角质酶的异源表达和超声辅助下降解PET塑料

Heterologous Expression of Cutinase and Degradation of Polyethylene Terephthalate with Ultrasound

聚对苯二甲酸乙二醇酯(PET)是一种典型的线性热塑性树脂材料,被广泛用于制造纤维、薄膜和饮料瓶。但是,PET为人类带来生活便利的同时也为全球环境造成不利影响,因为其难被降解,造成了大量的白色污染。本研究利用Escherichia.coli BL21(DE3)成功表达重组角质酶LCC,最优表达条件为:诱导剂异丙基-β-D-硫代半乳糖苷(IPTG)浓度0.2 mmol/L,诱导温度16℃,诱导时间20 h。经Ni离子亲和层析纯化后,LCC酶活为320 U/mg。采用超声辅助LCC降解PET,对其超声条件进行优化,温度为65℃,pH=8.0,超声功率为25 W,超声时间为5 h时,LCC动力学参数K_(m)最小,最大反应速度V_(max)最高,降解效率是未经处理的1.21倍,产物对苯二甲酸(TPA)质量浓度从0.72 g/L增加到0.87 g/L。通过扫描电镜发现,超声可以加速LCC对PET的降解。采用圆二色光谱和傅里叶变换红外光谱检测LCC二级结构,发现超声可能使LCC的α-螺旋和β-折叠向无规则卷曲转变,由此提高降解速率。这为超声加速降解反应提供了一定的理论依据,也为白色污染的生物降解开创了新思路。

Polyethylene terephthalate(PET)is a typical linear thermoplastic resin material,which is widely used in manufacture of fiber,film and beverage bottle.While PET brings many conveniences to human life,its property of difficult degradation has caused serious white pollution and threatened the global environment.In this research,the cutinase LCC was successfully expressed with Escherichia coli BL21(DE3),and the optimized condition in E.coli BL21(DE3)expression is:inducer isopropyl-β-D-thiogalactopyranoside(IPTG)=0.2 mmol/L,induction temperature=16℃,and induction time=20 h.After protein purification process via Ni-affinity chromatography,the enzyme activity of LCC is 320 U/mg.Subsequently,the purified LCC was used to degrade PET with ultrasound,and their reaction conditions were optimized.Under the conditions of reaction temperature=65℃,pH=8.0,ultrasonic power=25 W,ultrasonic time=5 h,K_(m) is the smallest and V_(max) is the largest,the degradation efficiency of LCC with ultrasound is 1.21 times higher than that of untreated,and the product terephthalic acid concentration of(TPA)increases from 0.72 g/L to 0.87 g/L.According to the scanning electron microscopy(SEM),the degradation of PET catalyzed by LCC can be accelerated by ultrasound.The circular dichroism(CD)spectroscopy and fourier transform infrared(FT-IR)spectroscopy show that theα-helix andβ-sheets of LCC secondary structure are possibly transformed into random coils with ultrasound.Thus,the degradation of PET catalyzed by LCC can be improved by ultrasonic process.This study provides some theoretical basis for the ultrasonic accelerated degradation reaction and opens up a new idea for the biodegradation of PET.