DataSheet1_Degradation kinetics of medium chain length Polyhydroxyalkanoate degrading enzyme: a quartz crystal microbalance study.docx

This study investigates the enzymatic degradation processes of different classes of polyhydroxyalkanoates (PHAs), a group of biopolymers naturally synthesized by various microorganisms. Medium chain length PHAs (mcl-PHAs) are distinguished biopolymers due to their biodegradability and diverse material properties. Using quartz crystal microbalance measurements as a valuable tool for accurate real-time monitoring of the enzymatic degradation process, the research provides detailed kinetic data, describing the interaction between enzymes and substrates during the enzymatic degradation process. Thin films of poly-3-hydroxybutyrate (PHB) and polyhydroxyoctanoate copolymer (PHO), containing molar fractions of about 84% 3-hydroxyoctanoate and 16% 3-hydroxyhexanoate, were exposed to scl-depolymerases from Pseudomonas lemoignei LMG 2207 and recombinant mcl-depolymerase produced in Escherichia coli DH5α harboring the plasmid pMAD8, respectively. Analyses based on a heterogeneous kinetic model for the polymer degradation indicated a six-fold stronger adsorption equilibrium constant of mcl-depolymerase to PHO. Conversely, the degradation rate constant was approximately twice as high for scl-depolymerases acting on PHB. Finally, the study highlights the differences in enzyme-substrate interactions and degradation mechanisms between the investigated scl- and mcl-PHAs.

本研究聚焦不同类别聚羟基脂肪酸酯（polyhydroxyalkanoates, PHAs）的酶促降解过程。聚羟基脂肪酸酯是一类可由多种微生物天然合成的生物聚合物。其中，中链长度聚羟基脂肪酸酯（medium chain length PHAs, mcl-PHAs）凭借优异的可生物降解性与多样的材料性能，成为极具特色的生物聚合物分支。本研究采用石英晶体微天平（quartz crystal microbalance, QCM）作为精准实时监测酶促降解过程的有效手段，获取了描述酶与底物在酶促降解过程中相互作用的详细动力学数据。实验中，研究人员将聚3-羟基丁酸酯（poly-3-hydroxybutyrate, PHB）薄膜，以及摩尔分数约为84% 3-羟基辛酸酯与16% 3-羟基己酸酯的聚羟基辛酸酯共聚物（polyhydroxyoctanoate copolymer, PHO）薄膜，分别置于来自莱蒙涅氏假单胞菌（Pseudomonas lemoignei）LMG 2207的短链解聚酶（scl-depolymerases），以及在携带质粒pMAD8的大肠杆菌（Escherichia coli）DH5α中重组表达的中链解聚酶（mcl-depolymerase）环境中进行孵育。基于聚合物降解的非均相动力学模型开展的分析结果显示，中链解聚酶与PHO的吸附平衡常数较其他体系高出6倍。与之相反，作用于PHB的短链解聚酶的降解速率常数则约为其2倍。最后，本研究明确了所探究的短链与中链聚羟基脂肪酸酯在酶-底物相互作用及降解机制层面的差异。