Recombinant rice xylanase inhibitor (RIXI) expressed in Escherichia coli and its inhibitory activity on family GH11endo-xylanases.
Recombinant rice xylanase inhibitor (RIXI) expressed in Escherichia coli and its inhibitory activity on family GH11endo-xylanases.
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DOI：
10.1016/j.ijbiomac.2017.11.177
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发表时间：
2017-12
影响因子：
8.2
通讯作者：
Wen-kang Huo;Ming-qi Liu;X. Weng;Y. Qi
中科院分区：
文献类型：
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作者：
Wen-kang Huo;Ming-qi Liu;X. Weng;Y. Qi
水稻木聚糖酶抑制剂基因rixi是从水稻基因组中克隆出来的。 Rixi的开放阅读框为915bp，编码304个氨基酸，理论分子量为33.9kDa。将Therixi插入新型表达载体pCold TF中，在大肠杆菌BL21（DE3）中高水平表达。 SDS-PAGE和Western blot分析表明重组水稻木聚糖酶抑制剂reERIXI的分子量约为89.8kDa。 reERIXI 对几种 GH11 木聚糖酶家族表现出显着的抑制活性。与reERIXI相互作用后，reBaxA50和TfxA_CD214的残余活性分别为59.24%和44.41%。 reERIXI对reBaxA50和TfxA_CD214的抑制活性的最适温度分别为60℃和50℃。热稳定性测定表明，reERIXI 在 60°C 以下稳定。当与 reBaxA50 和 TfxA_CD214 相互作用 30-60 分钟时，reERIXI 显示出高抑制性。 reBaxA50 和 TfxA_CD214 的本征荧光光谱随着 reERIXI 浓度的增加而猝灭。圆二色性测量显示，当与reERIXI相互作用时，reBaxA50的螺旋比率和TfxA_CD214的β比率显着降低。添加 reERIXI 后，山毛榉木聚糖水解产物的总浓度降低。
The rice xylanase inhibitor gene,rixi, was cloned from rice genome. The open reading frame ofrixiwas 915 bp and encoded 304 amino acids with the theoretical molecular mass of 33.9 kDa. Therixiwas inserted into the new-type expression vector pCold TF, and was high-level expressed inEscherichia coliBL21 (DE3). SDS-PAGE and Western blot analysis revealed that the molecular weight of the recombinant rice xylanase inhibitor, namely reERIXI, was approximately 89.8 kDa. The reERIXI exhibited significant inhibitory activities against several family GH11 xylanases. After interaction with reERIXI, the residual activity of reBaxA50 and TfxA_CD214 were 59.24% and 44.41%, respectively. The optimal temperature of reERIXI inhibitory activity to reBaxA50 and TfxA_CD214 were 60 °C and 50 °C, respectively. The thermostability assay revealed that reERIXI was stable below 60 °C. reERIXI showed high inhibitory when interacting with reBaxA50 and TfxA_CD214 for 30–60 min. The intrinsic fluorescence spectroscopy of reBaxA50 and TfxA_CD214 was quenched with increasing reERIXI concentration. Circular dichroism measurement revealed that ratio of helix of reBaxA50 and ratio of beta of TfxA_CD214 significantly decreased when interacting with reERIXI. The total concentration of hydrolytic products from beechwood xylan decreased when reERIXI was added.