In silico Screening and Heterologous Expression of a Polyethylene Terephthalate Hydrolase (PETase)-Like Enzyme (SM14est) With Polycaprolactone (PCL)-Degrading Activity, From the Marine Sponge-Derived Strain Streptomyces sp. SM14
In silico Screening and Heterologous Expression of a Polyethylene Terephthalate Hydrolase (PETase)-Like Enzyme (SM14est) With Polycaprolactone (PCL)-Degrading Activity, From the Marine Sponge-Derived Strain Streptomyces sp. SM14
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DOI：
10.3389/fmicb.2019.02187
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发表时间：
2019-10-01
影响因子：
5.2
通讯作者：
Dobson, Alan D. W.
中科院分区：
文献类型：
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作者：
Almeida, Eduardo L.;Rincon, Andres Felipe Carrillo;Dobson, Alan D. W.
塑料，例如聚对苯二甲酸乙二醇酯（PET），由于其在包装工业中特别有用的物理化学性质而广泛用于各种工业应用。然而，由于塑料废物管理不当和回收困难，消费后塑料废物已成为环境和人类健康的紧迫问题。因此，需要处理塑料废物的新技术和方法来解决这些问题。合成聚合物的酶辅助水解已被提出作为目前采用的方法的潜在更有效和环境友好的替代方案。最近，已经描述了许多PET水解酶，并且特别是来源于Ideonella sakaiensis 201-F6的PET酶（IsPET酶），其似乎是迄今为止发现的最有效和底物特异性的细菌PET水解酶。为了进一步研究这类PETase样酶，我们在生物技术相关的链霉菌属，包括陆地和海洋分离株，在寻找潜在的PETase同源物的硅基筛选方法。从总共分析的52个基因组中，我们能够鉴定出三种潜在的PETase样酶，所有这些酶都来自海洋海绵相关的链霉菌分离株。在链霉菌SM 14中鉴定了一个候选的PETase样基因（SM 14 est）。对SM 14 est蛋白序列及其预测的三维结构进行了进一步的计算机表征，并与充分表征的IsPETase进行了比较。丝氨酸水解酶基序Gly-x1-Ser-x2-Gly和催化三联体Ser、Asp、His在两个序列中都是保守的。分子对接实验表明，SM 14 est酶具有结合塑料作为底物的能力。最后，使用聚己内酯（PCL）平板清除试验（其是塑料降解的模型底物）确认聚酯酶活性;在大肠杆菌中异源表达SM 14 est后，天然链霉菌信号肽促进分泌。这些发现为这类重要的PETase样酶提供了进一步的见解。
Plastics, such as the polyethylene terephthalate (PET), are widely used for various industrial applications, due to their physicochemical properties which are particularly useful in the packaging industry. However, due to improper plastic waste management and difficulties in recycling, post-consumer plastic waste has become a pressing issue for both the environment and for human health. Hence, novel technologies and methods of processing plastic waste are required to address these issues. Enzymatic-assisted hydrolysis of synthetic polymers has been proposed as a potentially more efficient and environment-friendly alternative to the currently employed methods. Recently, a number of PET hydrolases have been described, and in particular a PETase derived from Ideonella sakaiensis 201-F6 (IsPETase), which appears to be the most efficient and substrate-specific bacterial PET hydrolase enzyme discovered to date. In order to further investigate this class of PETase-like enzymes, we employed an in silico-based screening approach on the biotechnologically relevant genus Streptomyces, including terrestrial and marine isolates; in a search for potential PETase homologs. From a total of 52 genomes analyzed, we were able to identify three potential PETase-like enzymes, all of which were derived from marine-sponge associated Streptomyces isolates. A candidate PETase-like gene (SM14est) was identified in Streptomyces sp. SM14. Further in silico characterization of the SM14est protein sequence and its predicted three-dimensional structure were performed and compared to the well-characterized IsPETase. Both the serine hydrolase motif Gly-x1-Ser-x2-Gly and the catalytic triad Ser, Asp, His are conserved in both sequences. Molecular docking experiments indicated that the SM14est enzyme possessed the capacity to bind plastics as substrates. Finally, polyesterase activity was confirmed using a polycaprolactone (PCL) plate clearing assay which is a model substrate for the degradation of plastics; following heterologous expression of SM14est in Escherichia coli, with secretion being facilitated by the native Streptomyces signal peptide. These findings provide further insights into this important class of PETase-like enzymes.