Homologous expression, purification, and characterization of a recombinant acetylxylan esterase from Aspergillus nidulans

Author(s)

Alves, Gabriela S. ; de Andrades, Diandra ; Salgado, Jose C. S. ; Mariano, Clara B. ; Berto, Gabriela Leila ; Segato, Fernando ; Ayub, Marco Antonio Zachia ; Ward, Richard J. ; Alnoch, Robson C. ; Polizeli, Maria de Lourdes T. M.

• Document type: Journal article
• Source: International Journal of Biological Macromolecules; v. 280, p. 12-pg., 2024-10-11.

Abstract

Acetylxylan esterases (AXEs) are essential enzymes that break down the acetyl groups in acetylated xylan found in plant cell walls polysaccharides. They work synergistically with backbone-depolymerizing xylanolytic enzymes to accelerate the degradation of complex polysaccharides. In this study, we cloned the gene axeA, which encodes the acetylxylan esterase from Aspergillus nidulans FGSC A4 (AxeAN), into the pEXPYR expression vector and introduced it into the high protein-producing strain A. nidulans A773. The purified AxeAN, with a molecular weight of 33.5 kDa as confirmed by SDS-PAGE, was found to be active on rho-nitrophenyl acetate (rho NPA), exhibiting a remarkably high specific activity (170 U mg(-1)) at pH 7.0 and 55 degrees C. AxeAN demonstrated stability over a wide pH range (5.5-9.0), retaining >80% of its initial activity after 24 h. The K-M and V-max were 0.098 mmol L-1 and 320 U mg(-1), respectively, using rho NPA as a substrate. We also evaluated the synergistic effect of AxeAN with an endo-1,4-beta-xylanase from Malbranchea pulchella (MpXyn10) in the hydrolysis of four different xylans (Birchwood, Beechwood, Oat spelt, and Arabinoxylan) to produce xylooligosaccharides (XOS). The best results were obtained using Birchwood xylan as substrate and MpXyn10-AxeAN as biocatalysts after 24 h of reaction (50 degrees C), with a XOS-yield of 91%, value 41% higher when compared to MpXyn10 (XOS-yield of 63%). These findings showed the potential of the application of AxeAN, together with other xylanases, to produce xylooligosaccharides with high purity and other products with high added value in the field of lignocellulosic biorefinery. (AU)

• FAPESP's process: 20/15510-8 - Immobilization and co-immobilization of lignocellulolytic enzymes in magnetic nanoparticles: a strategy for hydrolysis of biomass and industry waste
• Grantee: Diandra de Andrades
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 14/50884-5 - INCT 2014: National Institute of Science and Technology of Bioethanol
• Grantee: Marcos Silveira Buckeridge
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 18/07522-6 - Development of eco-friendly technologies for the hydrolysis of biomass and residues of cellulose pulp and paper industry
• Grantee: Maria de Lourdes Teixeira de Moraes Polizeli
• Support Opportunities: Regular Research Grants
• FAPESP's process: 19/21989-7 - Bioprospection of low molecular weight fungal enzymes involved in plant cell wall degradation
• Grantee: José Carlos dos Santos Salgado
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 23/09627-8 - Formulation of tailor-made enzymatic consortiums for the degradation of the cell wall of sugarcane varieties
• Grantee: Robson Carlos Alnoch
• Support Opportunities: Scholarships in Brazil - Support Program for Fixating Young Doctors
• FAPESP's process: 23/01547-5 - Formulation of tailor-made enzymatic consortiums for the degradation of the cell wall of sugarcane varieties
• Grantee: Maria de Lourdes Teixeira de Moraes Polizeli
• Support Opportunities: Regular Research Grants