Abstract

In this study, the catalytic activity of xynAm1 (a thermophilic xylanase family 11 variant) was significantly enhanced by fusing a carbohydrate-binding module (CBM) 9–2. First, CBM9–2 was fused to the N- and C-termini of xynAm1, generating the fusion enzymes C-X and X-C, respectively. The specific activity of C-X was 19.0 U/μM, which was 2.3-fold and 1.1-fold higher than that of xynAm1 and X-C, respectively. Then, the flexible linker (GGGGS) 2 was used to fuse CBM9–2 at the N-terminus of xynAm1, yielding C-F 2 -X with an additional 15.91-fold increase in the specific activity. By analyzing their modeled structures, the hydrogen bonds between xynAm1 and CBM in C-F 2 -X made the distance between the catalytic active site E379 and binding site W176 shorter by 8 Å than that in C-X. Meanwhile, the sugarcane xylan hydrolysis with C-F 2 -X produced 5.42 times higher amount of the xylose than that with xynAm1. When C-F 2 -X (200 U) and cellulase (200 U) were added to 30 mL suspension of wheat bran (1.5 g), the reducing sugar content and dry weight loss rate reached 143.23 nM and 82.7% after 24 h reaction, respectively. Therefore, the fusion enzyme C-F 2 -X could be a robust candidate enzyme for xylose production and lignocellulose degradation.