来源于海洋宏基因组塑料降解酶Ple629的耐热性提升改造

Engineering the plastic degradation enzyme Ple629 from marine consortium to improve its thermal stability

摘要:

石化来源的聚酯类塑料如聚对苯二甲酸乙二醇酯(polyethylene terephthalate, PET)以及聚己二酸/对苯二甲酸丁二醇酯(polybutylene adipate terephthalate, PBAT)等已被广泛使用，但由于它们在自然界中难以降解或生物降解周期较长导致了严重的环境污染，因此对这些塑料废弃物的处理是亟待解决的问题之一。从循环经济的角度考虑，利用生物酶法对聚酯类塑料如PET或PBAT等的废弃物进行解聚，再将解聚产物进行循环利用，是一个很有潜力的研究方向。探究近年来关于聚酯塑料降解酶的报道发现，高活性且耐高温的降解酶会有更大的潜在优势。来自海洋微生物宏基因组的中温塑料降解酶Ple629，在常温下对聚酯类塑料PET和PBAT均有较好的降解活力，但由于不耐受高温，限制了其潜在应用。在前期获得Ple629三维结构的基础上，本研究基于结构比对及能量设计，找到了一些潜在提升其热稳定性的位点进行改造设计，并对突变体进行了表达纯化和热稳定性测定。突变体V80C和D226C/S281C的熔点温度(T_m)值分别提升了5.2 °C和6.9 °C，突变体D226C/S281C的活性也比野生型酶提高了1.5倍，为后续对Ple629的进一步改造提供了思路和依据。

关键词:聚对苯二甲酸乙二醇酯;聚己二酸/对苯二甲酸丁二醇酯;水解酶;表达纯化;耐热性;突变

Abstract:

Petrochemical-derived polyester plastics such as polyethylene terephthalate (PET) and polybutylene adipate terephthalate (PBAT) have been widely used. However, the difficulty to be degraded in nature (PET) or the long biodegradation cycle (PBAT) resulted in serious environmental pollution. In this connection, treating these plastic wastes properly becomes one of the challenges of environment protection. From the perspective of circular economy, biologically depolymerizing the waste of polyester plastics and reusing the depolymerized products is one of the most promising directions. Recent years have seen many reports on polyester plastics degrading organisms and enzymes. Highly efficient degrading enzymes, especially those with better thermal stability, will be conducive to their application. The mesophilic plastic-degrading enzyme Ple629 from the marine microbial metagenome is capable of degrading PET and PBAT at room temperature, but it cannot tolerate high temperature, which hampers its potential application. On the basis of the three-dimensional structure of Ple629 obtained from our previous study, we identified some sites which might be important for its thermal stability by structural comparison and mutation energy analysis. We carried out transformation design, and performed expression, purification and thermal stability determination of the mutants. The melting temperature (T_m) values of mutants V80C and D226C/S281C were increased by 5.2 °C and 6.9 °C, respectively, and the activity of mutant D226C/S281C was also increased by 1.5 times compared with that of the wild-type enzyme. These results provide useful information for future engineering and application of Ple629 in polyester plastic degradation.

Key words:polyethylene terephthalate;polybutylene adipate terephthalate;hydrolase;expression and purification;thermal stability;mutagenesis