聚琥珀酸丁二醇酯的微生物降解机制及其降解酶的分子改造研究

Poly (butylene succinate) (PBS) is a new type polymer which prepared by polycondensation of butylene and succinate. It is now recognized as one of the best properties of biodegradable plastics. PBS has wide application because of its excellent performance and reasonable price. However, the microbial degradation mechanism of PBS is still unclear and it affects the further development of PBS. In this study, the PBS degrading enzyme would be purified and characterized basis on the screening of the PBS degrading strains. After cloning and expression of the degrading enzyme, the bioinformatics analysis and structure prediction should be done to explain the catalytic mechanisms of PBS degrading enzyme. The difference between pre and post PBS degradation would be analyzed and compared by polymer chemistry method. The production process of PBS monomer or oligomers would be investigated. The degradation mechanisms of interaction between degrading strains and degrading enzyme with its substrate (PBS) would be confirmed. Then the molecular modification of conserved and non-conserved part in PBS-degrading enzymes should be done. The structure and regularity of non-specific enzyme changed to specific enzyme should be confirmed. The molecular evolution basis of PBS-degrading enzymes should be described. This work would obtain efficient or new functional enzyme and provide the theoretical basis on the degradation of other non-natural polymers.

聚琥珀酸丁二醇酯（PBS）是由琥珀酸和丁二醇经缩聚而得的新型高分子材料，是目前公认的综合性能最好的生物降解塑料之一。PBS因其优异的综合性能和合理的性价比而具广泛用途。但目前有关PBS的生物降解机制仍然不清楚，以至于影响PBS产品的开发利用。本研究在完成PBS降解微生物选育和纯化表征PBS降解酶的基础上，完成降解酶的克隆表达。进一步在对降解酶序列的生物信息学分析和结构预测的基础上，解析PBS降解酶的催化机制。结合高分子化学分析手段，对PBS材料降解前后的差异进行分析和比较，并对PBS降解单体或寡聚物的生成过程进行研究，以充分掌握微生物及其降解酶对PBS的降解机制。继而对PBS降解酶分子中进化保守与非保守部分进行理性或定向改造，掌握由非专一性酶向专一高效酶转变的结构基础及其规律，阐述PBS降解酶的分子进化基础。本课题将为获得高效或具有新功能的酶类，实现其它相似非天然聚合物的降解提供理论基础。

聚琥珀酸丁二醇酯（PBS）是由琥珀酸和丁二醇经缩聚而得的新型高分子材料，是目前公认的综合性能最好的生物降解塑料之一。PBS因其优异的综合性能和合理的性价比而具广泛用途。但目前有关PBS的生物降解机制仍然不清楚，以至于影响PBS产品的开发利用。基于上述问题，本研究在完成PBS降解微生物选育和纯化表征PBS降解酶的基础上，对获得的PBS解聚酶进行解析，初步确定其属于角质酶，进而对其编码基因核苷酸进行优化，使其更适用于毕赤酵母的表达。再将优化后的基因序列表达于毕赤酵母，纯化获得了重组角质酶，借用高分子材料分析手段结合生物化学和微生物学实验方法，初步了解了该解聚酶PBS的生物降解机制。其次，采用化学合成法合成了一系列不同分子量大小的PBS以及与PBS有相似结构而单体碳原子数不同的PBS聚酯，进而使用重组角质酶进行降解。研究发现随着底物分子量的增加，聚酯失重率呈现先下降后增加的趋势。降解后剩余物质的主要成分仍为PBS,分子量也基本未发生改变。单体碳原子数的增加对聚酯降解效率有着较大的影响，无论是羟基单体还是羧基单体碳原子数的增加均会提升相同降解时间条件下聚酯的降解率。最后，开展了酶分子结构及活性位点的结构预测，进一步对酶分子进行定点突变，以进一步完整掌握降解酶的降解规律，并进一步获得高效的聚酯降解酶。