有机溶剂和聚电解质提升MHET水解酶催化性能

Catalytic Performance of Mono(2-Hydroxyethyl)Terephthalate Hydrolase Enhanced by Organic Solvents and Polyelectrolytes

聚对苯二甲酸乙二醇酯(PET)是一种广泛应用的塑料,但由于其难以自然降解导致严重的污染问题.PET水解酶和对苯二甲酸单乙二醇酯水解酶(MHETase)被认为是级联降解PET的关键酶,但尚未实现工业应用,本文针对MHETase开展稳定性研究以促进其工业开发.首先,选用4种不同的水溶性有机溶剂二甲基亚砜(DMSO)、N,N-二甲基甲酰胺、异丙醇和乙腈构建反应体系探寻最佳的MHETase反应条件,随后进一步研究有机溶剂和聚电解质预处理对MHETase催化活力和稳定性的影响.结果表明,当反应体系DMSO含量不高于10%时MHETase的活力几乎不受影响,其他条件下有机溶剂均会抑制MHETase活性,但这种抑制作用是可逆的,即该抑制可以通过去除有机溶剂来恢复.此外,低浓度的有机溶剂预处理能够提高MHETase的催化活力,且DMSO预处理能够提升MHETase的储存稳定性及热稳定性,MHETase在4℃下10%DMSO中储存96 h后的活力为原有活力的113%,在30℃下10%DMSO中孵育6 h后也能保持原有活力的103%.研究发现,MHETase活力可以通过阴、阳离子聚电解质孵育来提升,与等量阴、阳离子聚电解质共孵育的MHETase在4℃下10%DMSO中储存144 h也能保持118%的活力,但与DMSO预处理相比,聚电解质预处理对MHETase活力提升程度有限.本研究系统考察了有机溶剂和聚电解质对MHETase催化性能的影响,并指出DMSO和聚电解质的预处理有助于MHETase在常温下的储存和应用,为MHETase的工业应用提供了更多参考.

Polyethylene terephthalate(PET)is one of the most widely used plastics,whose wastes are hazardous and result in serious environmental problems because of its difficult self-degradation in the environment.PET hydrolase(PETase)and mono(2-hydroxyethyl)terephthalate hydrolase(MHETase)are considered as key enzymes for the cascade degradation of PET,but they have not yet achieved industrial applications.In this paper,the stability of MHETase was systematically studied to promote its industrial application.Four water-soluble organic solvents including dimethyl sulfoxide(DMSO),N,N-dimethylformamide(DMF),isopropanol(IPA)and acetonitrile(ACN)were used as cosolvents to explore the optimal reaction condition for MHETase.Then,the effects of organic solvents and polyelectrolyte pretreatment on the catalytic activity and stability of MHETase were studied.The experimental results showed that DMSO with a volume concentration below 10%did not impair the activity of MHETase,but the activity of MHETase was inhibited by adding other organic solvents into the reaction system.The inhibition on the activity of MHETase by adding organic solvents was reversible,i.e.,the activity of MHETase can be recovered by removing the organic solvents.Moreover,incubating pretreatment with a low concentration of organic solvent could improve the catalytic activity of MHETase,and pretreatment with DMSO significantly improved the storage stability and thermal stability of MHETase.MHETase showed 113%of the original relative activity after being stored in 10%DMSO at 4℃for 96 h,and 103%of that at 30℃for 6 h.In addition,the pretreatment of MHETase with amphoteric polyelectrolytes can enhance its activity,and the relative activity of MHETase was detected to be 118%of the original value when incubated with the same amounts of amphoteric polyelectrolytes in 10%DMSO at 4℃for 144 h.The effect of polyelectrolytes on the catalytic performance of MHETase was weaker than the 10%DMSO incubation.In conclusion,this study systematically investigated the effects of organic solvents and polyelectrolytes on the catalytic performance of MHETase,and pointed out that the pretreatment by DMSO and polyelectrolytes helps with the storage and application of MHETase at room temperature,providing more reference for the industrial application of MHETase.