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. T he 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 ßmercaptoethanol, 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.

Crecimiento in vitro y producción de enzimas degradadoras de pared celular vegetal de aislamientos argentinos de Macrophomina phaseolina, agente causal de la podredumbre carbonosa en maíz / In vitro growth and cell wall degrading enzyme production by Argentinean isolates of Macrophomina phaseolina, the causative agent of charcoal rot in corn

Macrophomina phaseolina is a polyphagous phytopathogen, causing stalk rot on many commercially important species. Damages caused by this pathogen in soybean and maize crops in Argentina during drought and hot weather have increased due its ability to survive as sclerotia in soil and crop debris under non-till practices. In this work, we explored the in vitro production of plant cell wall-degrading enzymes --#91;pectinases (polygalacturonase and polymethylgalacturonase); cellulases (endoglucanase); hemicellulases (endoxylanase) and the ligninolytic enzyme laccase--#93; by several Argentinean isolates of M. phaseolina, and assessed the pathogenicity of these isolates as a preliminary step to establish the role of these enzymes in M. phaseolina-maize interaction. The isolates were grown in liquid synthetic medium supplemented with glucose, pectin, carboxymethylcellulose or xylan as carbon sources and/or enzyme inducers and glutamic acid as nitrogen source. Pectinases were the first cell wall-degrading enzymes detected and the activities obtained (polygalacturonase activity was between 0.4 and 1.3 U/ml and polymethylgalacturonase between 0.15 and 1.3 U/ml) were higher than those of cellulases and xylanases, which appeared later and in a lesser magnitude. This sequence would promote initial tissue maceration followed by cell wall degradation. Laccase was detected in all the isolates evaluated (activity was between 36 U/l and 63 U/l). The aggressiveness of the isolates was tested in maize, sunflower and watermelon seeds, being high on all the plants assayed. This study reports for the first time the potential of different isolates of M. phaseolina to produce plant cell wall-degrading enzymes in submerged fermentation.

Macrophomina phaseolina es un fitopatógeno polífago, causante de podredumbre carbonosa. Los daños que genera en cultivos de soja y maíz bajo siembra directa en Argentina, en períodos secos y calurosos, se incrementaron por su habilidad para sobrevivir como esclerocios en suelos y restos de cosecha. El propósito del trabajo fue estudiar la producción in vitro de enzimas degradadoras de pared celular vegetal (pectinasas --#91;poligalacturonasa y polimetilgalacturonasa--#93;; celulasas --#91;endoglucanasa--#93;; hemicelulasas --#91;endoxilanasa--#93; y la enzima ligninolítica lacasa) de varios aislamientos argentinos de M. phaseolina y evaluar la patogenicidad de esos aislamientos, como paso preliminar para establecer el papel de estas enzimas en la interacción M. phaseolina-maíz. Se estudió la cinética de crecimiento del hongo y la de la producción de dichas enzimas en medios de cultivo líquidos sintéticos con ácido glutámico como fuente de nitrógeno y con pectina, carboximetilcelulosa (CMC) o xilano como fuentes de carbono. Las pectinasas fueron las primeras enzimas detectadas y los máximos títulos registrados (1,4 UE/ml --#91;poligalacturonasa--#93; y 1,2 UE/ml --#91;polimetilgalacturonasa--#93;, respectivamente) superaron a los de celulasas y xilanasas, que aparecieron más tardíamente y en menor magnitud. Esta secuencia promovería la maceración inicial del tejido, seguida luego por la degradación de la pared celular vegetal. Se detectó actividad lacasa en todos los aislamientos (36 a 63 U/l). La agresividad de todos los aislamientos resultó alta en los 3 hospedantes evaluados: semillas de maíz, de girasol y de melón. En este trabajo se investiga por primera vez el potencial de distintos aislamientos de M. phaseolina para producir enzimas degradadoras de pared celular vegetal en cultivo líquido.

Purification and characterization of xylanases from Trichoderma inhamatum

Background Two 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. Results The molecular masses of the purified enzymes estimated by SDS-PAGE and gel filtration were, respectively, 19 and 14 kDa for Xyl I and 21 and 14.6 kDa for Xyl II. The enzymes are glycoproteins with optimum activity at 50°C in pH 5.0-5.5 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, 1.6 mg·mL-1 and 2680.2 and 462.2 U·mg of protein-1 (Xyl I) and 10.7, 4.0 mg·mL-1 and 4553.7 and 1972.7 U·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. Conclusions The 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.

Production, purification, characterization and over-expression of xylanases from actinomycetes.

Xylanases are a group of depolymerizing enzymes often used for the hydrolysis of xylan (present in hemicellulose) to monomeric sugars and comprise endo-xylanases (EC 3.2.1.8) and β-xylosidases (EC 3.2.1.37). They often act in synergy with other enzymes for complete hydrolysis of hemicellulose. Xylanases find several industrial applications, for example in food and feed industries, paper and pulp industries and more recently have acquired a great role in biomass to biofuels program. Bacteria and fungi can best produce xylanases. Recent developments in rDNA technology have resulted in molecular cloning and expression of xylanases in heterologous and homologous hosts. In view of significance of the actinomycetes for the production of biotechnological products, attempts have been made in recent years to explore them for the production of industrial enzymes, including xylanses, aiming to find the enzyme with novel features. This review provides the state-of-art information and developments on the xylanases from actinomycetes, presenting the production, purification, characterization and over-expression from various actinomycetes cultures.

Optimization of extracellular xylanase production by Sclerotinia sclerotiorum S2 using factorial design.

The improvement of xylanase production by Sclerotinia sclerotiorum S2 using a liquid fermentation culture was investigated. The optimized process was divided into three basic steps: (i) evaluating xylanase inducers using different agricultural residues such as wheat bran, oat bran, orange peel and barley bran at 1% final concentration, and also filter paper. Among these, wheat bran showed the maximum activity (2.5 U/ml) at 12 days post-inoculation; (ii) for optimization, we determined the optimal concentration of inducer, the effect of phosphate anion (K2HPO4/KH2PO4) and culture aeration using a rotary shaker at 100 and 180 rpm. The optimal conditions for these three factors were determined in an experimental panel using factorial data, in which a mathematical model (Minitab software) was fitted; (iii) The optimized culture medium containing a high level of wheat bran (3%) without KH2PO4-K2HPO4 and submitted to a high agitation (180 rpm/min) increased the xylanase production from 2.5 U/ml to 4 U/ml (1.6-fold).