α-淀粉酶新亚家族GH13_37快速偏好降解大米生淀粉的机制研究

The study and application of rice starch play a key role in human life. Only a few raw starch digesting enzymes can hydrolyze raw rice starch that has more complex component and structure than other raw starches. Moreover, the processes of hydrolysis are slow and inefficient. Recently, we isolated a novel α-amylase (AmyP) from a marine metagenomic library, and the discovery led to the identification of a new subfamily GH13_37. The members of the subfamily GH13_37 were later shown to exhibit a unique and remarkable ability to preferentially and very rapidly digest raw rice starch, which has not been described before. AmyP is targeted for study in this project. Site-specific mutagenesis is used to obtain the mutants with slow speed of hydrolysis and disappearing preference toward raw rice starch. Typical mutants will be structurally characterized. A comparative study on enzymatic properties and the three-dimensional structures will be performed between the mutants and the wide type AmyP. The results will gain insights into the mechanism underlying the preferential and rapid degradation of raw rice starch by the members of the subfamily GH13_37. Then, rational protein design is used to obtain the mutant with an improved catalytic activity toward raw rice starch, which will provide a thorough understanding of the mechanism. This is the first study of a relationship between the structure of amylase and raw rice starch. This study will not only expand our horizons in the mechanisms for hydrolysis insoluble substrate by enzyme, but also extend our current understanding for raw starch digesting enzymes. The mutant with an improved activity may contribute to deep procession and industrial application of raw rice starch.

大米淀粉的研究和应用在人类生活中的地位举足轻重，但是由于大米生淀粉的成分和结构远比小麦等淀粉复杂，只有少数的生淀粉降酶能降解大米生淀粉，而且降解速度慢、效率不高。本课题组从海洋宏基因组文库中筛选到一个新型α-淀粉酶AmyP，并进而建立了新的亚家族GH13_37。随后，发现亚家族GH13_37成员都具有一种从未报道过的快速偏好降解大米生淀粉的能力。本项目以AmyP为研究对象，利用定点突变技术获得大米生淀粉降解速度和偏好性改变的突变体，并解析蛋白结构；对比研究突变体在酶学性质和蛋白结构方面的变化，揭示快速偏好降解大米生淀粉的机制。然后通过人工定向设计获得大米生淀粉降解能力提高的突变体，验证该机制。这是首次揭示淀粉酶结构与大米生淀粉之间的关系。预计结果不仅拓宽了对酶与不可溶底物作用机制的认识，而且丰富了对生淀粉降解酶的理解。获得的生淀粉降解能力提高的突变体也可能对大米生淀粉的深加工应用具有重要意

海洋宏基因组文库来源的新型α-淀粉酶AmyP的C端催化结构域代表了一个新的淀粉酶亚家族GH13_37，N端的淀粉结合结构域SBDAmyP代表了一个新的碳水化合物结合结构域CBM69家族。AmyP具有一种从未报道过的快速偏好降解大米生淀粉的能力。本课题围绕AmyP的催化特点，展开了系列研究。解析了AmyP△SBD的晶体结构和SBDAmyP的核磁结构。催化结构域中的氨基酸W269与AmyP的降解速度密切相关。没有发现能改变大米生淀粉的偏好性的氨基酸。但是我们发现，AmyP能更加高效的降解绿藻生淀粉。这说明绿藻生淀粉应该更加接近AmyP的天然底物，大米生淀粉也应该与AmyP的天然底物有一定的相似性。AmyP的结构显示了三个明显特点。非典型的Domain B区域会影响酶的整体结构和酶的电离。独有的Ca离子结合位点2参与了对底物中环状结构的识别。SBDAmyP是一个需要底物诱导才能形成完整三级结构的结构域，而且可以广谱的结合非淀粉类底物。通过置换SBD，我们获得了两个大米生淀粉降解能力显著增加的融合酶，其中的AmyP-Cr酶活和热稳定性均有增强，是一个具有巨大应用潜力的新型淀粉酶。此外，我们还测定了GH13_37家族另外一个没有SBD的α-淀粉酶AmyASS。