Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, USA.
BOTTLE Consortium, Golden, CO, USA.
Nat Commun. 2022 Dec 21;13(1):7850. doi: 10.1038/s41467-022-35237-x.
Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5-9.0 and temperatures from 30-70 °C. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.
由于水解酶能够在聚合物玻璃化转变温度附近将聚对苯二甲酸乙二醇酯 (PET) 解聚为其组成单体，因此对聚对苯二甲酸乙二醇酯 (PET) 的酶促解构受到了强烈关注。迄今为止，报道的 PET 水解酶来源于相对狭窄的序列空间。在这里，我们通过使用生物信息学和机器学习从自然多样性中鉴定出其他具有 PET 活性的生物催化剂，以挖掘 74 种可能的耐热 PET 水解酶。我们成功地表达、纯化并测定了来自七个不同系统发育群的 51 种酶；在 pH 值为 4.5-9.0 和温度为 30-70°C 的反应中，观察到 37 种酶对无定形 PET 薄膜的 PET 水解活性。我们对表现最好的酶进行了 PET 水解时程反应，其中我们观察到作为 PET 形态函数的底物选择性差异。我们采用 X 射线晶体学和 AlphaFold 检查了所有 74 个候选物的酶结构，揭示了以前与 PET 解构无关的蛋白质折叠和辅助结构域。总的来说，这项研究扩展了耐热酶解 PET 的数量和多样性。
