Cloning and expression of the 34 candidate xylanase genes

The 34 candidate xylanase genes (CDW-xyl-1 to CDW-xyl-34) were selected for cloning. The cloned xylanase genes were individually inserted into the pET-28a (+) expression vector by the One Step Cloning Kit (Vazyme, Nanjing, China). Consequently, 34 recombinant plasmids were individually used to transform the E. coli TOP10 strain. After verification, the correct recombinant plasmids were further introduced into E. coli BL21(DE3) strain for protein expression.

The E. coli BL21(DE3) strains carrying recombinant plasmids were individually inoculated into 100 mL LB medium with 50 µg/mL kanamycin in a 500-mL shake flask. The cultures were cultivated at 37 ℃ and 200 rpm. Once the OD600 value reached 0.6–0.8, IPTG was added to a final concentration of 200 µM to induce xylanase gene expression. After cultivation for 16 h at 20 ℃, the cell cultures were harvested by centrifugation at 12,000 rpm for 10 min. The harvested cell cultures were washed with phosphate buffered saline (PBS) buffer (pH 7.4) for 3 times, and then suspended in 40 mL lysis buffer with 1 mM phenylmethylsulfonyl fluoride (PMSF, protease inhibitor). The cells were disrupted by supersonic waves (Xiaomei, Kunshan, China) on ice at a power of 100 W for 15 min, with a pulse duration of 3 s and an interval of 5 s. Finally, the cell lysates were centrifuged at 12,000 rpm for 20 min, and the supernatants were used for the evaluation of crude xylanase activity.

To determine the crude xylanase activity, a substrate of 1% beechwood xylan was used, and the DNS method was employed. A total of 50 µL diluted cell supernatants and 50 µL 1% beechwood xylan were mixed and incubated at 20 ℃ for 20 min, 40 ℃ for 20 min, and 60 ℃ for another 20 min. Subsequently, 100 µL of DNS reagent was added to each tube to terminate the enzymatic reaction. The mixtures were then incubated at 95 ℃ for 5 min. The crude activity of the 34 expressed xylanases could be determined [30].