bacterial biomass degrading systems and structures of three enzymes from this family have been previously published. We report a new crystal structure of TfCel48A, a reducing end directed exocellulase from Thermobifida fusca, which shows that this enzyme shares important structural features with the other members of the GH48 family. The active site tunnel entrance of the known GH48 exocellulases is enriched in aromatic residues, which are known to interact well with anhydroglucose units of cellulose. We carried out site-directed mutagenesis studies of these aromatic residues (Y97, F195, Y213, and W313) along with two non-aromatic residues (N212 and S311) also located around the tunnel entrance and a W315 residue inside the active site tunnel. Only the aromatic residues located around the tunnel entrance appear to be important for the ability of TfCel48A to access individual cellulose chains on bacterial cellulose (BC), a crystalline substrate. Both Trp residues were found to be important for the processivity of TfCel48A on BC and phosphoric acid swollen cellulose (PASC), but only W313 appears to play a role in the ability of the enzyme to access individual cellulose chains in BC. When acting on BC, reduced processivity was found to correlate with reduced enzyme activity. The reverse, however, is true when PASC is the substrate. Presumably, higher density of available cellulose chain ends and the amorphous nature of PASC explain the increased initial activity of mutants with lower processivity.


Assuntos
Actinomycetales/enzimologia , Proteínas de Bactérias/química , Celulases/química , Actinomycetales/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Celulases/genética , Celulases/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica
19.
Science ; 342(6165): 1513-6, 2013 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-24357319

RESUMO

Most fungi and bacteria degrade plant cell walls by secreting free, complementary enzymes that hydrolyze cellulose; however, some bacteria use large enzymatic assemblies called cellulosomes, which recruit complementary enzymes to protein scaffolds. The thermophilic bacterium Caldicellulosiruptor bescii uses an intermediate strategy, secreting many free cellulases that contain multiple catalytic domains. One of these, CelA, comprises a glycoside hydrolase family 9 and a family 48 catalytic domain, as well as three type III cellulose-binding modules. In the saccharification of a common cellulose standard, Avicel, CelA outperforms mixtures of commercially relevant exo- and endoglucanases. From transmission electron microscopy studies of cellulose after incubation with CelA, we report morphological features that suggest that CelA not only exploits the common surface ablation mechanism driven by general cellulase processivity, but also excavates extensive cavities into the surface of the substrate. These results suggest that nature's repertoire of cellulose digestion paradigms remain only partially discovered and understood.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Celulase/química , Celulose/química , Proteínas de Bactérias/isolamento & purificação , Catálise , Domínio Catalítico , Celulase/isolamento & purificação , Temperatura Alta , Hidrólise , Especificidade por Substrato
20.
Fungal Genet Biol ; 61: 120-32, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24076077

RESUMO

Dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl α-1,3-mannosyltransferase (also known as "asparagine-linked glycosylation 3", or ALG3) is involved in early N-linked glycan synthesis and thus is essential for formation of N-linked protein glycosylation. In this study, we examined the effects of alg3 gene deletion (alg3Δ) on growth, development, pigment production, protein secretion and recombinant Trichoderma reesei cellobiohydrolase (rCel7A) expressed in Aspergillus niger. The alg3Δ delayed spore germination in liquid cultures of complete medium (CM), potato dextrose (PD), minimal medium (MM) and CM with addition of cAMP (CM+cAMP), and resulted in significant reduction of hyphal growth on CM, potato dextrose agar (PDA), and CM+cAMP and spore production on CM. The alg3Δ also led to a significant accumulation of red pigment on both liquid and solid CM cultures. The relative abundances of 54 of the total 215 proteins identified in the secretome were significantly altered as a result of alg3Δ, 63% of which were secreted at higher levels in alg3Δ strain than the parent. The rCel7A expressed in the alg3Δ mutant was smaller in size than that expressed in both wild-type and parental strains, but still larger than T. reesei Cel7A. The circular dichroism (CD)-melt scans indicated that change in glycosylation of rCel7A does not appear to impact the secondary structure or folding. Enzyme assays of Cel7A and rCel7A on nanocrystalline cellulose and bleached kraft pulp demonstrated that the rCel7As have improved activities on hydrolyzing the nanocrystalline cellulose. Overall, the results suggest that alg3 is critical for growth, sporulation, pigment production, and protein secretion in A. niger, and demonstrate the feasibility of this alternative approach to evaluate the roles of N-linked glycosylation in glycoprotein secretion and function.


Assuntos
Aspergillus niger/enzimologia , Aspergillus niger/crescimento & desenvolvimento , Celulose 1,4-beta-Celobiosidase/metabolismo , Regulação Fúngica da Expressão Gênica , Manosiltransferases/metabolismo , Pigmentos Biológicos/metabolismo , Aspergillus niger/genética , Aspergillus niger/metabolismo , Celulose 1,4-beta-Celobiosidase/química , Celulose 1,4-beta-Celobiosidase/genética , Dicroísmo Circular , Meios de Cultura/química , Deleção de Genes , Glicosilação , Humanos , Hifas/crescimento & desenvolvimento , Manosiltransferases/genética , Conformação Proteica , Dobramento de Proteína , Transporte Proteico , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , Trichoderma/enzimologia
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