Department of Biotechnology, Bandung Institute of Technology, Bandung, Indonesia.
Recent Pat Biotechnol. 2020;14(3):229-234. doi: 10.2174/1872208314666200311104541.
Polyethylene terephthalate (PET) is the most widely produced polyester plastic in the world. PET is very difficult to catalyze or biological depolymerization due to the limited access to ester bonds. Consequently, plastic will be stockpiled or flowed into the environment which is projected until hundreds of years. The most effective and environmental friendly plastic degradation method is biodegradation with microorganisms. Two specific enzyme for PET hydrolase, PETase and MHETase have been identified from Ideonella sakaiensis 201-F6. Recombinant genes are made to increase the effectiveness of enzymes in degrading PET. Previous studies of the PETase gene have been carried out, but to produce the final degradation PET product, the enzyme MHETase is needed. Thus, in this study the MHETase gene construction was carried out.
The goal of this study is to construct MHETase gene in pUCIDT plasmid with native signal peptide from I. sakaensis 201-F6 and constitutive promoter J23106 was expressed in Escherichia coli BL21 (DE3) by heats shock. Expression analysis using SDS-PAGE and activity of enzyme is analyzed by spectrophotometry method and SEM.
MHETase gene protein was successfully constructed in pUCIDT +Amp plasmid with native signal peptide from Ideonella sakaensis 201-F6, T7 terminator and constitutive promoter J23106. PCR analysis showed that the gene successfully contained in the cells by band size (1813 bp) in electrophoresis gel. Analysis using Snap Gene, pairwise alignment using MEGA X, and NCBI was demonstrated that MHETase sequence the gene was in-frame in pUCIDT plasmid.
MHETase gene was successfully constructed in plasmids by in silico method. Synthetic plasmids transformed in E. coli BL21 (DE3) contain MHETase gene sequences which were in frame. Hence, the E. coli BL21 (DE3) cells have the potential to produce MHETase proteins for the plastic degradation testing process. We will patent the construct of MHETase gene using constitutive promoter and signal peptide from native which expressed in E. coli BL21 (DE3). This patent refers to a more applicable plastic degradation system with a whole cell without the need for purification and environmental conditioning of pure enzymes.
聚对苯二甲酸乙二醇酯（PET）是世界上产量最大的聚酯塑料。由于酯键不易接近，因此 PET 很难进行催化或生物解聚。因此，塑料将被储存或流入环境中，预计将持续数百年。最有效和环保的塑料降解方法是微生物的生物降解。已经从 Ideonella sakaiensis 201-F6 中鉴定出两种用于 PET 水解酶的特定酶，即 PETase 和 MHETase。已经构建了重组基因以提高酶降解 PET 的效率。已经进行了以前对 PETase 基因的研究，但是要生产最终降解 PET 的产物，需要酶 MHETase。因此，在这项研究中进行了 MHETase 基因的构建。
本研究的目的是在 pUCIDT 质粒中构建 MHETase 基因，该基因带有来自 I. sakaensis 201-F6 的天然信号肽，并且使用热激在大肠杆菌 BL21（DE3）中表达组成型启动子 J23106。使用 SDS-PAGE 进行表达分析，并通过分光光度法和 SEM 分析酶的活性。
成功地在 pUCIDT+Amp 质粒中构建了带有天然信号肽的 MHETase 基因来自 Ideonella sakaensis 201-F6、T7 终止子和组成型启动子 J23106。PCR 分析表明，通过电泳凝胶中的条带大小（1813 bp）成功地将基因包含在细胞中。使用 Snap Gene 进行分析、使用 MEGA X 进行成对比对以及使用 NCBI 进行分析表明，MHETase 序列中的基因在 pUCIDT 质粒中是框内的。
成功地通过计算机方法在质粒中构建了 MHETase 基因。转化为大肠杆菌 BL21（DE3）的合成质粒包含 MHETase 基因序列，这些序列是框内的。因此，大肠杆菌 BL21（DE3）细胞具有生产用于塑料降解测试过程的 MHETase 蛋白的潜力。我们将使用组成型启动子和来自天然的信号肽对 MHETase 基因进行专利申请，该基因在大肠杆菌 BL21（DE3）中表达。该专利涉及一种更适用的塑料降解系统，该系统使用整个细胞，而无需对纯酶进行纯化和环境调节。
