PRODUCTION AND PURIFICATION OF RECOMBINANT XYLANASES FOR APPLICATION IN JUICE CLARIFICATION

PRODUCTION AND PURIFICATION OF RECOMBINANT XYLANASES FOR APPLICATION IN JUICE CLARIFICATION

Passarinho, Amanda Tafuri Paniago; Mamede, Mariana Siqueira Lacerda; Oliveira, Eduardo Basílio de; Guimaraes, Valeria Monteze

Resumo:

Endo-β-1,4-xylanases (EC 3.2.1.8) are able to hydrolyse xylan, the second most abundant polysaccharide in plant cell walls. In this work, two recombinant xylanases derived from Orpinomyces xynA gene were studied: one with mutations A226T and V135A (XM) and the other without mutations (XNM). They have been cloned in E. coli/pET24b, using IPTG as inducer and purified using ion exchange chromatography (Q-Sepharose). After purification, only one band of 25 kDa, corresponding to xylanases, was visualized on SDS-PAGE. Specific activities and Km against arabinoxylan were 12.844 U.mg-1 and 0.0021 mg.mL-1 for XNM and 10.794 U.mg-1 and 0.0014 mg.mL-1 for XM. The mutations improve thermo stability. The half-life of XNM was 220 min and 31 min at 50 °C and 60 °C, while for XM the values were 440 and 71 min at 50 oC and 60 oC, respectively. The clarification assays were conducted at 60 oC, 100 rpm for 60 min, using 50U xylanase/mL apple juice. The parameters available was pH, A660 (clarity), L* (luminosity), C (chroma) and h* (hue). All the juices tended to orange while only enzymatically treated with XM had an increase in color intensity (chroma / C) and higher content of soluble solids (sst) of 4,7 obrix. The results indicate that the processing of apple juice with xylanases can promote clarification and improvement of its properties.

Endo-β-1,4-xylanases (EC 3.2.1.8) are able to hydrolyse xylan, the second most abundant polysaccharide in plant cell walls. In this work, two recombinant xylanases derived from Orpinomyces xynA gene were studied: one with mutations A226T and V135A (XM) and the other without mutations (XNM). They have been cloned in E. coli/pET24b, using IPTG as inducer and purified using ion exchange chromatography (Q-Sepharose). After purification, only one band of 25 kDa, corresponding to xylanases, was visualized on SDS-PAGE. Specific activities and Km against arabinoxylan were 12.844 U.mg-1 and 0.0021 mg.mL-1 for XNM and 10.794 U.mg-1 and 0.0014 mg.mL-1 for XM. The mutations improve thermo stability. The half-life of XNM was 220 min and 31 min at 50 °C and 60 °C, while for XM the values were 440 and 71 min at 50 oC and 60 oC, respectively. The clarification assays were conducted at 60 oC, 100 rpm for 60 min, using 50U xylanase/mL apple juice. The parameters available was pH, A660 (clarity), L* (luminosity), C (chroma) and h* (hue). All the juices tended to orange while only enzymatically treated with XM had an increase in color intensity (chroma / C) and higher content of soluble solids (sst) of 4,7 obrix. The results indicate that the processing of apple juice with xylanases can promote clarification and improvement of its properties.

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DOI: 10.5151/biochem-jaibqi-0073

Como citar:

Passarinho, Amanda Tafuri Paniago; Mamede, Mariana Siqueira Lacerda; Oliveira, Eduardo Basílio de; Guimaraes, Valeria Monteze; "PRODUCTION AND PURIFICATION OF RECOMBINANT XYLANASES FOR APPLICATION IN JUICE CLARIFICATION", p-35-35. In: Anais da V Jornada Acadêmica Internacional de Bioquímica [= Blucher Biochemistry Proceedings, v.1, n.1]. São Paulo: Blucher, 2015.
ISSN 23595043, DOI 10.5151/biochem-jaibqi-0073