钙离子对木聚糖酶Xyn10A热稳定性的影响

木聚糖酶广泛应用于食品、饲料、纺织、能源等领域。在生产过程中木聚糖酶的热稳定性较为重要,它直接影响酶的反应温度及使用效率。添加Ca2+能够显著提高来源于Thermotoga thermarum DSM 5069的木聚糖酶Xyn10A在高温(85℃)条件下的热稳定性。为解析Xyn10A酶蛋白中的Ca2+结合区域及热稳定性机制,笔者采用蛋白质结构模拟和定点突变技术以确定该结合区域,并分析其对于酶热稳定性的影响机制。酶蛋白的建模和结构比对结果表明,GH10家族木聚糖酶的结构保守性远大于其序列保守性; 木聚糖酶Xyn10A中局部环区(712IYRDNATKYEIPP724)涉及Ca2+的结合功能,同时其热稳定性依赖于该环区与Ca2+之间的亲和力。对该环区的定点突变和删除突变导致Xyn10A无法有效地结合Ca2+。Ca2+可与酶蛋白中的(712IYRDNATKYEIPP724)环区形成配位键,显著降低Xyn10A酶催化结构域的柔性和自由度,使Xyn10A酶能够在高温下保持优良的热稳定性,进而有效地发挥其高温催化水解木聚糖的能力。
Xylanases are able to hydrolyze the β-1,4 glycosidic linkage of the backbone of xylan, participating in the hydrolysis process of hemicelluloses in nature. Due to their excellent catalytic abilities for the hydrolysis of xylan, xylanases are widely employed in many fields including food processing, feedstuff producing, textile processing and energy developing. Regarding the reaction temperature and efficiency of enzymes, the thermostability of xylanases is of great importance for industrial purposes. The results of our previous study showed that the addition of calcium divalent ion(Ca2+)can significantly enhance the thermostability of the xylanase(Xyn10A)from the thermophile Thermotoga thermarum DSM 5069 at 85℃. In order to find out the domain involved in the Ca2+ binding in the Xyn10A, protein structure modeling and site-directed mutagenesis were carried out in this study, and the mechanism of thermostability of Xyn10A was analyzed. The results of structural simulation and superposition of Xyn10A showed that the conservation of protein structure was much higher than that of amino acid sequence. The local loop region from 712 to 724(712IYRDNATKYEIPP724)was proven to be closely associated with Ca2+ binding in Xyn10A. The multi-point substitution and deletion mutation were introduced into the loop region(712IYRDNATKYEIPP724). Compared to original Xyn10A, all mutants showed poorer thermostability, and even lost their ability to bind with Ca2+. It indicated that the thermostability of Xyn10A mainly depended on the affinity and interaction between the loop region(712IYRDNATKYEIPP724)and Ca2+. In the presence of Ca2+, the side chains and backbone of loop region(712IYRDNATKYEIPP724)interacted with Ca2+ by forming a stable coordination complex, which greatly restricted the flexibility and freedom degree of the catalytic domain of Xyn10A. Therefore, the thermostability and the catalytic ability of Xyn10A were effectively maintained at higher temperature