高比活耐热普鲁兰酶(PulA)分子改造及耐酸机制的研究

Pullulanase(EC3.2.1.41) is an important starch debranching enzyme.It has been widely used in the production of glucose, maltose and other products. Until now, acid resistance still be the bottleneck of pullulanase application in the starch industry. The mechanism of the acid resistance of pullulanase remains to be revealed.In our previous study, we have obtained a pullulanase (PulA) with high specific activity. The crystal structure of PulA with high resolution has also been obtained. In addition, the thermostability of PulA has been enhanced significantly by semi-rational design. However, PulA has poor acid resistance. In this study, we intend to analyze the sequence and structure of PulA by a variety of bioinformatics methods. Site-directed mutagenesis or Iterative saturation mutagenesis(ISM) will be also performed to reveal critical sites related with acid resistance of PulA. A series of fusion recombinant pullulanases will be built by superposing the structures of PulA and BapulA, to reveal the critical domains related with acid resistance of PulA.Furthermore, mutants with better acid resistance and high catalytic efficiency under acidic conditions could be obtained. Discovery Studio™ and crystal structure will be helpful to analyze the key amino acids and domains, and further to illustrate the the mechanism of the acid resistance of PulA. The research will lay the foundation for the mechanism of the acid resistance of the pullulanase and provide new insights of molecular modification for improving the acid resistance of pullulanases and other enzymes.

普鲁兰酶（EC 3.2.1.41）是一种重要的淀粉脱支酶，广泛应用于葡萄糖、麦芽糖等产品的生产。然而大部分天然普鲁兰酶耐酸性差，限制了其在淀粉工业中的应用，而且目前普鲁兰酶的耐酸机制尚不明确。本课题组前期筛选获得了高比活普鲁兰酶(PulA)，并成功解析了PulA的晶体结构，并且通过半理性设计显著提高了该酶的热稳定性，但是该酶的耐酸性差。因此，本项目拟在晶体结构信息及前期改造基础上，应用多种生物信息学的方法对其序列和结构进行系统的分析，结合定点突变、迭代饱和突变(ISM)及融合蛋白的构建，揭示影响PulA耐酸性的关键氨基酸位点及结构域，获得耐酸性提高且高效的突变体，结合分子模拟软件（Discovery Studio™）及晶体结构的解析,分析关键氨基酸或结构域的结构变化,深入解析PulA耐酸性分子机制。通过本项目的开展，为普鲁兰酶甚至其他工业酶的耐酸性分子改造提供理论基础。

普鲁兰酶（EC 3.2.1.41）是一种重要的淀粉脱支酶，广泛应用于葡萄糖、麦芽糖等产品 的生产。然而大部分天然普鲁兰酶耐酸性差，限制了其在淀粉工业中的应用，而且目前普鲁兰酶的耐酸机制尚不明确。本课题组前期筛选获得了耐热I型普鲁兰酶(PulAR)，并通过半理性设计显著提高了该酶的热稳定性，但是该酶的耐酸性差。因此，本项目建立在对该普鲁兰酶的结构与序列与耐酸性I型普鲁兰酶进行比对分析的基础上，通过基于结构分析指导的氨基酸共义性的方法，结合定点突变与组合突变研究获得了普鲁兰酶的耐酸性提高的单点突变体与组合突变体PulAR-A365V、PulAR-V401C、PulAR-A365V/V401C与PulAR-A365V/V401C/T504V，酶学性质表明PulAR及其突变体PulAR-A365V、PulAR-V401C、PulAR-A365V/V401C与PulAR-A365V/V401C/T504V的最适温度分别为55、55、60、60、60与65 ℃，其中最佳突变体PulAR-A365V/V401C/T504V的最适温度比野生型PulAR提高10℃。此外，四突变体PulAR-A365V-V401C-T504V-H499A在60、65 ℃的半衰期(t1/2)分别比野生型PulAR提高了2.6与3.1倍；同时，四突变体PulAR-A365V-V401C-T504V-H499A在pH 4.5、pH5.0 t1/2的比野生型提高了1.6与1.8倍，而且最佳四突变体在pH 5.0、60℃的催化效率(kcat/KM)比野生型PulAR提高9.6倍，表明突变体在高温与酸性条件下的稳定性与催化效率显著提高。结构分析表明增加的疏水作用力、与水分子的氢键、减少溶剂可及表面积是普鲁兰酶PulAR耐热耐酸条件下稳定性提高的主要原因。