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1.
J Agric Food Chem ; 68(10): 3184-3194, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32105462

RESUMO

Enzymatic hydrolysis of xylan represents a promising way to produce xylooligosaccharide (XOS), which is a novel ingredient in functional food. However, the recalcitrance of xylan in natural lignocellulosic biomass entails effective and robust xylanases. In the present study, we reported the isolation of a thermophilic Streptomyces sp. B6 from mushroom compost producing high xylanase activity. Two xylanases of Streptomyces sp. B6 belonging to GH10 (XynST10) and GH11 (XynST11) families were thus identified and biochemically characterized to be robust enzymes with high alkaline- and thermostability. Direct hydrolysis of neutralized viscose fiber production waste using XynST10 and XynST11 showed that while XynST10 produced 23.22 g/L XOS with a degree of polymerization (DP) of 2-4 and 9.27 g/L xylose, XynST11 produced much less xylose (1.19 g/L) and a higher amounts of XOS with a DP = 2-4 (28.29 g/L). Thus, XynST11 holds great potential for the production of XOS from agricultural and industrial waste.


Assuntos
Proteínas de Bactérias/química , Endo-1,4-beta-Xilanases/química , Glucuronatos/química , Oligossacarídeos/química , Streptomyces/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Glucuronatos/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Resíduos Industriais/análise , Oligossacarídeos/metabolismo , Streptomyces/química , Streptomyces/genética , Xilose/química , Xilose/metabolismo
2.
Prep Biochem Biotechnol ; 50(1): 91-97, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31517567

RESUMO

Xylanases have gained increasing importance due to their diverse applications in the food, paper, and pharmaceutical industries, however, the production of these enzymes currently uses expensive substrates. It has already been estimated that more than 30% of the enzyme production cost originates from the substrate. The present study aimed to optimize the production of extracellular xylanases by the Bacillus sp. TC-DT 13 using solid-state fermentation with agro-industrial residues, with a view at reducing the production cost of these enzymes. All the agro-industrial residues were tested in submerged fermentation to select the best inductor to produce xylanase. Among these residues, wheat bran was selected as the best inducer of xylanase production with 1500 U/mL. Regarding solid-state fermentation, the use of wheat bran as the only fermentation substrate was used and a ratio of 1:4 moisture over a time of 144 hours induced higher amount of xylanase reaching 2943 U/g. The use of carbon and nitrogen sources did not result in the increase in production of xylanolitic enzymes. The use of agro-industrial residues in the solid-state fermentation, besides increasing the production of xylanase, reduces the cost of production and is an environmentally friendly alternative.


Assuntos
Bacillus/enzimologia , Fibras na Dieta/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Bacillus/metabolismo , Carbono/metabolismo , Fermentação , Microbiologia Industrial/economia , Microbiologia Industrial/métodos , Nitrogênio/metabolismo , Temperatura Ambiente
3.
Appl Biochem Biotechnol ; 190(1): 197-217, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31325025

RESUMO

Eucalyptus wood is the primary source of fibers to produce paper and cellulose in South American countries. The major by-product generated in the cellulose industry is sawdust derived from chip wood production, which is designated as Eucalyptus by-product (EB). The xylooligosaccharides (XOS) are xylose-based oligomers with proven effects over maintenance and stimulation of beneficial human gut bacteria. This study reported the EB extraction and characterization along with an assessment of hemicellulose hydrolysis using commercial xylanases to produce XOS. Hemicellulose derived from extracted and NaClO2 pretreated (HEEBPT) presented xylan content of 55%, which was similar to 58.5% found in commercial Birchwood hemicellulose (CBH). The enzymatic hydrolysis of HEEBPT and CBH presented 30% as maximum conversion of xylan into XOS without significant difference among the enzymatic extracts evaluated. The XOS production from EB was proven as a technically feasible alternative to recover a value-added product from hemicellulosic fraction generated in the cellulose industry. However, lignin removal with NaClO2 from EB affects the feasibility of an industrial process because they generate toxic compounds in the pretreatment step. Thus, further studies with alternative reagents, such as ionic liquids, are required to asses selectively lignin removal from EB. Graphical Abstract.


Assuntos
Endo-1,4-beta-Xilanases/metabolismo , Eucalyptus/metabolismo , Glucuronatos/biossíntese , Oligossacarídeos/biossíntese , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Hidrólise
4.
Recent Pat Biotechnol ; 14(1): 5-15, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31333132

RESUMO

BACKGROUND: Xylanases of thermophilic origin are more robust and stable and hence more suitable for industrial applications. The aim of the research was to develop a patent using a robust mutant exhibiting enhanced xylanase activity. The strain (Bacillus aestuarii SC-2014) subjected to mutagenesis is thermophilic in origin and hence it is envisioned that the enhancement of its catalytic potential will enhance its industrial applicability. OBJECTIVE: The main aim was to develop a stable and vigorous mutant having higher xylanase activity and improved thermostability. METHODS: The bacterial strain isolated from the Tattapani hot springs of Himachal Pradesh (India) was mutagenized by single separate exposure of Ethyl methane sulphonate (EMS) and N-methyl N-nitro N-nitrosoguanidine (MNNG). RESULTS: A mutant library was generated and extensive screening led to the identification of the most potent mutant strain selected and designated as Bacillus sp. SC-2014 EMS200 (MTCC number 25046) which displayed not only enhanced xylanase activity and thermo stability but also appreciable genetic stability. This strain displayed a 3-fold increase in enzyme activity and simultaneously, a significant reduction in fermentation time from 72 h to 48 h was also observed. The xylanase gene from wild and mutant strain was cloned, sequenced and subjected to molecular docking. Two mutations H121D and S123T were present inside the binding pocket. CONCLUSION: Mutation H121D made the binding pocket more acidic and charged, thus enhancing the xylanase activity for mutant protein. Mutations also resulted in charged amino acids (Y99K and H121D) which were identified as a probable cause for enhancing the thermostability of mutant protein.


Assuntos
Proteínas de Bactérias , Endo-1,4-beta-Xilanases , Engenharia de Proteínas/métodos , Bacillus/enzimologia , Bacillus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Fontes Termais/microbiologia , Temperatura Alta , Simulação de Acoplamento Molecular , Mutação
5.
Biosci Biotechnol Biochem ; 84(3): 640-650, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31746676

RESUMO

Corn fibre xylan (CX) shows high resistance to enzymatic hydrolysis due to its densely decorated side chains. To find enzymes capable of hydrolyzing CX, we isolated a bacterial strain (named H2C) from soil, by enrichment culture using non-starch polysaccharides of corn as the sole carbon source. Analysis based on the 16S rRNA sequence placed strain H2C within genus Paenibacillus. Enzymes were purified from supernatant of culture broth of strain H2C based on solubilizing activities toward CX. Four enzymes, Xyn5A, Xyn10B, Xyn11A, and Xyn30A, were successfully identified, which belong to glycoside hydrolase (GH) families, 5, 10, 11, and 30, respectively. Phylogenetic analysis classified Xyn5A in subfamily 35 of GH family 5, a subfamily of unknown function. Their activities toward beechwood xylan and/or wheat arabinoxylan indicated that these enzymes are ß-1,4-xylanases. They showed high solubilizing activities toward a feed material, corn dried distiller's grains with solubles, compared to five previously characterized xylanases.Abbreviations : CX: corn fibre xylan; DDGS: corn dried distiller's grains with solubles.


Assuntos
Endo-1,4-beta-Xilanases/isolamento & purificação , Endo-1,4-beta-Xilanases/metabolismo , Paenibacillus/enzimologia , Xilanos/metabolismo , Zea mays , Endo-1,4-beta-Xilanases/classificação , Hidrólise , Filogenia , Polissacarídeos/metabolismo
6.
J Appl Microbiol ; 128(1): 161-170, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31609034

RESUMO

AIMS: The utilization of micro-organisms in pulp and paper industries has proved biobleaching technology as an environmentally friendly alternative to the conventional approach. In this paper, the effect of actinobacterial fermentation broth on pulp biobleaching has been investigated. METHODS AND RESULTS: Actinobacterial colonies were isolated from lignocellulose-rich soil samples and screened for xylanase production and bleaching activity. The most efficient isolate in bleaching activity showed 100% similarity to Streptomyces rutgersensis. Pulp treatment with 5-day fermentation broth of this strain showed up to 7% increase in brightness (30°C for 6 h, pH (5-7)) compared to untreated (control) pulp. Also, after 60 min biotreatment, significant reduction (12·5%) in consumption of bleaching chemicals was achieved to obtain final brightness of 55%. CONCLUSION: Actinobacterial fermentation broth can be considered as a rich source of effective biobleaching agents which may be considered as environmental friendly and cost-effective technique in comparison with traditional method. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings showed ability of S. rutgersensis UTMC 2445 in bleaching chemomechanical paper pulp. Also, two strains of Saccharothrix, a rare actinobacterium, with biobleaching activity were introduced. In the proposed method, there is no need to use purified enzymes, and biobleaching process can be done using the fermentation broth.


Assuntos
Clareadores/metabolismo , Lignina/análise , Papel , Solo/química , Streptomyces/metabolismo , Actinomycetales/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , Lignina/metabolismo , Microbiologia do Solo , Streptomyces/enzimologia , Temperatura Ambiente
7.
Molecules ; 24(19)2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31569777

RESUMO

Most common industrial xylanases are produced from filamentous fungi. In this study, the codon-optimized xynA gene encoding xylanase A from the fungus Penicilium citrinum was successfully synthesized and expressed in the yeast Pichia pastoris. The levels of secreted enzyme activity under the control of glyceraldehyde-3-phosphate dehydrogenase (PGAP) and alcohol oxidase 1 (PAOX1) promoters were compared. The Pc Xyn11A was produced as a soluble protein and the total xylanase activity under the control of PGAP and PAOX1 was 34- and 193-fold, respectively, higher than that produced by the native strain of P. citrinum. The Pc Xyn11A produced under the control of the PAOX1 reached a maximum activity of 676 U/mL when induced with 1% (v/v) methanol every 24 h for 5 days. The xylanase was purified by ion exchange chromatography and then characterized. The enzyme was optimally active at 55 °C and pH 5.0 but stable over a broad pH range (3.0-9.0), retaining more than 80% of the original activity after 24 h or after pre-incubation at 40 °C for 1 h. With birchwood xylan as a substrate, Pc Xyn11A showed a Km(app) of 2.8 mg/mL, and a kcat of 243 s-1. The high level of secretion of Pc Xyn11A and its stability over a wide range of pH and moderate temperatures could make it useful for a variety of biotechnological applications.


Assuntos
Códon , Endo-1,4-beta-Xilanases/genética , Regulação da Expressão Gênica , Penicillium/enzimologia , Penicillium/genética , Pichia/genética , Proteínas Recombinantes , Sequência de Bases , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Ativação Enzimática , Vetores Genéticos/genética , Concentração de Íons de Hidrogênio , Temperatura Ambiente , Termodinâmica
8.
Microb Cell Fact ; 18(1): 159, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31542050

RESUMO

BACKGROUND: Xylanases randomly cleave the internal ß-1,4-glycosidic bonds in the xylan backbone and are grouped into different families in the carbohydrate-active enzyme (CAZy) database. Although multiple xylanases are detected in single strains of many filamentous fungi, no study has been reported on the composition, synergistic effect, and mode of action in a complete set of xylanases secreted by the same microorganism. RESULTS: All three xylanases secreted by Penicillium chrysogenum P33 were expressed and characterized. The enzymes Xyl1 and Xyl3 belong to the GH10 family and Xyl3 contains a CBM1 domain at its C-terminal, whereas Xyl2 belongs to the GH11 family. The optimal temperature/pH values were 35 °C/6.0, 50 °C/5.0 and 55 °C/6.0 for Xyl1, Xyl2, and Xyl3, respectively. The three xylanases exhibited synergistic effects, with the maximum synergy observed between Xyl3 and Xyl2, which are from different families. The synergy between xylanases could also improve the hydrolysis of cellulase (C), with the maximum amount of reducing sugars (5.68 mg/mL) observed using the combination of C + Xyl2 + Xyl3. Although the enzymatic activity of Xyl1 toward xylan was low, it was shown to be capable of hydrolyzing xylooligosaccharides into xylose. Xyl2 was shown to hydrolyze xylan to long-chain xylooligosaccharides, whereas Xyl3 hydrolyzed xylan to xylooligosaccharides with a lower degree of polymerization. CONCLUSIONS: Synergistic effect exists among different xylanases, and it was higher between xylanases from different families. The cooperation of hydrolysis modes comprised the primary mechanism for the observed synergy between different xylanases. This study demonstrated, for the first time, that the hydrolysates of GH11 xylanases can be further hydrolyzed by GH10 xylanases, but not vice versa.


Assuntos
Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/metabolismo , Penicillium chrysogenum/enzimologia , Polissacarídeos/metabolismo , Biocatálise , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Glucuronatos/metabolismo , Temperatura Alta , Hidrólise , Família Multigênica , Oligossacarídeos/metabolismo , Penicillium chrysogenum/química , Penicillium chrysogenum/genética , Domínios Proteicos , Xilanos/metabolismo
9.
J Agric Food Chem ; 67(40): 11198-11209, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31532988

RESUMO

The importance of inhibition sensitivity for xylanase functionality in bread making was investigated using mutants of the wild-type Bacillus subtilis xylanase (XBSTAXI), sensitive to Triticum aestivum xylanase inhibitor (TAXI). XBSNI, a mutant with reduced sensitivity to TAXI, and XBSTI, a mutant sensitive to all wheat endogenous proteinaceous inhibitors (TAXI, Xylanase Inhibiting Protein and Thaumatin-like Xylanase Inhibitor) were used. The higher inhibition sensitivity of XBSTAXI and XBSTI compared to XBSNI was associated with a respective 7- and 53-fold increase in enzyme dosage required for a maximal increase in bread loaf volume. XBSTI and XBSTAXI were only active during the mixing phase and the beginning of fermentation, while XBSNI was able to hydrolyze arabinoxylan until the end of fermentation. In spite of this difference in activity profile, no differences in loaf volume were observed for the different xylanases at optimal concentrations. Dough extensional viscosity analysis suggests that increased water availability as a result of xylanase activity favors starch-starch and starch-gluten interactions and drives the improvement in bread loaf volume.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Pão/análise , Endo-1,4-beta-Xilanases/antagonistas & inibidores , Endo-1,4-beta-Xilanases/química , Inibidores Enzimáticos/química , Proteínas de Plantas/química , Bacillus subtilis/química , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Inibidores Enzimáticos/metabolismo , Farinha/análise , Manipulação de Alimentos , Hidrólise , Mutação , Proteínas de Plantas/metabolismo , Triticum/química , Triticum/metabolismo , Viscosidade
10.
Appl Microbiol Biotechnol ; 103(19): 8035-8049, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31407040

RESUMO

Biotechnologies that aim to produce renewable fuels, chemicals, and bioproducts from residual ligno(hemi)cellulosic biomass mostly rely on enzymatic depolymerization of plant cell walls (PCW). This process requires an arsenal of diverse enzymes, including xylanases, which synergistically act on the hemicellulose, reducing the long and complex xylan chains to oligomers and simple sugars. Thus, xylanases play a crucial role in PCW depolymerization. Until recently, the largest xylanase family, glycoside hydrolase family 11 (GH11) has been exclusively represented by endo-catalytic ß-1,4- and ß-1,3-xylanases. Analysis of a metatranscriptome library from a microbial lignocellulose community resulted in the identification of an unusual exo-acting GH11 ß-1,4-xylanase (MetXyn11). Detailed characterization has been performed on recombinant MetXyn11 including determination of its low-resolution small-angle X-ray scattering (SAXS) molecular envelope in solution. Our results reveal that MetXyn11 is a monomeric globular enzyme that liberates xylobiose from heteroxylans as the only product. MetXyn11 has an optimal activity in a pH range from 6 to 9 and an optimal temperature of 50 °C. The enzyme maintained above 65% of its original activity in the pH range 5 to 6 after being incubated for 72 h at 50 °C. Addition of the enzyme to a commercial enzymatic cocktail (CelicCtec3) promoted a significant increase of enzymatic hydrolysis yields of hydrothermally pretreated sugarcane bagasse (16% after 24 h of hydrolysis).


Assuntos
Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Consórcios Microbianos , Dissacarídeos/metabolismo , Endo-1,4-beta-Xilanases/isolamento & purificação , Estabilidade Enzimática , Perfilação da Expressão Gênica , Concentração de Íons de Hidrogênio , Metagenômica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espalhamento a Baixo Ângulo , Temperatura Ambiente , Xilanos/metabolismo
11.
Enzyme Microb Technol ; 130: 109363, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31421720

RESUMO

GH11 xylanase XynJ from Bacillus sp. strain 41M-1 has a ß-jellyroll fold composed of eight ß strands with a deep active-site cleft. We hypothesized that the thermostability of XynJ will increase if the flexibility of the ß strands in the jellyroll structure is decreased without impairing activity. To verify this hypothesis, we introduced random mutations into Tyr13-Arg104 and Gly169-Tyr194, both of which are located in the ß-jellyroll fold of XynJ, to construct a site saturation mutagenesis library. By screening 576 clones followed by site saturation mutation analysis of Thr82, T82A was selected as the most thermostable variant. In the hydrolysis of beechwood xylan at pH 7.8, the temperatures required to reduce initial activity by 50% in 15 min were 61 °C for the wild-type XynJ (WT) and 65 °C for T82A. The optimum hydrolysis temperatures were 60 °C for WT and 65 °C for T82A. There was little difference in the kcat and Km values and the pH dependence of activity between WT and T82A. Crystallographic analysis of WT and T82A revealed that thermostabilization by the T82A mutation might result from the removal of unfavorable van der Waals interactions. Thus, a highly thermostable XynJ variant was generated without impairing activity using this mutation strategy.


Assuntos
Bacillus/enzimologia , Bacillus/genética , Endo-1,4-beta-Xilanases/genética , Temperatura Alta , Mutagênese , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Modelos Moleculares
12.
Enzyme Microb Technol ; 130: 109368, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31421728

RESUMO

In this work, the effect of particle size on alkali pretreatment of the almond shell was evaluated for recovery of hemicellulose. Further, endoxylanase from Thermomyces lanuginosus was immobilized on Fe-based magnetic nanoparticles to enable reuse of enzyme. Reduction in particle size significantly influences the recovery of hemicellulose as particle size below 120 µm enable recovery of 97% available hemicellulose in 1 h at 121 °C with 2 M alkali. The enzyme could retain 93.3% of enzymatic activity upon immobilization onto magnetic support using glutaraldehyde (25 mM) and was at par with the free enzyme in terms of pH and temperature profile. The measurement of reaction kinetics (Km and Vmax) indicates similar values for free and immobilized enzyme. The structural and morphological analysis indicates presence near spherical magnetic core and successful cross-linking of the enzyme without alteration of the magnetic core. The immobilized enzyme was able to hydrolyze hemicellulose to produce XOS, the yield equivalent to 67.4% of that obtained using free enzyme at 50 °C. The comparison of XOS production ability at 50 and 60 °C, suggests that the immobilized enzyme retains activity as similar yield was obtained at both temperatures, whereas, the yield for free enzyme decreases significantly. The XOS yield on recycling of immobilized enzyme for three successive cycles was found to reduce to 41% of the initial cycle. However, in all cycles of enzymatic hydrolysis, the percentage of xylobiose was found to be above 90%.


Assuntos
Endo-1,4-beta-Xilanases/metabolismo , Enzimas Imobilizadas , Glucuronatos/biossíntese , Oligossacarídeos/biossíntese , Prunus dulcis/química , Álcalis/química , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Polimerização , Polissacarídeos/metabolismo , Temperatura Ambiente
13.
J Anim Sci ; 97(8): 3535-3549, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31260526

RESUMO

This study investigated the effect of treatment of wheat straw using ammonia fiber expansion (AFEX) and exogenous fibrolytic enzymes (Viscozyme) on fiber digestibility, rumen fermentation, microbial protein synthesis, and microbial populations in an artificial rumen system [Rumen Simulation Technique (RUSITEC)]. Four treatments were assigned to 16 vessels (4 per treatment) in 2 RUSITEC apparatuses in a randomized block design. Treatments were arranged as a 2 × 2 factorial using untreated or AFEX-treated wheat straw with or without exogenous fibrolytic enzymes [0 or 500 µg of protein/g straw dry matter (DM)]. Fibrolytic enzymes were applied to straw, prior to sealing in nylon bags. The concentrate mixture was provided in a separate bag within each fermentation vessel. The RUSITECs were adapted for 8 d and disappearance of DM, neutral detergent fiber (NDF), acid detergent fiber (ADF), and crude protein (CP) was measured after 48 h of incubation. Ammonia fiber expansion increased (P < 0.01) the disappearance of wheat straw DM (69.6 vs. 38.3%), NDF (65.6 vs. 36.8%), ADF (61.4 vs. 36.0%), and CP (68.3 vs. 24.0%). Total dietary DM, organic matter (OM), and NDF disappearance was also increased (P ≤ 0.05) by enzymes. Total microbial protein production was greater (P < 0.01) for AFEX-treated (72.9 mg/d) than untreated straw (63.1 mg/d). Total gas and methane (CH4) production (P < 0.01) were also greater for AFEX-treated wheat straw than untreated straw, with a tendency for total gas to increase (P = 0.06) with enzymes. Ammonia fiber expansion increased (P < 0.01) total volatile fatty acid (VFA) production and the molar proportion of propionate, while it decreased (P < 0.01) acetate and the acetate-to-propionate ratio. The AFEX-treated straw had lower relative quantities of fungi, methanogens, and Fibrobacter succinogenes (P < 0.01) and fewer protozoa (P < 0.01) compared to untreated straw. The pH of fermenters fed AFEX-treated straw was lower (P < 0.01) than those fed untreated straw. Both AFEX (P < 0.01) and enzymes (P = 0.02) decreased xylanase activity. There was an enzyme × straw interaction (P = 0.02) for endoglucanase activity. Enzymes increased endoglucanase activity of AFEX-treated wheat straw, but had no effect on untreated straw. The addition of enzymes lowered the relative abundance of Ruminococcus flavefaciens, but increased F. succinogenes. These results indicate that AFEX increased the ruminal disappearance of wheat straw and improved fermentation and microbial protein synthesis in the RUSITEC.


Assuntos
Amônia/metabolismo , Fibras na Dieta/farmacologia , Ácidos Graxos Voláteis/metabolismo , Metano/metabolismo , Triticum/metabolismo , Ração Animal/análise , Animais , Bovinos , Celulase/metabolismo , Dieta/veterinária , Digestão/efeitos dos fármacos , Endo-1,4-beta-Xilanases/metabolismo , Feminino , Fermentação/efeitos dos fármacos , Rúmen/metabolismo , Silagem
14.
BMC Biotechnol ; 19(1): 51, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31345213

RESUMO

BACKGROUND: A mesophilic xylanase PjxA from Penicillium janthinellum MA21601 has high specific activity under acidic condition and holds great potential for applications in the animal feed industry. To enhance the thermostability of xylanase PjxA, two mutation strategies in the N-terminal region were examined and then integrated into the xylanase to further improvement. The recombinant xylanase PTxA-DB (The meaning of DB is disulfide-bridge.) was constructed by replacement of five residues in the mutated region in TfxA (T10Y, N11H, N12D, Y15F, N30 L), combined with an additional disulfide bridge in the N-terminal region. RESULTS: The Tm value of mutant PTxA-DB was improved from 21.3 °C to 76.6 °C, and its half-life was found to be 53.6 min at 60 °C, 107-fold higher than the wild type strain. The location of the disulfide bridge (T2C-T29C) was between the irregular loop and the ß-strand A2, accounting for most of the improvement in thermostability of PjxA. Further analysis indicated T2C, T29C, N30 L and Y15F lead to increase N-terminal hydrophobicity. Moreover, the specific activity and substrate affinity of PTxA-DB were also enhanced under the acidic pH values. CONCLUSIONS: These results indicated PTxA-DB could be a prospective additive to industrial animal feeds.


Assuntos
Endo-1,4-beta-Xilanases/genética , Proteínas Fúngicas/genética , Mutagênese , Penicillium/genética , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Dissulfetos/química , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Mutação , Penicillium/enzimologia , Conformação Proteica , Engenharia de Proteínas/métodos , Temperatura Ambiente
15.
Int J Mol Sci ; 20(15)2019 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-31357701

RESUMO

The thermophilic fungus Humicola insolens produces cellulolytic enzymes that are of great scientific and commercial interest; however, few reports have focused on its cellulase expression regulation mechanism. In this study, we constructed a creA gene (carbon catabolite repressor gene) disruption mutant strain of H. insolens that exhibited a reduced radial growth rate and stouter hyphae compared to the wild-type (WT) strain. The creA disruption mutant also expressed elevated pNPCase (cellobiohydrolase activities), pNPGase (ß-glucosidase activities), and xylanase levels in non-inducing fermentation with glucose. Unlike other fungi, the H. insolens creA disruption mutant displayed lower FPase (filter paper activity), CMCase (carboxymethyl cellulose activity), pNPCase, and pNPGase activity than observed in the WT strain when fermentation was induced using Avicel, whereas its xylanase activity was higher than that of the parental strain. These results indicate that CreA acts as a crucial regulator of hyphal growth and is part of a unique cellulase expression regulation mechanism in H. insolens. These findings provide a new perspective to improve the understanding of carbon catabolite repression regulation mechanisms in cellulase expression, and enrich the knowledge of metabolism diversity and molecular regulation of carbon metabolism in thermophilic fungi.


Assuntos
Carbono/metabolismo , Repressão Catabólica/genética , Sordariales/enzimologia , Ureo-Hidrolases/genética , Carbono/química , Carboximetilcelulose Sódica/metabolismo , Celulase/química , Celulase/genética , Celulase/metabolismo , Celulose/farmacologia , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Fermentação , Regulação Fúngica da Expressão Gênica/genética , Glucose/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Mutação/genética , Sordariales/metabolismo , Ureo-Hidrolases/química , beta-Glucosidase/química , beta-Glucosidase/metabolismo
16.
Microb Cell Fact ; 18(1): 122, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31286972

RESUMO

BACKGROUND: Acetyl xylan esterase plays an important role in the complete enzymatic hydrolysis of lignocellulosic materials. It hydrolyzes the ester linkages of acetic acid in xylan and supports and enhances the activity of xylanase. This study was conducted to identify and overexpress the acetyl xylan esterase (AXE) gene revealed by the genomic sequencing of the marine bacterium Ochrovirga pacifica. RESULTS: The AXE gene has an 864-bp open reading frame that encodes 287 aa and consists of an AXE domain from aa 60 to 274. Gene was cloned to pET-16b vector and expressed the recombinant AXE (rAXE) in Escherichia coli BL21 (DE3). The predicted molecular mass was 31.75 kDa. The maximum specific activity (40.08 U/mg) was recorded at the optimal temperature and pH which were 50 °C and pH 8.0, respectively. The thermal stability assay showed that AXE maintains its residual activity almost constantly throughout and after incubation at 45 °C for 120 min. The synergism of AXE with xylanase on beechwood xylan, increased the relative activity 1.41-fold. CONCLUSION: Resulted higher relative activity of rAXE with commercially available xylanase on beechwood xylan showed its potential for the use of rAXE in industrial purposes as a de-esterification enzyme to hydrolyze xylan and hemicellulose-like complex substrates.


Assuntos
Acetilesterase/metabolismo , Proteínas de Bactérias/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Fagus/química , Flavobacteriaceae/enzimologia , Xilanos/metabolismo , Acetilesterase/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Estabilidade Enzimática , Flavobacteriaceae/genética , Concentração de Íons de Hidrogênio , Hidrólise , Microbiologia Industrial , Fases de Leitura Aberta , Água do Mar/microbiologia , Especificidade por Substrato , Temperatura Ambiente
17.
Poult Sci ; 98(11): 5613-5621, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31222275

RESUMO

This study focused on analyzing the effects of inclusion of modern hybrid rye to corn-wheat diet on mechanical properties of bones and tendons. A total of 224 broiler chickens were fed a diet without rye inclusion or a diet containing 15% of hybrid rye cv. Brasetto. The diets were either unsupplemented or supplemented with xylanase (minimum activity 1000 FXU/g, dose 200 mg/kg of feed). Each dietary group consisted of 56 birds. On day 42, selected chickens (n = 7 from each group) were slaughtered. Tibia were analyzed for mineralization, geometry, and biomechanical characteristics of bone mid-diaphysis. The mechanical properties of digital flexor III tendon were also assessed. Bone mineral density and bone ash percentage did not differ when both diets were given without xylanase. Enzyme supplementation increased bone mineral density (P < 0.01) in both dietary groups, whereas bone ash percentage (P < 0.01) increased only for corn-wheat diet. Rye inclusion had no effect on bone mid-shaft geometrical traits related to tibia weight-bearing capacity (cross-sectional area, cortical index, and mean relative wall thickness). Performed bending test showed no effect of hybrid rye inclusion on bone mechanical endurance. When xylanase was supplemented, bone length (P < 0.01) and weight (P < 0.05) decreased, whereas yield load (P < 0.01), stiffness (P < 0.05), Young modulus (P < 0.05), elastics stress (P < 0.01), and ultimate stress (P < 0.01) increased, irrespective of rye presence. The tendon tensile strain test showed that in corn-wheat diet enzyme supplementation positively influenced rupture force (P < 0.05) and tendon stiffness (P < 0.01). Xylanase supplementation increased the value of energy required to tendon rupture, irrespective of rye inclusion (P < 0.05). Study showed that modern hybrid rye varieties can be introduced to corn-wheat diets of broiler chickens in the aspect of animal welfare related to the development and homeostasis of musculoskeletal system, irrespective of xylanase supplementation. The enzyme addition positively affected biomechanical properties of bones and tendons.


Assuntos
Osso e Ossos/fisiologia , Galinhas/fisiologia , Endo-1,4-beta-Xilanases/metabolismo , Secale/química , Tendões/fisiologia , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal/efeitos dos fármacos , Animais , Fenômenos Biomecânicos , Dieta/veterinária , Suplementos Nutricionais/análise , Endo-1,4-beta-Xilanases/administração & dosagem , Masculino , Distribuição Aleatória , Triticum/química , Zea mays/química
18.
Food Chem ; 297: 124945, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253310

RESUMO

Almond shell, a by-product obtained from the nut industry, was valorised into low degree of polymerisation xylooligosaccharides using alkaline pretreatment and enzymatic hydrolysis. The effect of particle size on hemicellulose recovery upon pretreatment was studied using 1 and 2 M NaOH. It was observed that particle size significantly influences hemicellulose recovery, as particles below 120 µm resulted in near complete recovery at 2 M NaOH. Enzymatic hydrolysis of hemicellulose was optimised using response surface methodology, to obtain efficient xylooligosaccharides production at low enzyme dose and high substrate concentration. For higher XOS yield, an enzyme dose of 10 U and substrate concentration <2% was optimal. The in-vitro human faecal fermentation study revealed no significant difference in gas and short chain fatty acid level among substrates evaluated. It was observed that short chain oligosaccharides produce higher level of acetate than medium chain oligosaccharides.


Assuntos
Fezes/microbiologia , Pentoses/química , Polissacarídeos/metabolismo , Técnicas de Cultura Celular por Lotes , Biomassa , Cromatografia Líquida de Alta Pressão , Endo-1,4-beta-Xilanases/metabolismo , Ácidos Graxos Voláteis/análise , Ácidos Graxos Voláteis/metabolismo , Fezes/química , Gases/química , Glucuronatos/análise , Glucuronatos/metabolismo , Humanos , Hidrólise , Oligossacarídeos/análise , Oligossacarídeos/metabolismo , Tamanho da Partícula , Polissacarídeos/química , Hidróxido de Sódio/química
19.
J Biosci Bioeng ; 128(6): 662-668, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31235414

RESUMO

Xylanases have useful applications in a wide range of industries. In this regard, Pichia pastoris has become one of the most attractive host platforms for large-scale production of xylanases. However, genomic engineering is still required for overexpression and efficient secretion. In this paper, we applied droplet-based method to screen directed evolved extracellular xylanase producing P. pastoris strain. Xylanase-producing P. pastoris cells were encapsulated in gel microdroplets with a fluorogenic reporter substrate. Improved production of xylanase increases fluorescence intensity of gel microdroplets, enabled accurate selection of evolved clones by droplet sorting. The screening strategy was validated by identifying yeast with improved xylanase production from a mixed sample with a positive selection accuracy of up to 98%. After three rounds of mutagenesis and selection, approximately 108 variants were screened, and a P. pastoris clone with more than 1.3-fold increase in xylanase activity was identified, representing cellular functions improvement of the production host. The throughput of this approach was at least 103-fold higher than that of the robot-assisted microtiter plate reader, and reagent consumption was reduced by ∼106-fold. Furthermore, the greatly shortened incubation time prior screening significantly accelerated the process of directed evolution.


Assuntos
Endo-1,4-beta-Xilanases/metabolismo , Pichia/enzimologia , Endo-1,4-beta-Xilanases/genética , Mutagênese , Pichia/genética
20.
BMC Plant Biol ; 19(1): 170, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31039733

RESUMO

BACKGROUND: Endo-ß-1,4-xylanase1 (EA), the key endoxylanase in plants, is involved in the degradation of arabinoxylan during grain germination. In barley (Hordeum vulgare L.), one gene (HvXYN-1) that encode a endo-beta-1,4-xylanase, has been cloned. However, the single nucleotide polymorphisms (SNPs) that affect the endoxylanase activity and total arabinoxylan (TAX) content have yet to be characterized. The investigation of genetic variation in HvXYN1 may facilitate a better understanding of the relationship between TAX content and EA activity in barley. RESULTS: In the current study, 56 polymorphisms were detected in HvXYN1 among 210 barley accessions collected from 34 countries, with 10 distinct haplotypes identified. The SNPs at positions 110, 305, 1045, 1417, 1504, 1597, 1880 bp in the genomic region of HvXYN1 were significantly associated with EA activity (P < 0.0001), and the sites 110, 305, and 1045 were highly significantly associated with TAX content. The amount of phenotypic variation in a given trait explained by each associated polymorphism ranged from 6.96 to 9.85%. Most notably, we found two variants at positions 1504 bp and 1880 bp in the second exon that significantly (P < 0.0001) affected EA activity; this result could be used in breeding programs to improve beer quality. In addition, African accessions had the highest EA activity and TAX content, and the richest germplasm resources were from Asia, indicating the high potential value of Asian barley. CONCLUSION: This study provided insight into understanding the relationship, EA activity, TAX content with the SNPs of HvXYN1 in barley. These SNPs can be applied as DNA markers in breeding programs to improve the quality of barley for beer brewing after further validation.


Assuntos
Endo-1,4-beta-Xilanases/metabolismo , Variação Genética , Hordeum/genética , Proteínas de Plantas/genética , Xilanos/metabolismo , Alelos , Endo-1,4-beta-Xilanases/genética , Haplótipos , Hordeum/enzimologia , Filogeografia , Proteínas de Plantas/metabolismo , Polimorfismo de Nucleotídeo Único
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