短须嗜热单孢菌聚羟基脂肪酸酯解聚酶的表达、热稳定性改造及在PHB降解中的应用  

Expression,thermal stability modification and application in PHB degradation of polyhydroxyalkanoate depolymerase from Thermomonospora umbrina

作　　者：李志刚[1,2] 陈世恒[1,2] 孔德民 陈晟 王蕾[1,2] 吴敬[1,2] LI Zhigang;CHEN Shiheng;KONG Demin;CHEN Sheng;WANG Lei;WU Jing(State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi 214122,Jiangsu,China;Key Laboratory of Industrial Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China)

机构地区：[1]江南大学食品科学与技术国家重点实验室,江苏无锡214122 [2]江南大学生物工程学院工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《生物工程学报》2023年第8期3351-3363,共13页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(2019YFA0706900);江苏省重点研发计划(BE2021625);江苏省政策引导类计划(BZ2020010)。

摘　　要：聚羟基脂肪酸酯解聚酶(polyhydroxyalkanoate depolymerase,PHAD)可用于聚羟基脂肪酸酯(polyhydroxyalkanoate,PHA)的降解回收,为开发热稳定性好的PHAD,本研究在大肠杆菌(Escherichiacoli)BL21(DE3)中成功表达了来自短须嗜热单孢菌(Thermomonospora umbrina)的PHA解聚酶(TumPHAD),并通过二硫键理性设计获得了热稳定性提升的突变体A190C/V240C,其最适温度为60℃,比野生型提高20℃,50℃半衰期为7h,是野生型酶的21倍。将突变体A190C/V240C用于典型PHA之一的聚羟基丁酸酯(polyhydroxybutyrate,PHB)降解,在50℃条件下,PHB的2 h和12 h降解率较野生型分别提高了2.1倍和3.8倍。本研究获得的TumPHAD突变体A190C/V240C具有耐高温、热稳定性好和PHB降解能力强的特点,对PHB的降解回收具有重要意义。Polyhydroxyalkanoate depolymerase(PHAD)can be used for the degradation and recovery of polyhydroxyalkanoate(PHA).In order to develop a PHAD with good stability under high temperature,PHAD from Thermomonospora umbrina(TumPHAD)was heterelogously expressed in Escherichia coli BL21(DE3).At the same time,a mutant A190C/V240C with enhanced stability was obtained via rational design of disulfide bonds.Characterization of enzymatic properties showed that the mutant A190C/V240C had an optimum temperature of 60℃,which was 20℃higher than that of the wild type.The half-life at 50℃was 7 hours,at 50℃which was 21 times longer than that of the wild type.The mutant A190C/V240C was used for the degradation of polyhydroxybutyrate(PHB),one of the typical PHA.At 50℃,the degradation rate of PHB being treated for 2 hours and 12 hours was 2.1 times and 3.8 times higher than that of the wild type,respectively.The TumPHAD mutant A190C/V240C obtained in this study shows tolerance to high temperature resistance,good thermal stability and strong PHB degradation ability,which may facilitate the degradation and recovery of PHB.

关 键 词：聚羟基脂肪酸酯 聚羟基脂肪酸酯解聚酶 热稳定性改造 二硫键 聚羟基丁酸酯 

分 类 号：Q936[生物学—微生物学]