温度对酶促聚对苯二甲酸乙二醇酯降解的影响分析  

On the temperature dependence of enzymatic degradation of poly(ethylene terephthalate)

作　　者：伊科拉木·艾克然木 曹宇飞 邢皓 丁于敬 罗宇政 韦韧 张一飞 Ekram Akram;Yufei Cao;Hao Xing;Yujing Ding;Yuzheng Luo;Ren Wei;Yifei Zhang(State Key Laboratory of Chemical Resources Engineering,Beijing University of Chemical Technology,Beijing 100029,China;Beijing Advanced Innovation Center for Soft Matter Science and Engineering,Beijing University of Chemical Technology,Beijing 100029,China;Laboratory of Applied Biocatalysis,School of Food Science and Technology,South China University of Technology,Guangzhou 510640,Guangdong,China;Junior Research Group Plastic Biodegradation,Institute of Biochemistry,University of Greifswald,Greifswald 17489,Germany)

机构地区：[1]北京化工大学化工资源有效利用国家重点实验室,北京100029 [2]北京化工大学北京软物质科学与工程高精尖创新中心,北京100029 [3]华南理工大学食品科学与工程学院应用生物催化实验室,广东广州510640 [4]格赖夫斯瓦尔德大学生物化学研究所,格赖夫斯瓦尔德,德国

出　　处：《Chinese Journal of Catalysis》2024年第5期284-293,共10页催化学报（英文）

基　　金：国家自然科学基金(32371325);中国石油化工集团公司种子基金项目(223260).

摘　　要：近年来,聚对苯二甲酸乙二酯(PET)的酶法降解及循环利用备受关注.PET的酶法降解是在不可溶聚合物表面发生的界面催化过程,酶通过水解PET聚合物表面上可及的酯键,将聚合物逐渐分解成单体.反应温度可以从酶的催化活性、热稳定性以及PET材料的生物可降解性等多个方面影响PET的降解过程,是影响PET酶法降解效果的主要因素之一.深入了解温度对PET酶降解过程的热力学和动力学调控机制,对PET降解酶的发掘与改造和酶法降解工艺的优化具有重要意义.本文以叶枝堆肥角质酶(LCC)的突变体WCCG为例,详细研究了温度对WCCG降解低结晶度PET(结晶度7.6%)和高结晶度(结晶度30%)PET微颗粒的影响.通常认为PET的酶促降解需要在其玻璃化转变温度(Tg,~70℃)附近或以上进行.实验测定了不同温度下的降解速率,并利用阿伦尼乌斯公式作图.结果表明,当反应温度在45℃(低于通常认为的Tg,b值约20℃)附近时,反应活化能突然降低,说明此时PET微颗粒表面的玻璃化转变已经开始,酶促降解已经能够有效进行,表现出酶解PET的界面催化特征.当温度高于75℃时,PET降解速率迅速降低,酶受热失活.研究还发现,WCCG存在热激活现象:通过将WCCG在不同温度下温育一段时间后在60℃下进行活性测定,WCCG在50-70℃范围内温育后活性显著提高;在70℃下温育4 h后其降解低结晶度PET的活性提高约1倍,同时对小分子底物4-硝基苯酚丁酯的活性也有明显提高.利用圆二色光谱和荧光光谱检测了WCCG的二级和三级结构变化.结果表明,热处理后的WCCG中的α-螺旋含量显著减少而β折叠含量增加,酶分子疏水性提高,这与分子动力学模拟结果一致.单分子力谱测试结果表明,热处理后的WCCG较处理前的酶与PET薄膜之间的粘附力从88 pN提高到150 pN.因此,WCCG的热激活是其本征催化活性改善和酶与PET表面吸附力增强共同作用的结果.WCCG�Enzymatic recycling of poly(ethylene terephthalate),PET,has attracted significant attention in recent years.Temperature is a governing factor in the enzymatic degradation of PET,influencing simultaneously the catalytic activity and thermal stability of enzymes,as well as the biodegradability of PET materials from many perspectives.Here we present a detailed examination of the complex and mutual effects of temperature on the degradation of low-crystallinity PET(LC-PET,7.6%)and high-crystallinity PET(HC-PET,30%)microparticles using the WCCG variant of the leaf-branch compost cutinase(LCC).The degradation velocity apparently increases exponentially with increasing temperature at temperatures below 65℃.Arrhenius plots show a sudden reduction in activation energy at temperatures higher than 40℃,suggesting the onset of the surface glass transition of PET particles.This is more than 20℃ lower than the bulk glass transition temperature,underscoring the interfacial catalytic nature of enzymatic PET degradation.WCCG undergoes substantial conformational changes upon thermal incubation at temperatures ranging from 50 to 70℃ and exhibits enhanced activity,owing to the increased intrinsic catalytic activity and improved adsorption on PET surface.Further increasing the temperature leads to the inactivation of enzymes alongside the rapid recrystallization of amorphous PET,impeding the enzymatic degradation.These findings offer a detailed mechanistic understanding of the temperature dependence of the enzymatic degradation of PET,and may have implications for the engineering of more powerful PET hydrolases and the selection of favorable conditions for industrially-related recycling processes.

关 键 词：聚对苯二甲酸乙二酯 酶法降解 酶的热激活 活化能 表面玻璃化转变 

分 类 号：TQ426[化学工程]