酿酒酵母孢子固定化聚对苯二甲酸乙二醇酯水解酶的酶学性质研究  

Enzymatic properties of polyethylene terephthalate hydrolase immobilized by yeast spores

作　　者：白佳文 王亚森 杜祥坤 佀辰钰 兰周格 中西秀树[1] 李子杰[1] BAI Jiawen;WANG Yasen;DU Xiangkun;SI Chenyu;LAN Zhouge;HIDEKI Nakanishi;LI Zijie(School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与发酵工业》2025年第5期78-84,共7页Food and Fermentation Industries

基　　金：国家自然科学基金项目(32171475)。

摘　　要：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 dit1Δdefective 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.

关 键 词：酿酒酵母 孢子表面展示 FAST-PETase BHET降解 酶学性质 

分 类 号：Q814.2[生物学—生物工程] TQ323.41[化学工程—合成树脂塑料工业] TQ426.97