Immobilization of a recombinant endo-1,5-arabinanase secreted by Aspergillus nidulans strain A773

• Published in: Journal of molecular catalysis. B, Enzymatic Enzymatic, 2012-05, Vol.77, p.39-45
• Main Authors: Damásio, André Ricardo de Lima, Pessela, Benevides Costa, Mateo, César, Segato, Fernando, Prade, Rolf Alexander, Guisan, Jose Manuel, de Lourdes Teixeira de Moraes Polizeli, Maria
• Format: Article
• Language: English
• Subjects: agarose; amino acid sequences; Aspergillus nidulans; Aspergillus niveus; Bioconversions. Hemisynthesis; Biological and medical sciences; Biotechnology; catalytic activity; cyanogen; Fundamental and applied biological sciences. Psychology; genes; half life; heat inactivation; hydrolysis; Immobilization; Methods. Procedures. Technologies; Over-expression; Purification; Recombinant endo-1,5-arabinanase; sequence analysis; temperature
• ISSN: 1381-1177; 1873-3158
• DOI: 10.1016/j.molcatb.2012.01.002

[Display omitted] ► pEXPYR plasmids carrying abnA gene was transformed into A. nidulans strain A773. ► The AbnA was successfully over-secreted to culture medium by maltose induction. ► Purified recombinant AbnA was immobilized in modified agarose supports. ► 10% glyoxyl agarose derivative was 10.3-fold more stable than the free enzyme at 70°C. ► Arabinotriose and arabinobiose were the major AbnA hydrolyses products from arabinan. An endo-1,5-arabinanase (abnA) encoding gene from Aspergillus niveus was identified, cloned and successfully expressed in Aspergillus nidulans strain A773. Based on amino acid sequence comparison, the 34-kDa enzyme could be assigned to CAZy GH family 43. Characterization of purified recombinant endo-1,5-arabinanase (AbnA) revealed that it is active at a wide pH range (pH 4.0–7.0) and an optimum temperature at 70°C. The immobilization of the AbnA was performed via covalent binding onto agarose-modified supports: glyoxyl iminodiacetic acid–Ni2+, glyoxyl amine, glyoxyl (4% and 10%) and cyanogen bromide activated sepharose. The yield of immobilization was similar on glyoxyl amine and glyoxyl (96%), and higher than glyoxyl iminodiacetic acid–Ni2+ (43%) support. The thermal inactivation of these immobilized preparations showed that the stability of the AbnA immobilized on glyoxyl 4 and 10% was improved by 4.0 and 10.3-fold factor at 70°C. The half-life of glyoxyl 4% derivative at 60°C was >48h (pH 5), 9h (pH 7) and 88min (pH 9). The major hydrolysis product of debranched arabinan or arabinopentaose by glyoxyl agarose-immobilized AbnA was arabinobiose.