Xilanases, ß-xilosidases de Penicillium janczewskii: purificação, caracterização e aplicação no branqueamento da polpa celulósica e para ração animal

Xilanases e β-xilosidases são as principais enzimas responsáveis pela degradação da xilana, o segundo principal constituinte da parede celular vegetal. Tanto a produção destas enzimas por micro-organismos, quanto a caracterização bioquímica das mesmas têm sido amplamente estudadas devido às suas inúmeras aplicações biotecnológicas. Neste trabalho, as principais xilanase e β-xilosidase produzidas por uma linhagem de Penicillium janczewskii em meio de cultura líquido foram purificadas por métodos clássicos de purificação de proteínas, sendo, posteriormente, caracterizadas bioquimicamente. A xilanase apresentou atividade ótima em pH 6,0 e a 65 °C e a β-xilosidase em pH 5,0 e a 75 °C. Ambas as enzimas apresentaram características interessantes principalmente com relação à atividade ótima de ambas as enzimas em elevadas temperaturas, a prolongada estabilidade da β-xilosidase a 60 °C, e a estabilidade da xilanase em pH mais alcalinos, considerando-se algumas de suas possíveis aplicações. Visando uma futura aplicação, a produção destas enzimas foi avaliada em cultivos sólidos utilizando-se como substrato o bagaço de malte, resíduo da indústria cervejeira. As melhores condições para produção enzimática foram: utilização da umidade inicial do substrato de 50% fornecida com solução de sais de Vogel, tempo de cultivo de sete dias a 25 °C para produção de xilanases e a 20 °C para produção de b-xilosidases. O material fermentado apresentou aumento no teor protéico, na quantidade de alguns aminoácidos essenciais e ausência de micotoxinas. Ainda com um enfoque ambiental o filtrado de cultura de P. janczewskii, produzido em condições anteriormente selecionadas, foi aplicado no biobranqueamento de uma polpa kraft de eucalipto pré-branqueada com oxigênio, sendo realizados ensaios para seleção da concentração de xilanases e do tempo de reaçãoXylanases and β-xylosidases are the main enzymes responsible for the degradation of xylan, the second main constituent of plant cell walls. The production of these enzymes by microorganisms, and their biochemical characterization has been extensively studied due to their wide range of biotechnological applications. In this work, the main xylanase and β-xylosidase produced in liquid cultures by a Penicillium janczewskii strain were purified by classical methods of protein purification, and further biochemically characterized. The xylanase showed optimal activity at pH 6.0 and 65 °C and β-xylosidases at pH 5.0 and 75 °C. Considering some possibilities of applications, the enzymes presented interesting characteristics especially in relation to the optimal activity at high temperatures, the prolonged stability of the β-xylosidase at 60 °C, and the stability of the xylanase in alkaline pH. Aiming at a future application, the production of these enzymes was investigated in solid state fermentation using brewer’s spent grain, a residue of the brewing industry, as substrate. The optimized conditions were: 50% initial substrate moisture supplied by Vogel’s salt solution, culturing for 7 days at 25 °C for xylanase production and at 20 °C for b-xylosidase production. The fermented substrate showed increase in protein content, in the amount of some essential amino acids, and absence of mycotoxins. Maintaining the environmental focus, the P. janczewskii crude filtrate, produced under previously selected conditions, was applied in the biobleaching of eucalyptus kraft pulp pre-bleached with oxygen. Trials were conducted for the selection of xylanase concentration and reaction timeFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Purification and characterization of xylanases from Trichoderma inhamatum

AbstractBackgroundTwo xylanases, Xyl I and Xyl II, were purified from the crude extracellular extract of a Trichoderma inhamatum strain cultivated in liquid medium with oat spelts xylan.ResultsThe molecular masses of the purified enzymes estimated by SDS-PAGE and gel filtration were, respectively, 19 and 14kDa for Xyl I and 21 and 14.6kDa for Xyl II. The enzymes are glycoproteins with optimum activity at 50°C in pH5.0–5.5 for Xyl I and 5.5 for Xyl II. The xylanases were very stable at 40°C and in the pH ranges from 4.5–6.5 for Xyl I and 4.0–8.0 for Xyl II. The ion Hg2+ and the detergent SDS strongly reduced the activity while 1,4-dithiothreitol stimulated both enzymes. The xylanases showed specificity for xylan, Km and Vmax of 14.5, 1.6mg·mL-1 and 2680.2 and 462.2U·mg of protein-1 (Xyl I) and 10.7, 4.0mg·mL-1 and 4553.7 and 1972.7U·mg of protein-1 (Xyl II) on oat spelts and birchwood xylan, respectively. The hydrolysis of oat spelts xylan released xylobiose, xylotriose, xylotetrose and larger xylooligosaccharides.ConclusionsThe enzymes present potential for application in industrial processes that require activity in acid conditions, wide-ranging pH stability, such as for animal feed, or juice and wine industries

Xylanase and β-xylosidase from Penicillium janczewskii: Purification, characterization and hydrolysis of substrates

Background: Xylanases and β-d-xylosidases are the most important enzymes responsible for the degradation of xylan, the second main constituent of plant cell walls. Results: In this study, the main extracellular xylanase (XYL I) and β-xylosidase (BXYL I) from the fungus Penicillium janczewskii were purified, characterized and applied for the hydrolysis of different substrates. Their molecular weights under denaturing and non-denaturing conditions were, respectively, 30.4 and 23.6 kDa for XYL I, and 100 and 200 kDa for BXYL I, indicating that the latter is homodimeric. XYL I is highly glycosylated (78%) with optimal activity in pH 6.0 at 65°C, while BXYL I presented lower sugar content (10.5%) and optimal activity in pH 5.0 at 75°C. The half-lives of XYL I at 55, 60 and 65°C were 125, 16 and 6 min, respectively. At 60°C, BXYL I retained almost 100% of the activity after 6 h. NH4+, Na+, DTT and β-mercaptoethanol stimulated XYL I, while activation of BXYL I was not observed. Interestingly, XYL I was only partially inhibited by Hg2+, while BXYL I was completely inhibited. Xylobiose, xylotriose and larger xylooligosaccharides were the main products from xylan hydrolysis by XYL I. BXYL I hydrolyzed xylobiose and larger xylooligosaccharides with no activity against xylans. Conclusion: The enzymes act synergistically in the degradation of xylans, and present industrial characteristics especially in relation to optimal activity at high temperatures, prolonged stability of BXYL I at 60°C, and stability of XYL I in wide pH range

Endophytic microorganisms isolated from coffee leaves, roots and branches as plant growth promoters and biocontrol agents of coffee leaf rust

Suppression of plant diseases and growth promotion due to the action of endophytic microorganisms has been demonstrated in several pathosystems. Experiments under controlled conditions involving 234 endophytic bacteria and fungi isolated from coffee leaves, roots and branches were conducted with the objective of evaluating the germination inhibition of Hemileia vastatrix urediniospores, the control of coffee leaf rust development in tests with leaf discs and on plastic bags seedling, and to promote growth of coffee seedlings. None of the fungal isolates induced plant growth or reduced disease severity. The bacterial isolates (identified by the fatty acids profile analysis) 85G (Escherichia fergusonii), 161G, 163G, 160G, 150G (Acinetobacter calcoaceticus) and 109G (Salmonella enterica) increased plant growth, the maximum being induced by 85G. This isolate produced in vitro phosphatase and indol acetic acid. In assay to control rust on coffee leaf disc, nine bacterial isolates, 64R, 137G, 3F (Brevibacillus choshinensis), 14F (Salmonella enterica), 36F (Pectobacterium carotovorum), 109G (Bacillus megaterium), 115G (Microbacterium testaceum), 116G and 119G (Cedecea davisae) significantly reduced disease severity, when applied 72 or 24h before challenging with the pathogen. In seedling tests most disease severity reduction was achieved by the isolates 109G and 119G. There was no correspondence between the organisms that promoted seedling growth and those that reduced rust severity on seedlings or leaf discs

Production, Purification, and Characterization of a Major Penicillium glabrum Xylanase Using Brewer's Spent Grain as Substrate

In recent decades, xylanases have been used in many processing industries. This study describes the xylanase production by Penicillium glabrum using brewer's spent grain as substrate. Additionally, this is the first work that reports the purification and characterization of a xylanase using this agroindustrial waste. Optimal production was obtained when P. glabrum was grown in liquid medium in pH 5.5, at 25 °C, under stationary condition for six days. The xylanase from P. glabrum was purified to homogeneity by a rapid and inexpensive procedure, using ammonium sulfate fractionation and molecular exclusion chromatography. SDS-PAGE analysis revealed one band with estimated molecular mass of 18.36 kDa. The optimum activity was observed at 60 °C, in pH 3.0. The enzyme was very stable at 50 °C, and high pH stability was verified from pH 2.5 to 5.0. The ion Mn2+ and the reducing agents β-mercaptoethanol and DTT enhanced xylanase activity, while the ions Hg2+, Zn2+, and Cu2+ as well as the detergent SDS were strong inhibitors of the enzyme. The use of brewer's spent grain as substrate for xylanase production cannot only add value and decrease the amount of this waste but also reduce the xylanase production cost

Agroindustrial biomass for xylanase production by Penicillium chrysogenum: Purification, biochemical properties and hydrolysis of hemicelluloses

Background: In this work, the xylanase production by Penicillium chrysogenum F-15 strain was investigated using agroindustrial biomass as substrate. The xylanase was purified, characterized and applied in hemicellulose hydrolysis. Results: The highest xylanase production was obtained when cultivation was carried out with sugar cane bagasse as carbon source, at pH 6.0 and 20°C, under static condition for 8 d. The enzyme was purified by a sequence of ion exchange and size exclusion chromatography, presenting final specific activity of 834.2 U·mg·prot-1. The molecular mass of the purified enzyme estimated by SDS-PAGE was 22.1 kDa. The optimum activity was at pH 6.5 and 45°C. The enzyme was stable at 40°C with half-life of 35 min, and in the pH range from 4.5 to 10.0. The activity was increased in the presence of Mg+2 and Mn+2 and reducing agents such as DTT and β-mercaptoethanol, but it was reduced by Cu+2 and Pb+2. The xylanase presented Km of 2.3 mM and Vmax of 731.8 U·mg·prot-1 with birchwood xylan as substrate. This xylanase presented differences in its properties when it was compared to the xylanases from other P. chrysogenum strains. Conclusion: The xylanase from P. chrysogenum F-15 showed lower enzymatic activity on commercial xylan than on hemicellulose from agroindustry biomass and its biochemistry characteristics, such as stability at 40°C and pH from 4.0 to 10.0, shows the potential of this enzyme for application in food, feed, pulp and paper industries and for bioethanol production.How to cite: Terrone CC, Freitas C, Terrasan CRF, et al. Agroindustrial biomass for xylanase production by Penicillium chrysogenum: purification, biochemical properties and hydrolysis of hemicelluloses. Electron J Biotechnol 2018;33. https://doi.org/10.1016/j.ejbt.2018.04.001. Keywords: Agroindustrial biomass, Enzyme production, Hemicellulose hydrolysis, Hemicelluloses, Penicillium chrysogenum, pH stability, Sugar cane bagasse, Thermal stability, Xylanase production, Xylanolytic enzym

Production, purification, and characterization of a major penicillium glabrum xylanase using brewer's spent grain as substrate

In recent decades, xylanases have been used in many processing industries. This study describes the xylanase production by Penicillium glabrum using brewer's spent grain as substrate. Additionally, this is the first work that reports the purification and characterization of a xylanase using this agroindustrial waste. Optimal production was obtained when P. glabrum was grown in liquid medium in pH 5.5, at 25 °C, under stationary condition for six days. The xylanase from P. glabrum was purified to homogeneity by a rapid and inexpensive procedure, using ammonium sulfate fractionation and molecular exclusion chromatography. SDS-PAGE analysis revealed one band with estimated molecular mass of 18.36 kDa. The optimum activity was observed at 60 °C, in pH 3.0. The enzyme was very stable at 50 °C, and high pH stability was verified from pH 2.5 to 5.0. The ion Mn2+ and the reducing agents β-mercaptoethanol and DTT enhanced xylanase activity, while the ions Hg2+, Zn2+, and Cu2+ as well as the detergent SDS were strong inhibitors of the enzyme. The use of brewer's spent grain as substrate for xylanase production cannot only add value and decrease the amount of this waste but also reduce the xylanase production cost. © 2013 Adriana Knob et al

Co-immobilization and stabilization of xylanase, β-xylosidase and α-L-arabinofuranosidase from Penicillium janczewskii for arabinoxylan hydrolysis

Differently activated agarose-based supports were evaluated for co-immobilization of a crude extract from Penicillium janczewskii containing xylanase, β-xylosidase and α-l-arabinofuranosidase activities. Adequately selecting support and immobilization conditions (8 h, using agarose with 10% crosslinking) increased enzyme levels substantially, mainly in relation to the xylanase (2-fold). A coating with dextran aldehyde MW 6000 Da, partially oxidized, covalently attached the enzymes to the support. Optimum activity was verified in the pH range 2-4, and at 50, 65 and 80 °C for the xylanase, α-l-arabinofuranosidase and β-xylosidase, respectively. The xylanase was highly thermostable retaining more than 70% of activity even after 24 h incubation at 60 and 70 °C; and at 80 °C its half-life was 1.7 h. The half-lives of the β-xylosidase and α-l-arabinofuranosidase at 50 °C were 2.3 and 3.8 h, respectively. The co-immobilization of the enzymes on a single support give raise to a functional multi-enzymatic biocatalyst acting in the complete hydrolysis of different and complex substrates such as oat spelt and wheat arabinoxylans, with xylose yield higher than 40%. The xylanase and the α-l-arabinofuranosidase presented high stability retaining 86.6 and 88.0% of activity after 10 reuse cycles.Peer Reviewe