Cloning, expression, and characterization of a novel alkali‐tolerant xylanase from alkaliphilic Bacillus sp. SN5

Abstract

A xylanase gene (xyn11A) was cloned from the genomic library of alkalophilic Bacillus sp. SN5. It encoded a polypeptide of 366 amino acids, consisting of a family 11 glycoside hydrolase, a short linker region, and a family 36 carbohydrate-binding module (CBM). The intact xylanase Xyn11A and the CBM-linker-truncated Xyn11A-LC were expressed in Escherichia coli BL21 (DE3). Both purified recombinant proteins exhibited the highest activity at 55 °C. The optimal pH for Xyn11A activity was 7.5, whereas Xyn11A-LC showed a broad pH profile (>80% activity at pH 5.5-8.5) with optimal activity at pH 5.5 and 7.5-8.0. They had high alkali tolerance, retaining over 80% residual activity after preincubation at pH 8.5-11.0 at 37 °C for 1 H. Xyn11A-LC showed better thermal stability, lower affinity, and lower catalytic activity to insoluble xylan than Xyn11A, whereas its specific activity for soluble beechwood xylan (4,511.9 U/mg) was greater than that of Xyn11A (3,136.4 U/mg). These results implied that the CBM of Xyn11A could change the enzymatic properties and play a role in degrading insoluble xylan. Xyn11A-LC is a family 11 alkali-tolerant cellulase-free xylanase with high specific activity, which qualifies it as a potential candidate for industrial applications, especially in the paper industry.

Improvement of alkalophilicity of an alkaline xylanase Xyn11A-LC from Bacillus sp. SN5 by random mutation and Glu135 saturation mutagenesis

“…The recombinant plasmid pET 28a-Xyn11A-LC was constructed by our research group [18]. E.coli BL21-Gold (DE3) strain was used as a host for the random mutagenesis library.…”

“…The PCR products were purified using the gel extraction kit (Omega), digested with Bam HI and Xho I, and then ligated into the pET28a between Bam HI and Xho I sites. The resulting plasmids were transformed into E.coli BL21-Gold (DE3) competent cells by electro transformation and plated on LKX medium (Luria-Bertani agar plate, 50 μg/ml kanamycin, 0.2 % RBB-xylan, pH 9.0) [18]. …”

“…SN5 was characterized [18]. It exhibited the highest catalytic activity at pH7.5, but little enzyme activity could be detected at pH 10.0.…”

Structural Insight into and Mutational Analysis of Family 11 Xylanases: Implications for Mechanisms of Higher pH Catalytic Adaptation

“…The expression and purification of the wild-type and mutant proteins was performed following the same procedure as described previously [ 21 ]. The purity of the proteins was analyzed by 12% SDS-PAGE.…”

“…We have previously characterized a family 11 xylanase, Xyn11A-LC from alkalophilic Bacillus sp. SN5, that exhibited maximal activity at pH 7.5–8.0 and 55°C [ 21 ]. We purified and crystallized this enzyme and solved the structure at 1.49 Å resolution [ 22 ].…”