n-Butyl levulinate (n-BL) has numerous applications in biofuel and green solvents. In this paper, we have reported mechanistic accounts of sonoenzymatic synthesis of n-BL using Novozym 435. Esterification parameters were optimized with statistical design of experiments. Application of 35 kHz sonication boosted n-BL yield from 70.9% to 92.22%. Mechanistic investigation using Ping-Pong Bi-Bi kinetics model and molecular docking simulations revealed interesting influence of sonication on esterification reaction. Exposure to sonication induced significant changes in the secondary structure of enzyme, as revealed in ATR-FTIR analysis. Sonication increased α-helix content of enzyme that increased enzyme activity by opening of flapping lid and widening the catalytic cavity. Sonication also caused unfolding of secondary structural motifs with rise in random coil content that favored formation of enzyme-ligand complexes. These effects enhanced reaction velocity and substrate affinity with reduction in inhibition and unfavorable dissociation of intermediate complexes, which ultimately enhanced n-BL yield.

乙酰丙酸正丁酯 (n-BL) 在生物燃料和绿色溶剂中有着广泛的应用。在本文中，我们报告了使用 Novozym 435 声酶法合成 n-BL 的机理。通过实验的统计设计优化了酯化参数。35 kHz 超声处理的应用将 n-BL 产量从 70.9% 提高到 92.22%。使用 Ping-Pong Bi-Bi 动力学模型和分子对接模拟的机理研究揭示了超声处理对酯化反应的有趣影响。正如 ATR-FTIR 分析所揭示的那样，超声处理会导致酶的二级结构发生显着变化。超声处理增加了酶的 α-螺旋含量，通过打开翻盖和扩大催化腔来增加酶的活性。超声处理还导致二级结构基序展开，随机卷曲含量增加，有利于酶-配体复合物的形成。这些影响提高了反应速度和底物亲和力，减少了中间复合物的抑制和不利的解离，最终提高了 n-BL 产量。