水稻XIP型抑制蛋白对11家族内切木聚糖酶催化活性的抑制作用及其机制研究

Endo-β-1, 4-xylanases (EC 3.2.1.8) are key enzymes that are involved in the degradation of xylans, which are made up of a main chain of β-1, 4-linked xylopyranose residues and are the major component of hemicelluloses in commelinoid monocot plants. Xylanase inhibitors are plant-produced proteinaceous inhibitors that inhibit the activity of endo-β-1, 4-xylanases. In recent year, the XIP-type endoxylanase inhibitors from rice were purified and characterized, which consist of RIXI、OsXIP-Ⅱand RiceXIP. The content of the XIP-type endoxylanase inhibitors in rice was relative high and they could inhibit the activity of most exogenous family 11 xylanase. The performance of xylanase used as additive in feed was affacted by the rice XIP-type xylanase inhibitors. Until now the mode of interaction and key amino acids between these inhibitors and xylanase are unclear.. Based on our previous study, the genes, rixi、osxip-Ⅱ and ricexip are cloned and expressed in Pichia pastoris GS115. The interactions between the rice XIP-type xylanase inhibitors (RIXI、OsXIP-Ⅱand RiceXIP) and family 11 xylanase (ATXX and the mutant ATXX113) are analysed using Yeast Two-hybrid technology. The interactions are tested by Co-IP and EMSA. Three inhibitor-xylanase compounds are characterized by FTIR, CD, and intrinsic/extrinsic fluorescence emission spectrum, respectively. The key amino acids and the mode of interaction between inhibitors and xylanases are revealed by sited/random mutation and assaying the inhibition level, substrate affinity and hydrolysis pattern of wild type and mutants. The mutants that show relative insensitivity to the rice XIP-type xylanase inhibitors are screened. Furthermore, the mutants are evaluted for the feasibility of feed application and preparactions of xylooligosaccharides (XOs).

水稻XIP 型木聚糖酶抑制蛋白是近年新发现的木聚糖酶抑制剂，能抑制大部分外源11家族木聚糖酶活性，显著影响木聚糖酶使用效果。目前，该类抑制蛋白与木聚糖酶的作用方式等尚不明确。. 本研究在前期工作基础上，克隆3种水稻XIP 型抑制蛋白基因，在酵母中高效表达；利用酵母双杂交系统分别研究RIXI、OsXIP-Ⅱ和RiceXIP 与木聚糖酶ATXX 及其突变体ATXX113 互作效应，检测3种抑制蛋白与ATXX，ATXX113 的作用水平及其差异，并采用体外免疫共沉淀和电泳迁移率技术验证；分析抑制蛋白—酶复合物结构特征，通过定点、随机突变，确定木聚糖酶与抑制蛋白作用的关键氨基酸位点，阐明水稻XIP型抑制蛋白与11 家族木聚糖酶的作用模式及其抑制酶活性的机制；构建出对水稻XIP型抑制蛋白高耐受性、高比活木聚糖酶，该酶对天然复合底物具有良好的结合水解性能，更适用于饲料添加剂和低聚木糖制备等领域。

本课题以探明水稻XIP型抑制蛋白与GH11家族木聚糖酶相互作用的结构基础和机制为主要目的，重点研究了该类抑制蛋白对GH11家族木聚糖酶ATXX、ATXX113 、BaxA、TfxA_CD及其突变体的竞争性抑制作用。初步明确了水稻XIP抑制蛋白-GH11家族木聚糖酶互作机制，同时获得了多个对抑制蛋白耐受性提高的木聚糖酶突变体。.1. 克隆、重组表达了3种水稻XIP型木聚糖酶抑制蛋白基因。reERIXI不同程度抑制多种11家族内切木聚糖酶活性。reERIXI对ATXX113、TfxA_CD214和reBaxA50抑制率分别为14.25%、35.13%，61.98%。reEriceXIP对ATXX、ATXX113的抑制率较低，对TfxA_CD214、reBaxA454和TfxA_CD526的抑制率分别为28.21%、26.19%和50.42%。reOsXIP对BaxA的突变体DS199(T31I)的抑制活性达到76.7%。三种抑制蛋白与 11家族木聚糖酶作用后均产生静态荧光猝灭。.2. 三种重组抑制蛋白对GH11家族木聚糖酶的抑制类型为竞争性抑制。随着reEriceXIP剂量的增加，TfxA_CD214的Km逐渐增加，而Vmax保持不变，抑制常数Ki为12.3 nM。reOsXIP剂量增加时DS199的Km增加，而Vmax不变，Ki为12.5 nM。reERIXI对ATXX、ATXX113的Ki分别为15.5 nM和 37.8 nM。在水解体系中添加3种重组抑制蛋白，木聚糖水解产物浓度明显降低，但水解产物类型无变化。.3. 利用酵母双杂交技术探究抑制蛋白与酶蛋白之间互作水平，结果表明RIXI、riceXIP与酶蛋白在胞内存在相互作用。与RIXI互作强度为reBaxA50>reBaxA>TfxA_CD214> TfxA_CD。riceXIP互作强度为TfxA_CD526> TfxA_CD214> TfxA_CD；reBaxA454 > reBaxA；该结果与DNS法测定结果一致。.4. 分子动力学模拟结果表明，抑制蛋白的Lα4/β5结构插入到GH11家族木聚糖酶催化口袋，与酶的活性中心、“拇指区”和“手掌区”具有较强的作用，阻碍底物与酶催化活性位点及其它关键氨基酸作用。抑制蛋白-酶作用界面的关键氨基酸之间以氢键和范德华力作用为主。