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1.
Enzyme Microb Technol ; 152: 109938, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34753033

RESUMO

Hydrolytic enzymes are highly demanded in the industry. Thermostability is an important property of enzymes that affects the economic costs of the industrial processes. The rational design of GH10 xylanase E (XylE) Penicillium canescens for the thermostability improvement was directed by ΔΔG calculations and structure analysis. Amino acid substitutions with stabilizing values of ΔΔG and providing an increase in side-chain volume of buried residues were performed experimentally. From the six designed substitutions, four substitutions appeared to be stabilizing, one - destabilizing, and one - neutral. For the improved XylE variants, values of Tm were increased by 1.1-3.1 °C, and times of half-life at 70 °C were increased in 1.3-1.7-times. Three of the four stabilizing substitutions were located in the N- or the C-terminus region. This highlights the importance of N- and C-terminus for the thermostability of GH10 xylanases and also enzymes with (ß/α)8 TIM barrel type of structure. The criteria of stabilizing values of ΔΔG and increased side-chain volume of buried residues for selection of substitutions may be applied in the rational design for thermostability improvement.


Assuntos
Penicillium , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Penicillium/genética , Penicillium/metabolismo , Temperatura
2.
Int J Mol Sci ; 22(19)2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34639149

RESUMO

Fungal enzymes degrading the plant cell wall, such as xylanases, can activate plant immune responses. The Fusarium graminearum FGSG_03624 xylanase, previously shown to elicit necrosis and hydrogen peroxide accumulation in wheat, was investigated for its ability to induce disease resistance. To this aim, we transiently and constitutively expressed an enzymatically inactive form of FGSG_03624 in tobacco and Arabidopsis, respectively. The plants were challenged with Pseudomonas syringae pv. tabaci or pv. maculicola and Botrytis cinerea. Symptom reduction by the bacterium was evident, while no reduction was observed after B. cinerea inoculation. Compared to the control, the presence of the xylanase gene in transgenic Arabidopsis plants did not alter the basal expression of a set of defense-related genes, and, after the P. syringae inoculation, a prolonged PR1 expression was detected. F. graminearum inoculation experiments of durum wheat spikes exogenously treated with the FGSG_03624 xylanase highlighted a reduction of symptoms in the early phases of infection and a lower fungal biomass accumulation than in the control. Besides, callose deposition was detected in infected spikes previously treated with the xylanase and not in infected control plants. In conclusion, our results highlight the ability of FGSG_03624 to enhance plant immunity, thus decreasing disease severity.


Assuntos
Arabidopsis/imunologia , Botrytis/patogenicidade , Resistência à Doença/imunologia , Endo-1,4-beta-Xilanases/metabolismo , Fusarium/enzimologia , Imunidade Vegetal , Pseudomonas syringae/patogenicidade , Tabaco/imunologia , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Tabaco/metabolismo , Tabaco/microbiologia
3.
Appl Microbiol Biotechnol ; 105(20): 7757-7767, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34553251

RESUMO

Xylanase is efficient for xylan degradation and widely applied in industries. We found a GH11 family xylanase (Xyn11A) with high thermostability and catalytic activity from compost metatranscriptome. This xylanase has the optimal reaction temperature at 80 °C with the activity of 2907.3 U/mg. The X-ray crystallographic structure shows a typical "right hand" architecture, which is the characteristics of the GH11 family enzymes. Comparing it with the mesophilic XYN II, a well-studied GH11 xylanase from Trichoderma reesei, Xyn11A is more compact with more H-bonds. Our mutagenic results show that the electrostatic interactions in the thumb and palm region of Xyn11A could result in its high thermostability and activity. Introducing a disulfide bond at the N-terminus further increased its optimal reaction temperature to 90 °C with augmented activity. KEY POINTS: • A hyperthermophilic xylanase with high activity was discovered using the metatranscriptomic method. • The mechanisms of thermophilicity and high activity were revealed using X-ray crystallography, mutagenesis, and molecular dynamics simulations. • The thermostability and activity were further improved by introducing a disulfide bond.


Assuntos
Compostagem , Endo-1,4-beta-Xilanases , Cristalografia por Raios X , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Hypocreales
4.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361767

RESUMO

This study describes the catalytic properties of a GH30_7 xylanase produced by the fungus Talaromyces leycettanus. The enzyme is an ando-ß-1,4-xylanase, showing similar specific activity towards glucuronoxylan, arabinoxylan, and rhodymenan (linear ß-1,3-ß-1,4-xylan). The heteroxylans are hydrolyzed to a mixture of linear as well as branched ß-1,4-xylooligosaccharides that are shorter than the products generated by GH10 and GH11 xylanases. In the rhodymenan hydrolyzate, the linear ß-1,4-xylooligosaccharides are accompanied with a series of mixed linkage homologues. Initial hydrolysis of glucuronoxylan resembles the action of other GH30_7 and GH30_8 glucuronoxylanases, resulting in a series of aldouronic acids of a general formula MeGlcA2Xyln. Due to the significant non-specific endoxylanase activity of the enzyme, these acidic products are further attacked in the unbranched regions, finally yielding MeGlcA2Xyl2-3. The accommodation of a substituted xylosyl residue in the -2 subsite also applies in arabinoxylan depolymerization. Moreover, the xylose residue may be arabinosylated at both positions 2 and 3, without negatively affecting the main chain cleavage. The catalytic properties of the enzyme, particularly the great tolerance of the side-chain substituents, make the enzyme attractive for biotechnological applications. The enzyme is also another example of extraordinarily great catalytic diversity among eukaryotic GH30_7 xylanases.


Assuntos
Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/metabolismo , Talaromyces/enzimologia , Xilanos/metabolismo , Sequência de Aminoácidos , Arabinose/química , Arabinose/metabolismo , Sequência de Carboidratos , Endo-1,4-beta-Xilanases/genética , Proteínas Fúngicas/genética , Expressão Gênica , Glucuronatos/química , Glucuronatos/metabolismo , Hidrólise , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Talaromyces/química , Talaromyces/genética , Xilanos/química
5.
Molecules ; 26(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34361682

RESUMO

Catalytic properties of GH30 xylanases belonging to subfamilies 7 and 8 were compared on glucuronoxylan, modified glucuronoxylans, arabinoxylan, rhodymenan, and xylotetraose. Most of the tested bacterial GH30-8 enzymes are specific glucuronoxylanases (EC 3.2.1.136) requiring for action the presence of free carboxyl group of MeGlcA side residues. These enzymes were not active on arabinoxylan, rhodymenan and xylotetraose, and conversion of MeGlcA to its methyl ester or its reduction to MeGlc led to a remarkable drop in their specific activity. However, some GH30-8 members are nonspecific xylanases effectively hydrolyzing all tested substrates. In terms of catalytic activities, the GH30-7 subfamily is much more diverse. In addition to specific glucuronoxylanases, the GH30-7 subfamily contains nonspecific endoxylanases and predominantly exo-acting enzymes. The activity of GH30-7 specific glucuronoxylanases also depend on the presence of the MeGlcA carboxyl, but not so strictly as in bacterial enzymes. The modification of the carboxyl group of glucuronoxylan had only weak effect on the action of predominantly exo-acting enzymes, as well as nonspecific xylanases. Rhodymenan and xylotetraose were the best substrates for exo-acting enzymes, while arabinoxylan represented hardly degradable substrate for almost all tested GH30-7 enzymes. The results expand current knowledge on the catalytic properties of this relatively novel group of xylanases.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/metabolismo , Fungos/enzimologia , Xilosidases/metabolismo , Catálise , Hidrólise , Especificidade por Substrato , Xilanos/metabolismo
6.
Braz J Biol ; 83: e243874, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34378658

RESUMO

In recent days, cheapest alternative carbon source for fermentation purpose is desirable to minimize production cost. Xylanases have become attractive enzymes as their potential in bio-bleaching of pulp and paper industry. The objective of the present study was to identify the potential ability on the xylanase production by locally isolated Bacillus pumilus BS131 by using waste fiber sludge and wheat bran media under submerged fermentation. Culture growth conditions were optimized to obtain significant amount of xylanase. Maximum xylanase production was recorded after 72 hours of incubation at 30 °C and 7 pH with 4.0% substrate concentration. In the nutshell, the production of xylanase using inexpensive waste fiber sludge and wheat-bran as an alternative in place of expensive xylan substrate was more cost effective and environment friendly.


Assuntos
Bacillus pumilus , Bacillus , Bacillus/metabolismo , Bacillus pumilus/metabolismo , Fibras na Dieta , Endo-1,4-beta-Xilanases/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , Esgotos , Temperatura
7.
Bioresour Technol ; 340: 125732, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34426240

RESUMO

The ancestor of ß-1,3-xylanases (AncXyl09) were reconstructed by the optimized ancestral sequences reconstruction strategy to solve the poor catalytic performances of existing ß-1,3-xylanases. The results showed that the half-life at 50 °C was 65.08 h, indicating good thermostability. The large number of hydrogen bonds and the disulfide bonds were the major attributes related with the thermal stability of Anxyl09. Interestingly, AncXyl09 could hydrolyze lichen besides the original substrate of ß-1, 3-xylan, which is the first reported ß-1,3-xylanase with substrate promiscuity. Moreover, the hydrolytic products are mainly disaccharides, the content of ß-1,3-xylobiose and lichoridiose more than 70% as determined by high performance liquid chromatography (HPLC), which could significantly facilitate the separation and purification of oligosaccharides. The successful design of AncXyl09 was the representative of the semi-rationally engineered ß-1, 3-xylanase, which will shield a new light on the ß-1,3-xylanase engineering, active oligosaccharide preparation and marine algae resource utilization.


Assuntos
Endo-1,4-beta-Xilanases , Xilanos , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Especificidade por Substrato , Temperatura
8.
Sci Rep ; 11(1): 16468, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389757

RESUMO

Paecilomyces variotii xylanase was, produced in stirred tank bioreactor with yield of 760 U/mL and purified using 70% ammonium sulfate precipitation and ultra-filtration causing 3.29-fold purification with 34.47% activity recovery. The enzyme purity was analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirming its monomeric nature as single band at 32 KDa. Zymography showed xylan hydrolysis activity at the same band. The purified enzyme had optimum activity at 60 °C and pH 5.0. The pH stability range was 5-9 and the temperature stability was up 70 °C. Fe2+and Fe3+ exhibited inhibition of xylanase enzyme while Cu2+, Ca2+, Mg2+ and Mn2+ stimulated its activity. Mercaptoethanol stimulated its activity; however, Na2-EDTA and SDS inhibited its activity. The purified xylanase could hydrolyze beechwood xylan but not carboxymethyl cellulose (CMC), avicel or soluble starch. Paecilomyces variotii xylanase Km and Vmax for beechwood were determined to be 3.33 mg/mL and 5555 U/mg, respectively. The produced xylanase enzyme applied on beech xylan resulted in different types of XOS. The antioxidant activity of xylo-oligosaccharides increased from 15.22 to 70.57% when the extract concentration was increased from 0.1 to 1.5 mg/mL. The enzyme characteristics and kinetic parameters indicated its high efficiency in the hydrolysis of xylan and its potential effectiveness in lignocellulosic hydrolysis and other industrial application. It also suggests the potential of xylanase enzyme for production of XOS from biomass which are useful in food and pharmaceutical industries.


Assuntos
Antioxidantes/metabolismo , Byssochlamys/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Glucuronatos/metabolismo , Oligossacarídeos/metabolismo , Reatores Biológicos , Byssochlamys/enzimologia , Eletroforese em Gel de Poliacrilamida , Endo-1,4-beta-Xilanases/isolamento & purificação , Concentração de Íons de Hidrogênio
9.
Enzyme Microb Technol ; 149: 109834, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34311879

RESUMO

The goal of this work was the autodisplay of the endo ß-1,4-xylanase (XynA) from Clostridium cellulovorans in Escherichia coli using the AIDA system to carry out whole-cell biocatalysis and hydrolysate xylans. For this, pAIDA-xynA vector containing a synthetic xynA gene was fused to the signal peptide of the toxin subunit B Vibro cholere (ctxB) and the auto-transporter of the synthetic aida gene, which encodes for the connector peptide and ß-barrel of the auto-transporter (AT-AIDA). E. coli TOP10 cells were transformed and the biocatalyst was characterized using beechwood xylans as substrate. Optimal operational conditions were temperature of 55 °C and pH 6.5, and the Michaelis-Menten catalytic constants Vmax and Km were 149 U/gDCW and 6.01 mg/mL, respectively. Xylanase activity was inhibited by Cu2+, Zn2+ and Hg2+ as well as EDTA, detergents, and organic acids, and improved by Ca2+, Co2+ and Mn2+ ions. Ca2+ ion strongly enhanced the xylanolytic activity up to 2.4-fold when 5 mM CaCl2 were added. Also, Ca2+ improved enzyme stability at 60 and 70 °C. Results suggest that pAIDA-xynA vector has the ability to express functional xylanase to perform whole-cell biocatalysis in order to hydrolysate xylans from hemicellulose feedstock.


Assuntos
Clostridium cellulovorans , Xilanos , Clostridium cellulovorans/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Temperatura
10.
Enzyme Microb Technol ; 149: 109854, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34311891

RESUMO

Xylanases are categorized into different family groups, two of which are glycoside hydrolases 10 (GH10) and 11 (GH11) families. These well-characterized xylanases demonstrate different modes of action in hydrolysis of xylans. Imitating certain types of microorganisms to produce bifunctional enzymes such as engineered xylanases has gained considerable attention among researchers. In this study, a recombinant chimeric enzyme (X11-10) was designed by fusing two thermostable xylanases through a peptide linker. The recombinant parental enzymes, xylanase 10 from fungus Bispora sp. MEY-1 (X10) and xylanase 11 from bacterium Thermobacillus xylanilyticus (X11), and their chimera were successfully expressed in Pichia pastoris (P. pastoris), purified, and characterized. Being active over a wide pH range, X11-10 chimera showed higher thermal stability, possessed a lower Km, and a higher catalytic efficiency (kcat/Km) in comparison to the parental enzymes. Also, molecular dynamics simulation (MDS) of X11-10 revealed that its active site residues were free to interact with substrate. This novel chimeric xylanase may have potential applications in different industrial processes since it can substitute two separate enzymes and therefore minimize the production costs.


Assuntos
Ascomicetos , Xilanos , Ascomicetos/metabolismo , Bacillales , Quimera/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Glicosídeo Hidrolases , Proteínas Recombinantes de Fusão/genética , Saccharomycetales , Especificidade por Substrato
11.
Molecules ; 26(14)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34299554

RESUMO

The present work reports the biobleaching effect on OPEFB pulp upon utilisation of extracellular xylano-pectinolytic enzymes simultaneously yielded from Bacillus amyloliquefaciens ADI2. The impacts of different doses, retention times, pH, and temperatures required for the pulp biobleaching process were delineated accordingly. Here, the OPEFB pulp was subjected to pre-treatment with xylano-pectinolytic enzymes generated from the same alkalo-thermotolerant isolate that yielded those of higher quality. Remarkable enhanced outcomes were observed across varying pulp attributes: for example, enzyme-treated pulp treated to chemical bleaching sequence generated improved brightness of 11.25%. This resulted in 11.25% of less chlorine or chemical consumption required for obtaining pulp with optical attributes identical to those generated via typical chemical bleaching processes. Ultimately, the reduced consumption of chlorine would minimise the organochlorine compounds found in an effluent, resulting in a lowered environmental effect of paper-making processes overall as a consequence. This will undoubtedly facilitate such environmentally-friendly technology incorporation in the paper pulp industry of today.


Assuntos
Bacillus amyloliquefaciens/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Frutas/metabolismo , Óleo de Palmeira/metabolismo , Concentração de Íons de Hidrogênio , Papel , Poligalacturonase/metabolismo , Temperatura
12.
Poult Sci ; 100(9): 101275, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34273648

RESUMO

Feed costs represent a significant portion of the cost of poultry production. This study, in 3 experiments, was conducted to evaluate the effectiveness of a heat-stable xylanase (XYL) as a dietary supplement and its effect on digesta viscosity, nitrogen-corrected apparent metabolizable energy (AMEn), and live performance in broiler chicks. Experiment 1: the objective was to determine the effects of the amount and type of enzyme supplementation on digesta viscosity, AMEn, and bird performance using 7 diets. The dietary treatments were: no supplementation (C), 5 levels of XYL (1 to 16 ppm), or supplementation with a carbohydrase cocktail (CC). Experiment 2: the objective was to determine the interaction of the dietary XYL and the energy content of the feed. There were 2 levels of XYL (0 and 20 ppm) and 3 dietary energy levels (2,770, 2,920, and 3,070 kcal/kg ME). Experiment 3: the objective was to determine the interaction of the dietary XYL and feed form. The treatments were: 5 levels of XYL (0 to 40 ppm) and 2 feed forms (mash and crumble). Broiler chicks were reared in battery cages to 21 d. Statistical analysis of the data was completed using Proc GLM of SAS (9.2) (SAS Institute, Cary, NC). In experiment 1, increasing XYL (0 to 16 ppm) resulted in a decrease in digesta viscosity and an increase in AMEn. The XYL included as low as 1 ppm resulted in a significant increase in AMEn which reached 5% with 16 ppm XYL. In contrast, increase in BWG (4%) above values with the basal diet was greatest with 1 ppm XYL. In experiment 2, the caloric content of the diet influenced the increase in AMEn with inclusion of XYL, 8% and 6% increases with 2,920 kcal/kg and 3,070 kcal/kg diets, respectively. Without addition of XYL, BWG was significantly lower when fed the diet with the highest energy content. In experiment 3, feed form x XYL influenced the effect of XYL on BWG. The BWG was greater when birds were fed the crumble diet with XYL vs when they were fed the mash feed with XYL. The xylanase proved effective for broilers to 21 d when fed the diets used herein with changes in digesta viscosity, increased dietary AMEn, and improved bird performance represented by either BW gain or FCR.


Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Galinhas , Ração Animal/análise , Animais , Galinhas/metabolismo , Dieta/veterinária , Suplementos Nutricionais/análise , Endo-1,4-beta-Xilanases/metabolismo , Metabolismo Energético , Temperatura Alta , Triticum/metabolismo , Viscosidade
13.
Microb Cell Fact ; 20(1): 129, 2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34238305

RESUMO

BACKGROUND: Xylanase-containing enzyme cocktails are used on an industrial scale to convert xylan into value-added products, as they hydrolyse the ß-1,4-glycosidic linkages between xylopyranosyl residues. In the present study, we focused on xynS1, the glycoside hydrolase (GH) 11 xylanase gene derived from the Streptomyces sp. strain J103, which can mediate XynS1 protein synthesis and lignocellulosic material hydrolysis. RESULTS: xynS1 has an open reading frame with 693 base pairs that encodes a protein with 230 amino acids. The predicted molecular weight and isoelectric point of the protein were 24.47 kDa and 7.92, respectively. The gene was cloned into the pET-11a expression vector and expressed in Escherichia coli BL21(DE3). Recombinant XynS1 (rXynS1) was purified via His-tag affinity column chromatography. rXynS1 exhibited optimal activity at a pH of 5.0 and temperature of 55 °C. Thermal stability was in the temperature range of 50-55 °C. The estimated Km and Vmax values were 51.4 mg/mL and 898.2 U/mg, respectively. One millimolar of Mn2+ and Na+ ions stimulated the activity of rXynS1 by up to 209% and 122.4%, respectively, and 1 mM Co2+ and Ni2+ acted as inhibitors of the enzyme. The mixture of rXynS1, originates from Streptomyces sp. strain J103 and acetyl xylan esterase (AXE), originating from the marine bacterium Ochrovirga pacifica, enhanced the xylan degradation by 2.27-fold, compared to the activity of rXynS1 alone when Mn2+ was used in the reaction mixture; this reflected the ability of both enzymes to hydrolyse the xylan structure. The use of an enzyme cocktail of rXynS1, AXE, and commercial cellulase (Celluclast® 1.5 L) for the hydrolysis of lignocellulosic biomass was more effective than that of commercial cellulase alone, thereby increasing the relative activity 2.3 fold. CONCLUSION: The supplementation of rXynS1 with AXE enhanced the xylan degradation process via the de-esterification of acetyl groups in the xylan structure. Synergetic action of rXynS1 with commercial cellulase improved the hydrolysis of pre-treated lignocellulosic biomass; thus, rXynS1 could potentially be used in several industrial applications.


Assuntos
Acetilesterase/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Lignina/metabolismo , Streptomyces/enzimologia , Xilanos/metabolismo , Biomassa , Celulase/metabolismo , Clonagem Molecular , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Hidrólise , Metais/farmacologia , Proteínas Recombinantes/metabolismo , Temperatura
14.
Biochemistry ; 60(26): 2071-2083, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34156819

RESUMO

Xylan and cellulose are the two major constituents of numerous types of lignocellulose. The bifunctional enzyme that exhibits xylanase/cellulase activity has attracted a great deal of attention in biofuel production. Previously, a thermostable GH10 family enzyme (XynA) from Bacillus sp. KW1 was found to degrade both xylan and cellulose. To improve bifunctional activity on the basis of structure, we first determined the crystal structure of XynA at 2.3 Å. Via molecular docking and activity assays, we revealed that Gln250 and His252 were indispensable to bifunctionality, because they could interact with two conserved catalytic residues, Glu182 and Glu280, while bringing the substrate close to the activity pocket. Then we used a structure-based engineering strategy to improve xylanase/cellulase activity. Although no mutants with increased bifunctional activity were obtained after much screening, we found the answer in the N-terminal 36-amino acid truncation of XynA. The activities of XynA_ΔN36 toward beechwood xylan, wheat arabinoxylan, filter paper, and barley ß-glucan were significantly increased by 0.47-, 0.53-, 2.46-, and 1.04-fold, respectively. Furthermore, upon application, this truncation released more reducing sugars than the wild type in the degradation of pretreated corn stover and sugar cane bagasse. These results showed the detailed molecular mechanism of the GH10 family bifunctional endoxylanase/cellulase. The basis of these catalytic performances and the screened XynA_ΔN36 provide clues for the further use of XynA in industrial applications.


Assuntos
Proteínas de Bactérias/química , Celulase/química , Endo-1,4-beta-Xilanases/química , Enzimas Multifuncionais/química , Sequência de Aminoácidos , Bacillus/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Celulase/genética , Celulase/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Simulação de Acoplamento Molecular , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Oligossacarídeos/metabolismo , Ligação Proteica , Engenharia de Proteínas , Xilanos/metabolismo
15.
PLoS One ; 16(6): e0251556, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086701

RESUMO

A diverse range of monocot and dicot grains and their by-products are commonly used in the animal feed industry. They all come with complex and variable cell wall structures which in turn contribute significant fiber to the complete feed. The cell wall is a highly interconnected matrix of various polysaccharides, proteins and lignin and, as such, requires a collaborative effort of different enzymes for its degradation. In this regard, we investigated the potential of a commercial multicomponent carbohydrase product from a wild type fermentation of Trichoderma reesei (T. reesei) (RONOZYME® MultiGrain) in degrading cell wall components of wheat, barley, rye, de-oiled rice bran, sunflower, rapeseed and cassava. A total of thirty-one different enzyme proteins were identified in the T. Reesei carbohydrase product using liquid chromatography with tandem mass spectrometry LC-MS/MS including glycosyl hydrolases and carbohydrate esterases. As measured by in vitro incubations and non-starch polysaccharide component analysis, and visualization by immunocytochemistry and confocal microscopy imaging of immuno-labeled samples with confocal microscopy, the carbohydrase product effectively solubilized cellulolytic and hemicellulolytic polysaccharides present in the cell walls of all the feed ingredients evaluated. The T. reesei fermentation also decreased viscosity of arabinoxylan, xyloglucan, galactomannan and ß-glucan substrates. Combination of several debranching enzymes including arabinofuranosidase, xylosidase, α-galactosidase, acetyl xylan esterase, and 4-O-methyl-glucuronoyl methylesterase with both GH10 and GH11 xylanases in the carbohydrase product resulted in effective hydrolyzation of heavily branched glucuronoarabinoxylans. The different ß-glucanases (both endo-ß-1,3(4)-glucanase and endo-ß-1,3-glucanase), cellulases and a ß-glucosidase in the T. reesei fermentation effectively reduced polymerization of both ß-glucans and cellulose polysaccharides of viscous cereals grains (wheat, barley, rye and oat). Interestingly, the secretome of T. reesei contained significant amounts of an exceptional direct chain-cutting enzyme from the GH74 family (Cel74A, xyloglucan-specific ß-1,4-endoglucanase), that strictly cleaves the xyloglucan backbone at the substituted regions. Here, we demonstrated that the balance of enzymes present in the T. reesei secretome is capable of degrading various cell wall components in both monocot and dicot plant raw material used as animal feed.


Assuntos
Ração Animal/análise , Parede Celular/metabolismo , Glicosídeo Hidrolases/metabolismo , Hypocreales/metabolismo , Acetilesterase/metabolismo , Celulases/metabolismo , Cromatografia Líquida/métodos , Endo-1,4-beta-Xilanases/metabolismo , Glucanos/metabolismo , Espectrometria de Massas em Tandem/métodos , Xilanos/metabolismo , Xilosidases/metabolismo
16.
Biomolecules ; 11(5)2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33946575

RESUMO

Endo-ß-1,4-xylanase is a key enzyme in the degradation of ß-1,4-d-xylan polysaccharides through hydrolysis. A glycoside hydrolase family 10 (GH10) endo-ß-1,4-xylanase (XylR) from Duganella sp. PAMC 27433, an Antarctic soil bacterium, was identified and functionally characterized. The XylR gene (1122-bp) encoded an acidic protein containing a single catalytic GH10 domain that was 86% identical to that of an uncultured bacterium BLR13 endo-ß-1,4-xylanase (ACN58881). The recombinant enzyme (rXylR: 42.0 kDa) showed the highest beechwood xylan-degrading activity at pH 5.5 and 40 °C, and displayed 12% of its maximum activity even at 4 °C. rXylR was not only almost completely inhibited by 5 mM N-bromosuccinimide or metal ions (each 1 mM) including Hg2+, Ca2+, or Cu2+ but also significantly suppressed by 1 mM Ni2+, Zn2+, or Fe2+. However, its enzyme activity was upregulated (>1.4-fold) in the presence of 0.5% Triton X-100 or Tween 80. The specific activities of rXylR toward beechwood xylan, birchwood xylan, oat spelts xylan, and p-nitrophenyl-ß-d-cellobioside were 274.7, 103.2, 35.6, and 365.1 U/mg, respectively. Enzymatic hydrolysis of birchwood xylan and d-xylooligosaccharides yielded d-xylose and d-xylobiose as the end products. The results of the present study suggest that rXylR is a novel cold-adapted d-xylobiose- and d-xylose-releasing endo-ß-1,4-xylanase.


Assuntos
Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Oxalobacteraceae/enzimologia , Oxalobacteraceae/genética , Sequência de Aminoácidos , Regiões Antárticas , Clonagem Molecular , DNA Bacteriano , Dissacarídeos/metabolismo , Endo-1,4-beta-Xilanases/química , Concentração de Íons de Hidrogênio , Hidrólise , Oxalobacteraceae/classificação , Oxalobacteraceae/isolamento & purificação , Filogenia , Reação em Cadeia da Polimerase , RNA Ribossômico 16S , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Microbiologia do Solo , Especificidade por Substrato , Temperatura , Xilanos/metabolismo , Xilose/metabolismo
17.
Appl Environ Microbiol ; 87(15): e0059321, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34047636

RESUMO

The filamentous fungus Trichoderma reesei is a model strain for cellulase production. Cellulase gene expression in T. reesei is controlled by multiple transcription factors. Here, we identified by comparative genomic screening a novel transcriptional activator, ACE4 (activator of cellulase expression 4), that positively regulates cellulase gene expression on cellulose in T. reesei. Disruption of the ace4 gene significantly decreased expression of four main cellulase genes and the essential cellulase transcription factor-encoding gene ace3. Overexpression of ace4 increased cellulase production by approximately 22% compared to that in the parental strain. Further investigations using electrophoretic mobility shift assays, DNase I footprinting assays, and chromatin immunoprecipitation assays indicated that ACE4 directly binds to the promoter of cellulase genes by recognizing the two adjacent 5'-GGCC-3' sequences. Additionally, ACE4 directly binds to the promoter of ace3 and, in turn, regulates the expression of ACE3 to facilitate cellulase production. Collectively, these results demonstrate an important role for ACE4 in regulating cellulase gene expression, which will contribute to understanding the mechanism underlying cellulase expression in T. reesei. IMPORTANCE T. reesei is commonly utilized in industry to produce cellulases, enzymes that degrade lignocellulosic biomass for the production of bioethanol and bio-based products. T. reesei is capable of rapidly initiating the biosynthesis of cellulases in the presence of cellulose, which has made it useful as a model fungus for studying gene expression in eukaryotes. Cellulase gene expression is controlled through multiple transcription factors at the transcriptional level. However, the molecular mechanisms by which transcription is controlled remain unclear. In the present study, we identified a novel transcription factor, ACE4, which regulates cellulase expression on cellulose by binding to the promoters of cellulase genes and the cellulase activator gene ace3. Our study not only expands the general functional understanding of the novel transcription factor ACE4 but also provides evidence for the regulatory mechanism mediating gene expression in T. reesei.


Assuntos
Celulase/genética , Transativadores/genética , Trichoderma/genética , Celulase/metabolismo , Celulose/metabolismo , Celulose 1,4-beta-Celobiosidase/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Regulação Fúngica da Expressão Gênica , Trichoderma/crescimento & desenvolvimento , Trichoderma/metabolismo
18.
Plant Sci ; 308: 110792, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34034860

RESUMO

Arabinoxylans are cell wall polysaccharides whose re-modelling and degradation during plant development are mediated by several classes of xylanolytic enzymes. Here, we present the identification and new annotation of twelve putative (1,4)-ß-xylanase and six ß-xylosidase genes, and their spatio-temporal expression patterns during vegetative and reproductive growth of barley (Hordeum vulgare cv. Navigator). The encoded xylanase proteins are all predicted to contain a conserved carbohydrate-binding module (CBM) and a catalytic glycoside hydrolase (GH) 10 domain. Additional domains in some xylanases define three discrete phylogenetic clades: one clade contains proteins with an additional N-terminal signal sequence, while another clade contains proteins with multiple CBMs. Homology modelling revealed that all fifteen xylanases likely contain a third domain, a ß-sandwich folded from two non-contiguous sequence segments that bracket the catalytic GH domain, which may explain why the full length protein is required for correct folding of the active enzyme. Similarly, predicted xylosidase proteins share a highly conserved domain structure, each with an N-terminal signal peptide, a split GH 3 domain, and a C-terminal fibronectin-like domain. Several genes appear to be ubiquitously expressed during barley growth and development, while four newly annotated xylanase and xylosidase genes are expressed at extremely high levels, which may be of broader interest for industrial applications where cell wall degradation is necessary.


Assuntos
Endo-1,4-beta-Xilanases/genética , Genes de Plantas , Hordeum/genética , Proteínas de Plantas/genética , Xilosidases/genética , Sequência de Aminoácidos , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Perfilação da Expressão Gênica , Hordeum/enzimologia , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alinhamento de Sequência , Análise Espaço-Temporal , Xilosidases/química , Xilosidases/metabolismo
19.
Appl Microbiol Biotechnol ; 105(11): 4561-4576, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34014347

RESUMO

Xylanases have a broad range of applications in industrial biotechnologies, which require the enzymes to resist the high-temperature environments. The majority of xylanases have maximum activity at moderate temperatures, which limited their potential applications in industries. In this study, a thermophilic GH10 family xylanase XynAF1 from the high-temperature composting strain Aspergillus fumigatus Z5 was characterized and engineered to further improve its thermostability. XynAF1 has the optimal reaction temperature of 90 °C. The crystal structure of XynAF1 was obtained by X-ray diffraction after heterologous expression, purification, and crystallization. The high-resolution X-ray crystallographic structure of the protein-product complex was obtained by soaking the apo-state crystal with xylotetraose. Structure analysis indicated that XynAF1 has a rigid skeleton, which helps to maintain the hyperthermophilic characteristic. The homologous structure analysis and the catalytic center mutant construction of XynAF1 indicated the conserved catalytic center contributed to the high optimum catalytic temperature. The amino acids in the surface of xylanase XynAF1 which might influence the enzyme thermostability were identified by the structure analysis. Combining the rational design with the saturation mutation at the high B-value regions, the integrative mutant XynAF1-AC with a 6-fold increase of thermostability was finally obtained. This study efficiently improved the thermostability of a GH10 family xylanase by semi-rational design, which provided a new biocatalyst for high-temperature biotechnological applications. KEY POINTS: • Obtained the crystal structure of GH10 family hyperthermophilic xylanase XynAF1. • Shed light on the understanding of the GH10 family xylanase thermophilic mechanism. • Constructed a 6-fold increased thermostability recombinant xylanase.


Assuntos
Endo-1,4-beta-Xilanases , Temperatura Alta , Cristalografia por Raios X , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Modelos Moleculares , Temperatura
20.
Sci Rep ; 11(1): 10440, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001974

RESUMO

Metagenomic data mining of the Nellore cattle rumen microbiota identified a new bifunctional enzyme, endo-1,4-ß-xylanase/esterase, which was subsequently overexpressed in E. coli BL21 (DE3). This enzyme was stable at pH intervals of 5 to 6.5 and temperatures between 30 and 45 °C, and under the test conditions, it had a Vmax of 30.959 ± 2.334 µmol/min/mg, Km of 3.6 ± 0.6 mM and kcat of 2.323 ± 175 s-1. Additionally, the results showed that the enzyme is tolerant to NaCl and organic solvents and therefore is suitable for industrial environments. Xylanases are widely applicable, and the synergistic activity of endo-1,4-ß-xylanase/esterase in a single molecule will improve the degradation efficiency of heteroxylans via the creation of xylanase binding sites. Therefore, this new molecule has the potential for use in lignocellulosic biomass processing and as an animal feed food additive and could improve xylooligosaccharide production efficiency.


Assuntos
Proteínas de Bactérias/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Esterases/metabolismo , Microbioma Gastrointestinal , Rúmen/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Bovinos , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/isolamento & purificação , Ensaios Enzimáticos , Esterases/genética , Esterases/isolamento & purificação , Glucuronatos/biossíntese , Microbiologia Industrial/métodos , Lignina/metabolismo , Metagenoma , Oligossacarídeos/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Energia Renovável
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