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.
作者
李志刚
陈世恒
孔德民
陈晟
王蕾
吴敬
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)
出处
《生物工程学报》
CAS
CSCD
北大核心
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)。
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
polyhydroxyalkanoate
polyhydroxyalkanoate depolymerase
thermal stability modification
disulfide bond
polyhydroxybutyrate