石油降解酶的筛选、固定化及添加表面活性剂强化其对石油的降解

在石油开采和加工过程中产生的含油污泥是石油化工行业中主要的污染源，会对周边生态环境造成持续性的危害。生物法降解被认为是一种有效且可持续的技术而备受关注。目前的研究多聚焦在微生物法降解石油，而酶法降解鲜有报道。【目的】本研究旨在优选石油高效降解酶，并深入分析其降解特性，以期构建一套酶法降解石油的体系。【方法】基于分子对接模拟技术，分析酶与石油类常见底物的结合可能性及结合模式，通过石油降解实验优选降解酶；通过添加表面活性剂及酶固定化进一步提高酶对石油的降解效果。【结果】利用分子对接模拟及实验验证获得5种具有石油降解能力的生物酶，其中枯草芽孢杆菌(Bacillus subtilis)来源的漆酶BsLac对石油的降解率最高，72 h可降解34.1%的石油；进一步研究发现，表面活性剂的添加均可促进BsLac对石油的降解，其中槐糖脂的促进作用尤为显著。当槐糖脂终浓度为1 000 mg/L时，石油的降解率最高，为46.3%；然而，2,2''-联氮-双(3-乙基苯并噻唑啉-6-磺酸)二铵盐的添加对BsLac降解石油的促进作用并不明显；固定化结果表明，以花生壳为吸附载体固定化的BsLac对石油的降解率最高，为56.3%。【结论】本研究基于分子对接模拟和实验筛选获得了石油降解酶BsLac，固定化的BsLac可实现石油的高效降解，为生物酶法降解石油的进一步探索奠定了实验和理论基础。

Petroleum-degrading enzymes: screening and performance improvement by immobilization and surfactants

The oily sludge produced in petroleum exploitation and processing is the main pollution source in the petrochemical industry, causing continuous harm to the surrounding eco-environment. Bioremediation as an effective and sustainable technology has attracted much attention. However, the current studies focus on the microbial degradation of petroleum in oily sludge and rarely report enzymatic degradation. Objective] This study aims to screen petroleum-degradation enzymes by computer simulation and experimental techniques and improve the degradation effect by adding surfactants and enzyme immobilization. Methods] Molecular docking was employed to analyze the possibility and mode of binding of target enzymes to common substrates in petroleum and the strongest degrading enzyme was screened out by enzymatic degradation experiments. Furthermore, the degradation conditions of the enzyme screened out were optimized, and the degradation effect on petroleum was further improved by immobilization and addition of surfactants. Results] A total of five petroleum-degrading enzymes were obtained by molecular docking simulation and experimental verification. Among them, Bacillus subtilis laccase (BsLac) exhibited the highest degradation rate of petroleum, which reached 34.1% at the time point of 72 h. Surfactants improved the degradation of BsLac on petroleum, and sophorolipid showcased the strongest promoting effect, with the highest degradation rate of 46.3% at the sophorolipid concentration of 1 000 mg/L. However, 2,2''-azinoo-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS) did not present promoting effect on BsLac for petroleum degradation. The BsLac immobilized by peanut shell as the adsorption carrier exhibited the highest petroleum degradation rate (56.3%). Conclusion] We screened out the strongest petroleum-degrading enzyme by molecular docking and experimental verification. Furthermore, the immobilization of BsLac can improve the degradation performance on petroleum. The findings lay an experimental and theoretical foundation for further exploring the enzymatic degradation of petroleum.