Enhancement of the degradation capacity of IsPETase by acidic amino acids insertion and carbohydrate-binding module fusion.

IF 2.6 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
3 Biotech Pub Date : 2024-09-01 Epub Date: 2024-08-07 DOI:10.1007/s13205-024-04041-3
Chuang Li, Qingqing Zheng, Wei Liu, Quanyu Zhao, Ling Jiang
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引用次数: 0

Abstract

The biocatalytic degradation of poly(ethylene terephthalate) (PET) through enzymatic methods has garnered considerable attention due to its environmentally friendly and non-polluting nature, as well as its high specificity. While previous efforts in enhancing IsPETase performance have focused on amino acid substitutions in protein engineering, we introduced an amino acid insertion strategy in this work. By inserting a negatively charged acidic amino acid, Glu, at the right-angle bend of IsPETase, the binding capability between the enzyme's active pocket and PET was improved. The resulted mutant IsPETase9394insE exhibited enhanced hydrolytic activity towards PET at various temperatures ranging from 30 to 45 ℃ compared with the wild-type IsPETase. Notably, a 10.04-fold increase was observed at 45 ℃. To further enhance PET hydrolysis, different carbohydrate-binding modules (CBMs) were incorporated at the C-terminus of IsPETase9394insE. Among these, the fusion of CBM from Verrucosispora sioxanthis exhibited the highest enhancement, resulting in a 1.82-fold increase in PET hydrolytic activity at 37 ℃ compared with the IsPETase9394insE. Finally, the engineered variant was successfully employed for the degradation of polyester filter cloth, demonstrating its promising hydrolytic capacity. In conclusion, this research presents an alternative enzyme engineering strategy for modifying PETases and enriches the pool of potential candidates for industrial PET degradation.

Abstract Image

通过插入酸性氨基酸和融合碳水化合物结合模块提高 IsPETase 的降解能力。
通过酶法生物催化降解聚对苯二甲酸乙二酯(PET)因其环境友好、无污染以及高特异性而备受关注。以往提高 IsPET 酶性能的方法主要是在蛋白质工程中进行氨基酸置换,而我们在这项工作中引入了氨基酸插入策略。通过在 IsPETase 的直角弯曲处插入一个带负电荷的酸性氨基酸 Glu,提高了酶的活性口袋与 PET 之间的结合能力。与野生型 IsPETase 相比,得到的突变体 IsPETase9394insE 在 30 至 45 ℃ 的不同温度下对 PET 的水解活性都有所提高。值得注意的是,在 45 ℃ 时,活性提高了 10.04 倍。为了进一步加强 PET 的水解,在 IsPETase9394insE 的 C 端加入了不同的碳水化合物结合模块(CBM)。其中,融合来自 Verrucosispora sioxanthis 的 CBM 的效果最好,与 IsPETase9394insE 相比,37 ℃ 下的 PET 水解活性提高了 1.82 倍。最后,该工程变体被成功用于降解聚酯滤布,证明其具有良好的水解能力。总之,这项研究为改造 PET 酶提供了另一种酶工程策略,并丰富了工业 PET 降解的潜在候选酶库。
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来源期刊
3 Biotech
3 Biotech Agricultural and Biological Sciences-Agricultural and Biological Sciences (miscellaneous)
CiteScore
6.00
自引率
0.00%
发文量
314
期刊介绍: 3 Biotech publishes the results of the latest research related to the study and application of biotechnology to: - Medicine and Biomedical Sciences - Agriculture - The Environment The focus on these three technology sectors recognizes that complete Biotechnology applications often require a combination of techniques. 3 Biotech not only presents the latest developments in biotechnology but also addresses the problems and benefits of integrating a variety of techniques for a particular application. 3 Biotech will appeal to scientists and engineers in both academia and industry focused on the safe and efficient application of Biotechnology to Medicine, Agriculture and the Environment.
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