Institute of Bioprocess Science and Engineering, BOKU University, Muthgasse 18, Vienna, 1190, Austria.
Microb Cell Fact. 2024 Oct 10;23(1):272. doi: 10.1186/s12934-024-02551-6.
The global plastic waste crisis requires combined recycling strategies. One approach, enzymatic degradation of PET waste into monomers, followed by re-polymerization, offers a circular economy solution. However, challenges remain in producing sufficient amounts of highly active PET-degrading enzymes without costly downstream processes.
Using the growth-decoupled enGenes e-press V2 E. coli strain, pH, induction strength and feed rate were varied in a factorial-based optimization approach, to find the best-suited production conditions for the PHL7 enzyme. This led to a 40% increase in activity of the fermentation supernatant. Optimization of the expression construct resulted in a further 4-fold activity gain. Finally, the identified improvements were applied to the production of the more active and temperature stable enzyme variant, PHL7mut3. The unpurified fermentation supernatant of the PHL7mut3 fermentation was able to completely degrade our PET film sample after 16 h of incubation at 70 °C at an enzyme loading of only 0.32 mg enzyme per g of PET.
In this research, we present an optimized process for the extracellular production of thermophile and highly active PETases PHL7 and PHL7mut3, eliminating the need for costly purification steps. These advancements support large-scale enzymatic recycling, contributing to solving the global plastic waste crisis.
全球塑料废物危机需要综合回收策略。一种方法是利用酶将 PET 废物降解为单体，然后再聚合，从而形成循环经济解决方案。然而，在不进行昂贵的下游处理的情况下，生产足够数量的高活性 PET 降解酶仍然存在挑战。
利用生长解耦的 enGenes e-press V2 E. coli 菌株，通过基于因子的优化方法来改变 pH 值、诱导强度和进料速率，以找到最适合 PHL7 酶生产的条件。这使得发酵上清液的活性提高了 40%。对表达构建体的优化又使活性提高了 4 倍。最后，将确定的改进应用于更具活性和温度稳定性的酶变体 PHL7mut3 的生产中。在 70°C 下孵育 16 小时，仅用 0.32mg 酶/g PET 的酶用量，未纯化的 PHL7mut3 发酵上清液就能完全降解我们的 PET 薄膜样品。
在这项研究中，我们提出了一种优化的方法，用于在细胞外生产耐热和高活性的 PETase PHL7 和 PHL7mut3，无需进行昂贵的纯化步骤。这些进展支持大规模的酶法回收，有助于解决全球塑料废物危机。
