RESUMO
Xylooligosaccharides (XOS) are widely used in the food industry as prebiotic components. XOS with high purity are required for practical prebiotic function and other biological benefits, such as antioxidant and inflammatory properties. In this work, we immobilized the recombinant endo-1,4-ß-xylanase of Malbranchea pulchella (MpXyn10) in various chemical supports and evaluated its potential to produce xylooligosaccharides (XOS) from hydrothermal liquor of eucalyptus wood chips. Values >90% of immobilization yields were achieved from amino-activated supports for 120 min. The highest recovery values were found on Purolite (142%) and MANAE-MpXyn10 (137%) derivatives, which maintained more than 90% residual activity for 24 h at 70 °C, while the free-MpXyn10 maintained only 11%. In addition, active MpXyn10 derivatives were stable in the range of pH 4.0−6.0 and the presence of the furfural and HMF compounds. MpXyn10 derivatives were tested to produce XOS from xylan of various sources. Maximum values were observed for birchwood xylan at 8.6 mg mL−1 and wheat arabinoxylan at 8.9 mg mL−1, using Purolite-MpXyn10. Its derivative was also successfully applied in the hydrolysis of soluble xylan present in hydrothermal liquor, with 0.9 mg mL−1 of XOS after 3 h at 50 °C. This derivative maintained more than 80% XOS yield after six cycles of the assay. The results obtained provide a basis for the application of immobilized MpXyn10 to produce XOS with high purity and other high-value-added products in the lignocellulosic biorefinery field.
Assuntos
Eucalyptus , Xilanos , Madeira , Glucuronatos , Oligossacarídeos/química , Endo-1,4-beta-Xilanases , Prebióticos , HidróliseRESUMO
Enzyme immobilization can promote several advantages for their industrial application. In this work, a lipase from Hypocrea pseudokoningii was efficiently linked to four chemical supports: agarose activated with cyanogen bromide (CNBr), glyoxyl-agarose (GX), MANAE-agarose activated with glutaraldehyde (GA) and GA-crosslinked with glutaraldehyde. Results showed a more stable lipase with both the GA-crosslinked and GA derivatives, compared to the control (CNBr), at 50 °C, 60 °C and 70 °C. Moreover, all derivatives were stabilized when incubated with organic solvents at 50%, such as ethanol, methanol, n-propanol and cyclohexane. Furthermore, lipase was highly activated (4-fold) in the presence of cyclohexane. GA-crosslinked and GA derivatives were more stable than the CNBr one in the presence of organic solvents. All derivatives were able to hydrolyze sardine, açaí (Euterpe oleracea), cotton seed and grape seed oils. However, during the hydrolysis of sardine oil, GX derivative showed to be 2.3-fold more selectivity (eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) ratio) than the control. Additionally, the types of immobilization interfered with the lipase enantiomeric preference. Unlike the control, the other three derivatives preferably hydrolyzed the R-isomer of 2-hydroxy-4-phenylbutanoic acid ethyl ester and the S-isomer of 1-phenylethanol acetate racemic mixtures. On the other hand, GX and CNBr derivatives preferably hydrolyzed the S-isomer of butyryl-2-phenylacetic acid racemic mixture while the GA and GA-crosslink derivatives preferably hydrolyzed the R-isomer. However, all derivatives, including the control, preferably hydrolyzed the methyl mandelate S-isomer. Moreover, the derivatives could be used for eight consecutive cycles retaining more than 50% of their residual activity. This work shows the importance of immobilization as a tool to increase the lipase stability to temperature and organic solvents, thus enabling the possibility of their application at large scale processes.
Assuntos
Enzimas Imobilizadas/química , Hypocrea/química , Lipase/química , Reagentes de Ligações Cruzadas/química , Brometo de Cianogênio/química , Ácidos Docosa-Hexaenoicos/química , Ácido Eicosapentaenoico/química , Ativação Enzimática , Estabilidade Enzimática , Glutaral/química , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Óleos/química , Desnaturação Proteica , Estabilidade Proteica , Sefarose/química , Solventes , Estereoisomerismo , Especificidade por Substrato , TemperaturaRESUMO
Fibrolytic enzyme production by Aspergillus japonicus C03 was optimized in a medium containing agro-industrial wastes, supplemented with peptone and yeast extract. A 2(3) full factorial composite and response surface methodology were used to design the experiments and analysis of results. Tropical forages were hydrolyzed by A. japonicus C03 enzymatic extract in different levels, and they were also tested as enzymatic substrate. Optimal production to xylanase was obtained with soybean bran added to crushed corncob (1:3), 0.01% peptone, and 0.2% yeast extract, initial pH 5.0, at 30 °C under static conditions for 5 days of incubation. Optimal endoglucanase production was obtained with wheat bran added to sugarcane bagasse (3:1), 0.01% peptone, and 0.2% yeast extract, initial pH 4.0, at 30 °C, for 6 days, under static conditions. Addition of nitrogen sources as ammonium salts either inhibited or did not influence xylanase production. This enzymatic extract had a good result on tropical forage hydrolyzes and showed better performance in the Brachiaria genera, due to their low cell wall lignin quantity. These results represent a step forward toward the use of low-cost agricultural residues for the production of valuable enzymes with potential application in animal feed, using fermentation conditions.