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1.
Int J Biol Macromol ; 185: 1-19, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34146557

RESUMO

Nanostructured materials along with an added value of polymers-based support carriers have gained high interest and considered ideal for enzyme immobilization. The recently emerged nanoscience interface in the form of nanostructured materials combined with immobilized-enzyme-based bio-catalysis has now become research and development frontiers in advance and applied bio-catalysis engineering. With the involvement of nanoscience, various polymers have been thoroughly developed and exploited to nanostructured engineer constructs as ideal support carriers/matrices. Such nanotechnologically engineered support carriers/matrix possesses unique structural, physicochemical, and functional attributes which equilibrate principal factors and strengthen the biocatalysts efficacy for multipurpose applications. In addition, nano-supported catalysts are potential alternatives that can outstrip several limitations of conventional biocatalysts, such as reduced catalytic efficacy and turnover, low mass transfer efficiency, instability during the reaction, and most importantly, partial, or complete inhibition/deactivation. In this context, engineering robust and highly efficient biocatalysts is an industrially relevant prerequisite. This review comprehensively covered various biopolymers and nanostructured materials, including silica, hybrid nanoflower, nanotubes or nanofibers, nanomembranes, graphene oxide nanoparticles, metal-oxide frameworks, and magnetic nanoparticles as robust matrices for cellulase immobilization. The work is further enriched by spotlighting applied and industrially relevant considerations of nano-immobilized cellulases. For instance, owing to the cellulose-deconstruction features of nano-immobilized cellulases, the applications like lignocellulosic biomass conversion into industrially useful products or biofuels, improved paper sheet density and pulp beat in paper and pulp industry, fruit juice clarification in food industry are evident examples of cellulases, thereof are discussed in this work.


Assuntos
Biopolímeros/química , Celulases/metabolismo , Celulose/química , Biocatálise , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Indústria Alimentícia , Nanoestruturas
2.
Nat Commun ; 12(1): 3847, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158485

RESUMO

Enzyme reactions, both in Nature and technical applications, commonly occur at the interface of immiscible phases. Nevertheless, stringent descriptions of interfacial enzyme catalysis remain sparse, and this is partly due to a shortage of coherent experimental data to guide and assess such work. In this work, we produced and kinetically characterized 83 cellulases, which revealed a conspicuous linear free energy relationship (LFER) between the substrate binding strength and the activation barrier. The scaling occurred despite the investigated enzymes being structurally and mechanistically diverse. We suggest that the scaling reflects basic physical restrictions of the hydrolytic process and that evolutionary selection has condensed cellulase phenotypes near the line. One consequence of the LFER is that the activity of a cellulase can be estimated from its substrate binding strength, irrespectively of structural and mechanistic details, and this appears promising for in silico selection and design within this industrially important group of enzymes.


Assuntos
Algoritmos , Celulases/metabolismo , Celulose/metabolismo , Simulação de Dinâmica Molecular , Biocatálise , Celulases/química , Hidrólise , Cinética , Ligação Proteica , Domínios Proteicos , Especificidade por Substrato
3.
Methods Mol Biol ; 2290: 115-127, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34009586

RESUMO

Glycerol is a promising low-cost solvent for biomass pretreatment since a large amount of glycerol is generated as a by-product in the biodiesel industry. Pretreatment is a method of disintegration of the recalcitrant structure of biomass to enhance the accessibility of cellulose and hemicelluloses to enzymes for complete saccharification. During pretreatment, glycerol breaks the lignin carbohydrate complex and selectively solubilizes lignin. Thus, the glycerol pretreatment improves the accessibility of cellulose to cellulases leading to higher sugar yields. The glycerol pretreatment is carried out at high temperature (>190 °C) to disintegrate the structure of biomass. The glycerol pretreatment in the presence of acid or base catalyst such as H2SO4 or NaOH results in lower pretreatment temperature and higher glucan hydrolysis. This chapter describes the methodology to carry out glycerol pretreatment of sorghum biomass with or without acid/alkali as catalyst and the basic calculations to determine the efficiency of the pretreatment.


Assuntos
Celulases/isolamento & purificação , Glicerol/química , Sorghum/química , Biocombustíveis/análise , Biomassa , Celulases/metabolismo , Celulose/metabolismo , Fermentação , Hidrólise , Lignina/metabolismo , Polissacarídeos/metabolismo , Solventes , Açúcares
4.
Carbohydr Polym ; 262: 117943, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33838820

RESUMO

Aspergillus spp. are well-known producers of pectinases commonly used in the industry. Aspergillus aculeatinus is a recently identified species but poorly characterized. This study aimed at giving a comprehensive characterization of the enzymatic potential of the O822 strain to produce Rhamnogalacturonan type I (RGI)-degrading enzymes. Proteomic analysis identified cell wall degrading enzymes (cellulases, hemicellulases, and pectinases) that accounted for 92 % of total secreted proteins. Twelve out of fifty proteins were identified as RGI-degrading enzymes. NMR and enzymatic assays revealed high levels of arabinofuranosidase, arabinanase, galactanase, rhamnogalacturonan hydrolases and rhamnogalacturonan acetylesterase activities in aqueous extracts. Viscosity assays carried out with RGI-rich camelina mucilage confirmed the efficiency of enzymes secreted by O822 to hydrolyze RGI, by decreasing viscosity by 70 %. Apple juice trials carried out at laboratory and pilot scale showed an increase in filtration flow rate and yield, paving the way for an industrial use of enzymes derived from A. aculeatinus.


Assuntos
Aspergillus/enzimologia , Filtração/métodos , Sucos de Frutas e Vegetais , Proteínas Fúngicas/metabolismo , Ramnogalacturonanos/metabolismo , Metabolismo dos Carboidratos , Celulases/metabolismo , Manipulação de Alimentos/métodos , Glicosídeo Hidrolases/metabolismo , Hidrolases/metabolismo , Malus , Pectinas/metabolismo , Poligalacturonase/metabolismo , Proteômica
5.
Int J Mol Sci ; 22(9)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925273

RESUMO

BACKGROUND: The filamentous fungus Trichoderma reesei is used on an industrial scale to produce enzymes of biotechnological interest. This fungus has a complex cellulolytic system involved in the degradation of lignocellulosic biomass. However, several aspects related to the regulation of the expression of holocellulolytic genes and the production of cellulases by this fungus are still understood. METHODS: Here, we constructed a null mutant strain for the xyloglucanase cel74a gene and performed the characterization of the Δcel74a strain to evaluate the genetic regulation of the holocellulases during sugarcane bagasse (SCB) cultivation. RESULTS: Our results demonstrate that the deletion of xyloglucanase cel74a may impact the regulation of holocellulase expression during SCB cultivation. The expression of cellulases cel7a, cel7b, and cel6a was reduced in Δcel74a strain, while the hemicellulases xyn1 and xyn2 were increased in the presence of SCB. The cel74a mutation also affected the xyloglucan hydrolysis patterns. In addition, CEL74A activity was modulated in the presence of calcium, suggesting that this ion may be required for efficient degradation of xyloglucan. CONCLUSIONS: CEL74A affects the regulation of holocellulolytic genes and the efficient degradation of SCB in T. reesei. This data makes a significant contribution to our understanding of the carbon utilization of fungal strains as a whole.


Assuntos
Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Hypocreales/genética , Biomassa , Celulases/genética , Celulases/metabolismo , Celulose/metabolismo , Proteínas Fúngicas/metabolismo , Hidrólise , Hypocreales/metabolismo , Saccharum/metabolismo , Trichoderma/genética , Trichoderma/metabolismo
6.
Molecules ; 26(5)2021 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-33803082

RESUMO

Cocoa pod husks are a waste generated during the processing of cocoa beans. We aimed to explore the enzymatic extraction of pectin using cellulases. The extraction process was optimized using a central composite design (CCD) and analyzed by response surface methodology (RSM). The parameters optimized were feedstock concentration (%), enzyme dosage (µL/g), and time (h). Three dependent variables were studied: pectin yield (g/100 g dry husk) (R2 = 97.02), galacturonic acid content (g/100 g pectin) (R2 = 96.90), and galacturonic acid yield (g/100 g feedstock) (R2 = 95.35). The optimal parameters were 6.0% feedstock concentration, 40 µL g-1 of enzyme, and 18.54 h, conditions that produced experimentally a pectin yield of 10.20 g/100 g feedstock, 52.06 g galacturonic acid/100 g pectin, and a yield 5.31 g galacturonic acid/100 g feedstock. Using the chemical extraction method, a yield of 8.08 g pectin/100 g feedstock and a galacturonic acid content of 60.97 g/100 g pectin were obtained. Using assisted sonication, a pectin yield of 8.28 g/100 g feedstock and a galacturonic acid content of 42.77 g/100 g pectin were obtained. Enzymatically optimized pectin has rheological and physicochemical features typical of this biomaterial, which provides an interesting alternative for the valorization of cocoa husks.


Assuntos
Cacau/química , Pectinas/química , Pectinas/isolamento & purificação , Cacau/metabolismo , Celulases/metabolismo , Esterificação , Ácidos Hexurônicos/análise , Concentração de Íons de Hidrogênio , Peso Molecular , Sonicação , Temperatura , Resíduos
7.
Carbohydr Polym ; 264: 118059, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-33910709

RESUMO

Processive cellulases are highly efficient molecular engines involved in the cellulose breakdown process. However, the mechanism that processive bacterial enzymes utilize to recruit and retain cellulose strands in the catalytic site remains poorly understood. Here, integrated enzymatic assays, protein crystallography and computational approaches were combined to study the enzymatic properties of the processive BlCel48B cellulase from Bacillus licheniformis. Hydrolytic efficiency, substrate binding affinity, cleavage patterns, and the apparent processivity of bacterial BlCel48B are significantly impacted by the cellulose size and its surface morphology. BlCel48B crystallographic structure was solved with ligands spanning -5 to -2 and +1 to +2 subsites. Statistical coupling analysis and molecular dynamics show that co-evolved residues on active site are critical for stabilizing ligands in the catalytic tunnel. Our results provide mechanistic insights into BlCel48B molecular-level determinants of activity, substrate binding, and processivity on insoluble cellulose, thus shedding light on structure-activity correlations of GH48 family members in general.


Assuntos
Bacillus licheniformis/enzimologia , Celulase/química , Celulase/metabolismo , Celulose/metabolismo , Bacillus licheniformis/química , Domínio Catalítico , Celulases/química , Celulases/metabolismo , Celulose/química , Cristalografia por Raios X/métodos , Hidrólise , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Especificidade por Substrato
8.
Carbohydr Polym ; 260: 117814, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33712158

RESUMO

Lytic polysaccharide monooxygenases (LPMOs), monocopper enzymes that oxidatively cleave recalcitrant polysaccharides, have important biotechnological applications. Thermothelomyces thermophilus is a rich source of biomass-active enzymes, including many members from auxiliary activities family 9 LPMOs. Here, we report biochemical and structural characterization of recombinant TtLPMO9H which oxidizes cellulose at the C1 and C4 positions and shows enhanced activity in light-driven catalysis assays. TtLPMO9H also shows activity against xyloglucan. The addition of TtLPMO9H to endoglucanases from four different glucoside hydrolase families (GH5, GH12, GH45 and GH7) revealed that the product formation was remarkably increased when TtLPMO9H was combined with GH7 endoglucanase. Finally, we determind the first low resolution small-angle X-ray scattering model of the two-domain TtLPMO9H in solution that shows relative positions of its two functional domains and a conformation of the linker peptide, which can be relevant for the catalytic oxidation of cellulose and xyloglucan.


Assuntos
Celulases/metabolismo , Celulose/metabolismo , Ativação Enzimática/efeitos da radiação , Proteínas Fúngicas/metabolismo , Luz , Oxigenases de Função Mista/metabolismo , Sordariales/enzimologia , Biomassa , Catálise , Celulose/química , Proteínas Fúngicas/química , Proteínas Fúngicas/classificação , Proteínas Fúngicas/genética , Glucanos/química , Glucanos/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/classificação , Oxigenases de Função Mista/genética , Oxirredução , Filogenia , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espalhamento a Baixo Ângulo , Estereoisomerismo , Especificidade por Substrato , Difração de Raios X , Xilanos/química , Xilanos/metabolismo
9.
J Biosci Bioeng ; 131(6): 622-630, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33676867

RESUMO

Traditionally, filamentous fungi and actinomycetes are well-known cellulolytic microorganisms that have been utilized in the commercial production of cellulase enzyme cocktails for industrial-scale degradation of plant biomass. Noticeably, the Ktedonobacteria lineage (phylum Chloroflexi) with actinomycetes-like morphology was identified and exhibited diverse carbohydrate utilization or degradation abilities. In this study, we performed genome-wide profiling of carbohydrate-active enzymes (CAZymes) in the filamentous Ktedonobacteria lineage. Numerous CAZymes (153-290 CAZymes, representing 63-131 glycoside hydrolases (GHs) per genome), including complex mixtures of endo- and exo-cellulases, were predicted in 15 available Ktedonobacteria genomes. Of note, 4-28 CAZymes were predicted to be extracellular enzymes, whereas 3-29 CAZymes were appended with carbohydrate-binding modules (CBMs) that may promote their binding to insoluble carbohydrate substrates. This number far exceeded other Chloroflexi lineages and were comparable to the cellulolytic actinomycetes. Six multi-modular extracellular GHs were cloned from the thermophilic Thermosporothrix hazakensis SK20-1T strain and heterologously expressed. The putative endo-glucanases of ThazG5-1, ThazG9, and ThazG12 exhibited strong cellulolytic activity, whereas the putative exo-glucanases ThazG6 and ThazG48 formed weak but observable halos on carboxymethyl cellulose plates, indicating their potential biotechnological application. The purified recombinant ThazG12 had near-neutral pH (optimal 6.0), high thermostability (60°C), and broad specificity against soluble and insoluble polysaccharide substrates. It also represented described a novel thermostable bacterial ß-1,4-glucanase in the GH12 family. Together, this research revealed the underestimated cellulolytic potential of the Ktedonobacteria lineage and highlighted its potential biotechnological utility as a promising microbial resource for the discovery of industrially useful cellulases.


Assuntos
Metabolismo dos Carboidratos/genética , Celulases/genética , Celulose/metabolismo , Chloroflexi , Bactérias/metabolismo , Celulases/metabolismo , Chloroflexi/classificação , Chloroflexi/enzimologia , Chloroflexi/genética , Chloroflexi/metabolismo , Mapeamento Cromossômico , Fungos/metabolismo , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Engenharia Metabólica , Organismos Geneticamente Modificados , Plantas/metabolismo , Polissacarídeos/metabolismo
10.
J Microbiol Biotechnol ; 31(5): 740-746, 2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-33746194

RESUMO

Efficient cellulolytic enzyme production is important for the development of lignocellulose-degrading enzyme mixtures. However, purification of cellulases from their native hosts is time- and labor-consuming. In this study, a constitutive expression system was developed in Penicillium oxalicum for the secreted production of proteins. Using a constitutive polyubiquitin gene promoter and cultivating with glucose as the sole carbon source, nine cellulolytic enzymes of different origins with relatively high purity were produced within 48 h. When supplemented to a commercial cellulase preparation, cellobiohydrolase I from P. funiculosum and cellobiohydrolase II from Talaromyces verruculosus showed remarkable enhancing effects on the hydrolysis of steam-exploded corn stover. Additionally, a synergistic effect was observed for these two cellobiohydrolases during the hydrolysis. Taken together, the constitutive expression system provides a convenient tool for the production of cellulolytic enzymes, which is expected to be useful in the development of highly efficient lignocellulose-degrading enzyme mixtures.


Assuntos
Celulases/genética , Celulases/metabolismo , Lignina/metabolismo , Penicillium/metabolismo , Biomassa , Meios de Cultura/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Glucose/metabolismo , Hidrólise , Penicillium/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
11.
J Biol Chem ; 296: 100431, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33610545

RESUMO

Efficient enzymatic saccharification of cellulosic biomass into fermentable sugars can enable production of bioproducts like ethanol. Native crystalline cellulose, or cellulose I, is inefficiently processed via enzymatic hydrolysis but can be converted into the structurally distinct cellulose III allomorph that is processed via cellulase cocktails derived from Trichoderma reesei up to 20-fold faster. However, characterization of individual cellulases from T. reesei, like the processive exocellulase Cel7A, shows reduced binding and activity at low enzyme loadings toward cellulose III. To clarify this discrepancy, we monitored the single-molecule initial binding commitment and subsequent processive motility of Cel7A enzymes and associated carbohydrate-binding modules (CBMs) on cellulose using optical tweezers force spectroscopy. We confirmed a 48% lower initial binding commitment and 32% slower processive motility of Cel7A on cellulose III, which we hypothesized derives from reduced binding affinity of the Cel7A binding domain CBM1. Classical CBM-cellulose pull-down assays, depending on the adsorption model fitted, predicted between 1.2- and 7-fold reduction in CBM1 binding affinity for cellulose III. Force spectroscopy measurements of CBM1-cellulose interactions, along with molecular dynamics simulations, indicated that previous interpretations of classical binding assay results using multisite adsorption models may have complicated analysis, and instead suggest simpler single-site models should be used. These findings were corroborated by binding analysis of other type-A CBMs (CBM2a, CBM3a, CBM5, CBM10, and CBM64) on both cellulose allomorphs. Finally, we discuss how complementary analytical tools are critical to gain insight into the complex mechanisms of insoluble polysaccharides hydrolysis by cellulolytic enzymes and associated carbohydrate-binding proteins.


Assuntos
Celulases/metabolismo , Celulose/metabolismo , Hypocreales/enzimologia , Adsorção , Proteínas de Transporte/metabolismo , Domínio Catalítico , Celulase/química , Celulases/química , Celulose 1,4-beta-Celobiosidase/química , Hidrólise , Hypocreales/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Trichoderma/enzimologia
12.
J Biol Chem ; 296: 100385, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33556371

RESUMO

Glycoside hydrolases (GHs) are involved in the degradation of a wide diversity of carbohydrates and present several biotechnological applications. Many GH families are composed of enzymes with a single well-defined specificity. In contrast, enzymes from the GH16 family can act on a range of different polysaccharides, including ß-glucans and galactans. SCLam, a GH16 member derived from a soil metagenome, an endo-ß-1,3(4)-glucanase (EC 3.2.1.6), can cleave both ß-1,3 and ß-1,4 glycosidic bonds in glucans, such as laminarin, barley ß-glucan, and cello-oligosaccharides. A similar cleavage pattern was previously reported for other GH16 family members. However, the molecular mechanisms for this dual cleavage activity on (1,3)- and (1,4)-ß-D-glycosidic bonds by laminarinases have not been elucidated. In this sense, we determined the X-ray structure of a presumably inactive form of SCLam cocrystallized with different oligosaccharides. The solved structures revealed general bound products that are formed owing to residual activities of hydrolysis and transglycosylation. Biochemical and biophysical analyses and molecular dynamics simulations help to rationalize differences in activity toward different substrates. Our results depicted a bulky aromatic residue near the catalytic site critical to select the preferable configuration of glycosidic bonds in the binding cleft. Altogether, these data contribute to understanding the structural basis of recognition and hydrolysis of ß-1,3 and ß-1,4 glycosidic linkages of the laminarinase enzyme class, which is valuable for future studies on the GH16 family members and applications related to biomass conversion into feedstocks and bioproducts.


Assuntos
Proteínas de Bactérias/metabolismo , Celulases/metabolismo , Glucanos/metabolismo , Proteínas de Bactérias/química , Sequência de Carboidratos , Domínio Catalítico , Celulases/química , Cristalografia por Raios X/métodos , Glucanos/classificação , Glicosídeos/química , Glicosídeos/metabolismo , Hidrólise , Simulação de Dinâmica Molecular , Microbiologia do Solo , Especificidade por Substrato
13.
Arch Insect Biochem Physiol ; 106(3): e21766, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33590531

RESUMO

Among termites, lower termites need symbiotic microorganisms in the digestive tract for digestion and cellulose metabolism. In this symbiotic relationship, the decomposition of cellulose is initiated by endoglucanase in termite salivary glands and completed by ß-glycosidase of symbiotic microorganisms in the hindgut. The expression of ß-glycosidase in lower termites has been reported in recent studies. The expression of two endoglucanases and one ß-glycosidase gene related to cellulose degradation was identified in Reticulitermes speratus, a lower termite, through transcriptomic analysis. The proposed enzyme activities of three identified cellulose degradation genes were confirmed by heterologous expression in Escherichia coli. In addition to the endoglucanase expressed in the salivary gland, additional endoglucanase and ß-glycosidase genes suggest that R. speratus performs the overall cellulose digestion using its own enzymes at all stages.


Assuntos
Celulases/genética , Isópteros/genética , Animais , Celulase/metabolismo , Celulases/metabolismo , Celulose/metabolismo , Trato Gastrointestinal/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Genes de Insetos , Glicosídeo Hidrolases/metabolismo , Isópteros/metabolismo
14.
Food Chem ; 342: 128564, 2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33223299

RESUMO

Thermal processing (TP) and high hydrostatic pressure (HHP) are two important puree processing methods. In this study, the polyphenol oxidase (PPO) and ß-glucosidase activities, chromatic values, peroxide radical scavenging capacities (PSCs), cellular antioxidant activities (CAAs), and anthocyanin profiles were evaluated in blueberry puree following TP and HHP treatments. Nine anthocyanins were identified and cyanidin glycosides were the most abundant compounds in the blueberry puree sample. Petunidin-3-O-arabinoside, malvidin-3-O-galactoside, and malvidin-3-O-glucoside concentrations increased at temperatures of 70-90 °C (TP) and a pressure of 300 MPa (HHP). The highest total anthocyanin concentration (503.5 µg/mL) and PSC (13.45 µg VCE/mL) were observed following the TP (90 °C) treatment. Furthermore, a positive correlation was observed between the anthocyanin content and PSC (R2 = 0.655, P < 0.05). Finally, HHP treatment resulted in better puree color retention than TP treatment. The results of this study could provide valuable information for optimizing the processing methods for anthocyanin-rich products.


Assuntos
Antocianinas/análise , Antioxidantes/metabolismo , Catecol Oxidase/metabolismo , Celulases/metabolismo , Manipulação de Alimentos/métodos , Vaccinium/química , Antocianinas/química , Antocianinas/farmacologia , Antioxidantes/química , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Cor , Células Hep G2 , Humanos , Pressão Hidrostática , Espectrometria de Massas em Tandem , Temperatura , Vaccinium/metabolismo
15.
J Biochem ; 169(1): 109-117, 2021 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-32810224

RESUMO

Lignocellulosic biomass conversion using cellulases/polygalacturonases is a process that can be progressively influenced by several determinants involved in cellulose microfibril degradation. This article focuses on the kinetics and thermodynamics of thermal inactivation of recombinant Escherichia coli cellulases, cel12B, cel8C and a polygalacturonase, peh 28, derived from Pectobacterium carotovorum sub sp. carotovorum. Several consensus motifs conferring the enzymes' thermal stability in both cel12B and peh28 model structures have been detailed earlier, which were confirmed for the three enzymes through the current study of their thermal inactivation profiles over the 20-80°C range using the respective activities on carboxymethylcellulose and polygalacturonic acid. Kinetic constants and half-lives of thermal inactivation, inactivation energy, plus inactivation entropies, enthalpies and Gibbs free energies, revealed high stability, less conformational change and protein unfolding for cel12B and peh28 due to thermal denaturation compared to cel8C. The apparent thermal stability of peh28 and cel12B, along with their hydrolytic efficiency on a lignocellulosic biomass conversion as reported previously, makes these enzymes candidates for various industrial applications. Analysis of the Gibbs free energy values suggests that the thermal stabilities of cel12B and peh28 are entropy-controlled over the tested temperature range.


Assuntos
Biocombustíveis , Celulases/metabolismo , Escherichia coli/enzimologia , Poligalacturonase/metabolismo , Termodinâmica , Carboximetilcelulose Sódica/metabolismo , Estabilidade Enzimática , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Pectinas/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Temperatura
16.
Fungal Biol ; 125(1): 1-11, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33317771

RESUMO

As the universal methyl donor for methylation reactions, S-adenosylmethionine (AdoMet) plays an indispensable role in most cellular metabolic processes. AdoMet is synthesized by AdoMet synthetase. We identified the only one AdoMet synthetase (PoSasA) in filamentous fungus Penicillium oxalicum. PoSasA was widely distributed in mycelium at different growth stages. The absence of PoSasA was lethal for P. oxalicum. The misregulation of the PoSasA encoding gene affected the synthesis of extracellular cellulolytic enzymes. The expression levels of cellobiohydrolase encoding gene cbh1/cel7A, ß-1-4 endoglucanase eg1/cel7B, and xylanase encoding gene xyn10A were remarkably downregulated as a result of decreased PosasA gene expression. The production of extracellular cellulases and hemicellulases was also reduced. By contrast, the overexpression of PosasA improved the production of extracellular cellulases and hemicellulases. A total of 133 putative interacting proteins with PoSasA were identified using tandem affinity purification and mass spectrometry. The results of functional enrichment on these proteins showed that they were mainly related to ATP binding, magnesium ion binding, and ATP synthetase activity. Several methyltransferases were also observed among these proteins. These results were consistent with the intrinsic feature of AdoMet synthetase. This work reveals the indispensable role of PoSasA in various biological processes.


Assuntos
Regulação Fúngica da Expressão Gênica , Metionina Adenosiltransferase , Viabilidade Microbiana , Penicillium , Celulases/genética , Celulases/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Viabilidade Microbiana/genética , Penicillium/enzimologia , Penicillium/genética
17.
Plant Signal Behav ; 16(2): 1846928, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33315514

RESUMO

Brassicaceae plants, including Arabidopsis thaliana, develop endoplasmic reticulum (ER)-derived structures called ER bodies, which are involved in chemical defense against herbivores. NAI1 is a basic helix-loop-helix (bHLH) type transcription factor that regulates two downstream genes, NAI2 and BGLU23, that are responsible for the ER body formation and function. Here, we examined the transcription factor function of NAI1, and found that NAI1 binds to the promoter region of NAI2 and activates the NAI2 promoter. The recombinant NAI1 protein recognizes the canonical and non-canonical G-box motifs in the NAI2 promoter. Furthermore, we examined the DNA binding activity of NAI1 toward several E-box motifs in the NAI2 and BGLU23 promoters and found that NAI1 binds to a DNA fragment that includes an E-box motif from the BGLU23 promoter. Subcellular localization of NAI1 was evident in the nucleus, which is consistent with its transcription factor function. Transient expression experiments in Nicotiana benthamiana leaves showed that GFP-NAI1 protein activated the NAI2 promoter by binding to the two G-boxes of the promoter. Disruption of the G-boxes abolished the NAI1-dependent activation of the NAI2 promoter. These results indicate that NAI1 has a DNA binding activity in a motif-dependent manner and suggest that NAI1 regulates NAI2 and BGLU23 gene expressions through binding to these DNA motifs in their promoters.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Celulases/genética , Celulases/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologia , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
18.
Anal Chem ; 92(24): 15719-15725, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33259186

RESUMO

Determination of the intracellular location of proteins is one of the fundamental tasks of microbiology. Conventionally, label-based microscopy and super-resolution techniques are employed. In this work, we demonstrate a new technique that can determine intracellular protein distribution at nanometer spatial resolution. This method combines nanoscale spatial resolution chemical imaging using the photothermal-induced resonance (PTIR) technique with multivariate modeling to reveal the intracellular distribution of cell components. Here, we demonstrate its viability by imaging the distribution of major cellulases and xylanases in Trichoderma reesei using the colocation of a fluorescent label (enhanced yellow fluorescence protein, EYFP) with the target enzymes to calibrate the chemometric model. The obtained partial least squares model successfully shows the distribution of these proteins inside the cell and opens the door for further studies on protein secretion mechanisms using PTIR.


Assuntos
Celulases/análise , Endo-1,4-beta-Xilanases/análise , Hypocreales/enzimologia , Celulases/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Microscopia de Força Atômica , Tamanho da Partícula , Espectrofotometria Infravermelho , Propriedades de Superfície
19.
J Agric Food Chem ; 68(45): 12671-12682, 2020 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-33140639

RESUMO

The filamentous fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is widely used as a cellulase producer in the industry. Herein, we describe the rational engineering of the publicly available T. reesei QM9414 strain to achieve a remarkable high-level production of cellulase on glucose. Overexpression of the key cellulase regulator XYR1 by the copper-repressible promoter Ptcu1 was first implemented to achieve a full cellulase production in the context of catabolite repression (CCR) while eliminating the requirement of inducing sugars for enzyme production. The T. reesei bgl1 gene was further overexpressed to compensate for its low ß-glucosidase activity on glucose. This overexpression resulted in a 102% increase in FPase activity compared with the CCR-released RUT-C30 strain cultured on Avicel. Moreover, the saccharification efficiency toward pretreated corncob residues by crude enzymes from the engineered strain on glucose increased by 85% compared with that treated by enzymes from RUT-C30 cultivated on Avicel. The engineered T. reesei strain thus shows great potential as a viable alternative to deliver commercial cellulases after further optimization for efficient saccharification of agricultural waste.


Assuntos
Celulases/metabolismo , Proteínas Fúngicas/metabolismo , Glucose/metabolismo , Hypocreales/enzimologia , Hypocreales/genética , Zea mays/microbiologia , Biodegradação Ambiental , Celulases/genética , Proteínas Fúngicas/genética , Engenharia Genética , Hypocreales/metabolismo , Resíduos/análise , Zea mays/metabolismo
20.
Mol Biol Rep ; 47(11): 8739-8746, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33099759

RESUMO

Stevia rebaudiana Bertoni is an important economic crop that is well known for its secondary metabolites, steviol glycosides (SGs), found in leaves. Because the enzymes of deglycosylation (glycoside hydrolases) play important roles in SGs biosynthetic processes, our study is focused on the functions of ß-glucosidases in SGs catabolism in stevia. We cloned and characterized 19 stevia GH1 genes based on transcriptomic sequences. The 19 genes were divided into five putative subfamilies in Arabidopsis. Conserved motifs in the SrGH1 proteins were analysed using the online motif-based sequence analysis tool, MEME. Most of the identified proteins contain the conserved 'TFNEP' motif (contains the catalytic acid/base) and 'ITENG' motif (contains the catalytic nucleophile). Furthermore, the steviol glycoside content and expression of these 19 genes were characterized under constant darkness. The dark treatment lowered the steviol glycoside content significantly, while SrBGLU16 responded to darkness and was markedly upregulated. This study is the first transcriptome-wide analysis of the GH1 family in Stevia rebaudiana. The sequences of 19 SrGH1 members and their expression when grown in darkness were characterized. Among the 19 genes, SrBGLU16 was markedly upregulated by darkness. Thus, we identified SrBGLU16 for further investigation as a possible steviol glycoside beta-glucosidase.


Assuntos
Celulases , Escuridão , Genes de Plantas , Stevia , Celulases/genética , Celulases/metabolismo , Diterpenos do Tipo Caurano/metabolismo , Regulação da Expressão Gênica de Plantas , Glucosídeos/metabolismo , Stevia/enzimologia , Stevia/genética , Transcriptoma
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