基于酿酒酵母孢子表面展示系统,实现聚对苯二甲酸乙二醇酯(polyethylene terephthalate,PET)水解酶FAST-PETase的孢子固定化。使用整合型质粒pRS306-TEF1pr-ss-FAST-PETase,将编码FAST-PETase的基因插入到酿酒酵母dit1Δ缺陷型菌株的基因组中,使蛋白更加稳定的表达。以双-2-(羟乙基)对苯二甲酸酯[bis(2-hydroxyethyl) terephthalate,BHET]为底物,对孢子固定化FAST-PETase的最适反应条件、热稳定性、pH耐受性、洗涤剂/有机试剂耐受性和重复使用能力进行研究。孢子固定化FAST-PETase的最适反应条件为50 ℃,pH 8.0。在30~80 ℃孵育12 h后,能保留82%以上的活性。同样地,在pH 5.0~9.0孵育12 h后,也能保持78%以上的活性。与游离酶相比,孢子固定化FAST-PETase对有机试剂也有一定的抗性,在重复使用7次后仍然保留初始活性的35%。该研究利用酿酒酵母孢子表面展示系统成功实现孢子固定化FAST-PETase,并将其应用于BHET的水解中,为PET塑料的长期降解提供了新的平台。
Based on the surface display system of Saccharomyces cerevisiae spores, the immobilization of polyethylene terephthalate (PET) hydrolase FAST-PETase was achieved.By using the integrated plasmid pRS306-TEF1pr-ss-FAST-PETase, the gene encoding FAST-PETase was inserted into the genome of the dit1Δ defective strain of S.cerevisiae to enable more stable protein expression.Using bis(2-hydroxyethyl) terephthalate (BHET) as the substrate, the optimal reaction conditions, thermal stability, pH tolerance, detergent/organic reagent tolerance, and reusability of spore-immobilized FAST-PETase were studied.The optimal reaction conditions for spore-immobilized FAST-PETase were 50 ℃ and pH 8.0.After incubation at temperatures ranging from 30 ℃ to 80 ℃ for 12 h, more than 82% of the original activity could be retained.Similarly, after incubation at pH 5.0-9.0 for 12 h, more than 78% of the original activity could be maintained.Compared with the free enzyme, spore-immobilized FAST-PETase also showed resistance to organic reagents, retaining 35% of its initial activity after seven reuse cycles.In this study, the immobilization of FAST-PETase was successfully realized by using the spore surface display system of S.cerevisiae and applied to the hydrolysis of BHET, providing a new platform for the long-term degradation of PET plastics.
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