In-fusion expression and characterization of β-xylanase and β-1,3-1,4-glucanase in Pichia pastoris

Abstract

Two chimeric genes, XynA-Bs-Glu-1 and XynA-Bs-Glu-2, encoding Aspergillus sulphureus β-xylanase (XynA, 26 kDa) and Bacillus subtilis β-1,3-1,4-glucanase (Bs-Glu, 30 kDa), were constructed via in-fusion by different linkers and expressed successfully in Pichia pastoris. The fusion protein (50 kDa) exhibited both β-xylanase and β-1,3-1,4-glucanase activities. Compared with parental enzymes, the moiety activities were decreased in fermentation supernatants. Parental XynA and Bs-Glu were superior to corresponding moieties in each fusion enzymes because of lower Km and higher kcat. Despite some variations, common optima were generally 50• C and pH 3.4 for the XynA moiety and parent, and 40• C and pH 6.4 for the Bs-Glu counterparts. Thus, the fusion enzyme XynA-Bs-Glu-1 and XynA-Bs-Glu-2 were bifunctional.

A review on chimeric xylanases: methods and conditions

“…Accordingly, access to solvent for long (average length of 21.0 ± 7.6 residues), medium (9.1 ± 2.4) and short (4.5 ± 0.7) linkers are about 42.5, 26.3 and 14.4%, respectively (Chen et al 2013;George and Heringa 2003). Although a linker should be long enough so that each enzyme acts independently, the possibility of breakage increases if the linker be too long (Qiao and Cao 2012). It has been demonstrated that a 31 amino acids linker is suitable not only to prevent collision of two enzymes, but also to make great flexibility to provide free access and binding to the substrate (Diogo et al 2015).…”