Synergism for xylo-oligosaccharides, ρ-coumaric and ferulic acid production, and thermostability modulation of GH 62 α-l-arabinofuranosidase

Thermostability Enhancement of GH 62 α-l-Arabinofuranosidase by Directed Evolution and Rational Design

“…We verified the purity of Af_afu62 and its variants by SDS-Page analysis (Figure S1). The molecular weights of the protein samples were around 37 kDa, as expected based on previous characterization . Af_afu62 and its variants presented similar purity profiles and protein concentrations.…”

“…Notably, the kinetic parameters for the GH 62_1 subfamily from Scytalidium thermophilus represent the sole available data for this specific subfamily (see Table S5). For an in-depth analysis of the kinetic parameters of Af_afu62 and other GH 62 enzymes, readers are encouraged to refer to our previous work …”

“…We optimized de codon of the Af_afu62 gene (Genbank ID Afu_2G12770, 948 pb, without a signal peptide and TAG codon) from Aspergillus fumigatus and M5 Bacillus subtilis signal peptide (MNDLNDFLKTISLSFGFFLLLSLPTVAEA) for Escherichia coli expression. We synthesized the gene and the artificial recognition sites EcoRI and XhoI.…”

“…Detecting arabinofuranosidase activity in the crude supernatant was determined by enzymatic hydrolysis of wheat arabinoxylan (Megazyme International Ireland, Wicklow, Ireland). In short, 10 μL of culture supernatant was added to a 90 μL reaction volume containing 1% (w/v) wheat arabinoxylan and 5 mM MnCl 2 in McIlvaine buffer pH 5.0 (unless otherwise specified), in triplicate. The released arabinose was detected using the l -arabinose/ d -galactose assay kit (Megazyme International Ireland, Co., Wicklow, Ireland).…”

“…For an in-depth analysis of the kinetic parameters of Af_afu62 and other GH 62 enzymes, readers are encouraged to refer to our previous work. 4 The catalytic efficiency (k cat /K M ) of Af_afu62 outperformed that of all species, except for AnAbf62A from Aspergillus nidulans, which contains a carbohydrate binding module and optimum temperature between 60 and 65 °C. 31,32 However, AnAbf62A also presented its melting temperature at 60 °C and no residual activity data were available for comparison.…”