Wei Ren, Oeser Thorsten, Then Johannes, Kühn Nancy, Barth Markus, Schmidt Juliane, Zimmermann Wolfgang
Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, University of Leipzig, Johannisallee 21-23, Leipzig, D-04103, Germany.
AMB Express. 2014 Jun 3;4:44. doi: 10.1186/s13568-014-0044-9. eCollection 2014.
Thermomonospora curvata is a thermophilic actinomycete phylogenetically related to Thermobifida fusca that produces extracellular hydrolases capable of degrading synthetic polyesters. Analysis of the genome of T. curvata DSM43183 revealed two genes coding for putative polyester hydrolases Tcur1278 and Tcur0390 sharing 61% sequence identity with the T. fusca enzymes. Mature proteins of Tcur1278 and Tcur0390 were cloned and expressed in Escherichia coli TOP10. Tcur1278 and Tcur0390 exhibited an optimal reaction temperature against p-nitrophenyl butyrate at 60°C and 55°C, respectively. The optimal pH for both enzymes was determined at pH 8.5. Tcur1278 retained more than 80% and Tcur0390 less than 10% of their initial activity following incubation for 60 min at 55°C. Tcur0390 showed a higher hydrolytic activity against poly(ε-caprolactone) and polyethylene terephthalate (PET) nanoparticles compared to Tcur1278 at reaction temperatures up to 50°C. At 55°C and 60°C, hydrolytic activity against PET nanoparticles was only detected with Tcur1278. In silico modeling of the polyester hydrolases and docking with a model substrate composed of two repeating units of PET revealed the typical fold of α/β serine hydrolases with an exposed catalytic triad. Molecular dynamics simulations confirmed the superior thermal stability of Tcur1278 considered as the main reason for its higher hydrolytic activity on PET.
弯曲嗜热放线菌是一种与栖热栖热放线菌在系统发育上相关的嗜热放线菌，它能产生可降解合成聚酯的细胞外水解酶。对弯曲嗜热放线菌DSM43183的基因组分析表明，有两个基因编码假定的聚酯水解酶Tcur1278和Tcur0390，它们与栖热栖热放线菌的酶具有61%的序列同一性。Tcur1278和Tcur0390的成熟蛋白在大肠杆菌TOP10中进行了克隆和表达。Tcur1278和Tcur0390对丁酸对硝基苯酯的最佳反应温度分别为60°C和55°C。两种酶的最佳pH值均为8.5。在55°C孵育60分钟后，Tcur1278保留了超过80%的初始活性，而Tcur0390保留的初始活性不到10%。在高达50°C的反应温度下，与Tcur1278相比，Tcur0390对聚（ε-己内酯）和聚对苯二甲酸乙二酯（PET）纳米颗粒表现出更高的水解活性。在55°C和60°C时，仅用Tcur1278检测到对PET纳米颗粒的水解活性。对聚酯水解酶进行计算机模拟，并与由PET的两个重复单元组成的模型底物对接，揭示了具有暴露催化三联体的典型α/β丝氨酸水解酶折叠结构。分子动力学模拟证实了Tcur1278具有卓越的热稳定性，这被认为是其对PET具有更高水解活性的主要原因。
