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

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.