Molecular and biochemical characterization of a novel cold-active and metal ion-tolerant GH10 xylanase from frozen soil
2017
Qiu, Haiyan | Li, Zhongyuan | Wang, Hui | Zhang, Haiying | Li, Shuang | Luo, Xuegang | Song, Yajian | Wang, Nan | He, Hongpeng | Zhou, Hao | Ma, Wenjian | Zhang, Tongcun
A novel xylanase gene of family 10 (xyn27) was obtained from the genomic DNA of frozen soil of Daxinganling in China by Touch-down polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL) PCR methods. Xyn27 contained a putative signal peptide (20 residues) and a catalytic motif of GH10 (327 residues) and shared the highest similarity (83%) with the reported GH10 xylanase (XM_003662144). The recombinant xyn27 was successfully expressed in Pichia pastoris GS115 and the optimal induction condition was 30 °C for 48 h. Xyn27 was demonstrated to be a cold-active xylanase, which shows its highest activity at 35 °C and still had 60.25%, 38.70% and 10.8% relative activity at 20 °C, 10 °C and 0 °C. Further analysis showed that Xyn27 has fewer arginine and more alanine residues compared with its mesophilic or thermophilic counterparts. The optimal pH of Xyn27 was 7.0, and it was stable after incubating under the pH range from 3.0 to 9.0 for 1 h. Besides, Xyn27 exhibited superior metal ion tolerance than other GH10 cold-active xylanases, being tolerant to most metal ions and organic solvents, and significantly enhanced by Ca²⁺, Mn²⁺ and Zn²⁺ metal ions. In addition, the Kₘ, Vₘₐₓ, kcₐₜ and kcₐₜ Kₘ⁻¹ against beechwood xylan were 13.42 mg mL⁻¹, 9.07 μmol min⁻¹ mg⁻¹, 192.98 min⁻¹ and 14.38 mL min⁻¹ mg⁻¹, and Xyn27 could completely degrade xylan into xylobiose. The features of cold activity and metal-ion tolerance suggested that xylanase Xyn27 could have potential application in basic research and various industries like food possessing.
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