Abstract Bacillus subtilis lipase A (BSLA) has been extensively studied through protein engineering; however, its immobilization and behavior as an insoluble biocatalyst have not been extensively explored. In this work, for the first time, a direct immobilization of recombinant BSLA from microbial culture supernatant was reported, using chemically modified porous with different electrostatic, hydrophobic, hydrophilic, and hydrophilic−hydrophobic enzyme-support interactions. The resulting biocatalysts were evaluated based on their immobilization kinetics, activity expression (pH 7.4), thermal stability (50 °C), solvent resistance and substrate preference. Biocatalysts obtained using glyoxyl silica support resulted in the selective immobilization of BSLA, resulting in an activity recovery of 50 % and an outstanding aqueous stabilization factor of 436, and 9.5 in isopropyl alcohol, compared to the free enzyme. This selective immobilization methodology of BSLA allows to efficiently generate immobilized biocatalysts, thus avoiding laborious purification steps from cell culture supernatant, which is usually a limiting step when large amounts of enzyme variants or candidates are assessed as immobilized biocatalysts. Direct enzyme immobilization from cell supernatant provides an interesting tool which can be used to facilitate the development and assessment of immobilized biocatalysts from engineered enzyme variants and mutant libraries, especially in harsh conditions, such as high temperatures or non-aqueous solvents, or against non-water-soluble substrates. Furthermore, selective immobilization approaches from cell culture supernatant or clarified lysates could help bridging the gap between protein engineering and enzyme immobilization, allowing for the implementation of immobilization steps in high throughput enzyme screening platforms for their potential use in directed evolution campaigns.

中文翻译：

---

从细胞培养上清液中选择性固定枯草芽孢杆菌脂肪酶 A：提高催化性能和耐热性

摘要 枯草芽孢杆菌脂肪酶 A (BSLA) 已通过蛋白质工程得到广泛研究。然而，其作为不溶性生物催化剂的固定化和行为尚未得到广泛探索。在这项工作中，首次报道了使用具有不同静电、疏水、亲水和亲水-疏水酶-支持相互作用的化学修饰多孔直接固定微生物培养上清液中的重组 BSLA。根据其固定动力学、活性表达 (pH 7.4)、热稳定性 (50 °C)、耐溶剂性和底物偏好对所得生物催化剂进行评估。使用乙醛基二氧化硅载体获得的生物催化剂导致 BSLA 的选择性固定，与游离酶相比，活性恢复率为 50%，水溶液稳定因子为 436，在异丙醇中为 9.5。BSLA 的这种选择性固定化方法允许有效地生成固定化生物催化剂，从而避免从细胞培养上清液中进行费力的纯化步骤，当大量酶变体或候选酶被评估为固定化生物催化剂时，这通常是一个限制步骤。来自细胞上清液的直接酶固定提供了一种有趣的工具，可用于促进来自工程酶变体和突变文库的固定化生物催化剂的开发和评估，特别是在恶劣条件下，如高温或非水溶剂，或针对非-水溶性基质。此外，