Engineering and evaluation of thermostable IsPETase variants for PET degradation

被引:76
作者
Brott, Stefan [1 ]
Pfaff, Lara [1 ]
Schuricht, Josephine [1 ]
Schwarz, Jan-Niklas [1 ]
Boettcher, Dominique [1 ]
Badenhorst, Christoffel P. S. [1 ]
Wei, Ren [1 ]
Bornscheuer, Uwe T. [1 ]
机构
[1] Univ Greifswald, Dept Biotechnol & Enzyme Catalysis, Inst Biochem, Felix Hausdorff Str 4, D-17489 Greifswald, Germany
来源
ENGINEERING IN LIFE SCIENCES | 2022年 / 22卷 / 3-4期
基金
欧盟地平线“2020”;
关键词
PET hydrolysis; PETase; polyethylene terephthalate; protein engineering; thermostability; ENZYMATIC SURFACE HYDROLYSIS; IDEONELLA-SAKAIENSIS; TEREPHTHALATE; CUTINASE; POLY(ETHYLENE-TEREPHTHALATE); BIODEGRADATION; MOISTURE;
D O I
10.1002/elsc.202100105
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Polyethylene terephthalate (PET) is a mass-produced petroleum-based synthetic polymer. Enzymatic PET degradation using, for example, Ideonella sakaiensis PETase (IsPETase) can be a more environmentally friendly and energy-saving alternative to the chemical recycling of PET. However, IsPETase is a mesophilic enzyme with an optimal reaction temperature lower than the glass transition temperature (T-g) of PET, where the amorphous polymers can be readily accessed for enzymatic breakdown. In this study, we used error-prone PCR to generate a mutant library based on a thermostable triple mutant (TM) of IsPETase. The library was screened against the commercially available polyester-polyurethane Impranil DLN W 50 for more thermostable IsPETase variants, yielding four variants with higher melting points. The most promising IsPETaseTM(K95N/F201I) variant had a 5.0 degrees C higher melting point than IsPETaseTM. Although this variant showed a slightly lower activity on PET at lower incubation temperatures, its increased thermostability makes it a more active PET hydrolase at higher reaction temperatures up to 60 degrees C. Several other variants were compared and combined with selected previously published IsPETase mutants in terms of thermostability and hydrolytic activity against PET nanoparticles and amorphous PET films. Our findings indicate that thermostability is one of the most important characteristics of an effective PET hydrolase.
引用
收藏
页码:192 / 203
页数:12
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