Engineering access tunnel dynamics of Candida antarctica lipase B enhances esterification of multiple bulky substrates.

IF 4 2区农林科学 Q1 AGRICULTURE, MULTIDISCIPLINARY

Journal of the Science of Food and Agriculture Pub Date : 2026-10-27 DOI:10.1002/jsfa.71204

Wenjun Cheng, Wenya Chong, Binbin Nian, Xingjiang Li, Xing Sun

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引用次数: 0

Abstract

Background: Lipase-catalyzed esterification in organic and solvent-rich systems offers a sustainable route for producing high-value esters, but conversion is often limited by accumulation of water generated during esterification and the steric demands of bulky substrates.

Results: Here, molecular dynamics (MD)-guided access-tunnel engineering was combined with a copper-soap high-throughput colorimetric assay to improve Candida antarctica lipase B (CALB)-catalyzed esterification. The previously reported thermostable variant M1 (L147S/T244P/S250P/T256D/N292D) was used as template to obtain an improvement of both activity and thermostability. M4 (L147S/T244P/S250P/T256D/N292D/L140W/L144F/I285F/V286L) was the best-performing variant and showed more than 4.5-fold higher volume-normalized apparent activity than wild-type (WT) in bulky-substrate esterifications, including phytosteryl oleate, glucose laurate, sucrose laurate, and α-tocopheryl succinate synthesis. Because succinic-anhydride acylation does not generate stoichiometric water, α-tocopheryl succinate synthesis was used only as a bulky-substrate-access probe. Structural and MD analyses indicated that substitutions near Trp140/Phe144 increased lid-region flexibility and remodeled the tunnel microenvironment, consistent with a putative relay-style water-transport model involving active-site Ser105 and tunnel-entrance Trp140. Immobilized M4@AOMR further achieved 98.6% conversion in preparative sucrose stearate synthesis.

Conclusion: These results show that coordinated regulation of tunnel dynamics, interactions with reaction-generated water, and immobilized-enzyme performance is a potential strategy for ester synthesis; however, the steric and water-transport contributions were not quantitatively separated. © 2026 Society of Chemical Industry.

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南极假丝酵母脂肪酶B的工程通道动力学增强了多种大体积底物的酯化。

背景：脂肪酶催化的酯化反应在有机和溶剂丰富的体系中为生产高价值酯提供了一条可持续的途径，但转化往往受到酯化过程中产生的水的积累和大体积底物的空间需求的限制。结果：本研究将分子动力学（MD）引导下的通道工程与铜皂高通量比色法相结合，改善了南极念珠菌脂肪酶B （CALB）催化的酯化反应。先前报道的耐热型M1 （L147S/T244P/S250P/T256D/N292D）被用作模板，以获得活性和热稳定性的改进。M4 （L147S/T244P/S250P/T256D/N292D/L140W/L144F/I285F/V286L）表现出比野生型（WT）高4.5倍以上的体积标准化表观活性，包括油酸植物甾醇酯、葡萄糖月桂酸盐、月桂酸蔗糖和α-生育酚琥珀酸酯的合成。由于琥珀酸酐酰化不产生化学计量水，α-生育酚琥珀酸酯合成仅用作大体积底物进入探针。结构和MD分析表明，Trp140/Phe144附近的替换增加了盖子区域的灵活性，并重塑了隧道微环境，这与假设的接力式水运模型一致，该模型涉及活性位点Ser105和隧道入口Trp140。固定化M4@AOMR进一步达到98.6%的转化率制备硬脂酸蔗糖合成。结论：这些结果表明，协调调节隧道动力学、与反应生成水的相互作用和固定化酶的性能是一种潜在的酯合成策略；然而，空间和水运输的贡献并没有在数量上分开。©2026化学工业协会。

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来源期刊

Journal of the Science of Food and Agriculture 工程技术-农业综合

CiteScore

8.10

自引率

4.90%

发文量

634

审稿时长

3.1 months

期刊介绍： The Journal of the Science of Food and Agriculture publishes peer-reviewed original research, reviews, mini-reviews, perspectives and spotlights in these areas, with particular emphasis on interdisciplinary studies at the agriculture/ food interface. Published for SCI by John Wiley & Sons Ltd. SCI (Society of Chemical Industry) is a unique international forum where science meets business on independent, impartial ground. Anyone can join and current Members include consumers, business people, environmentalists, industrialists, farmers, and researchers. The Society offers a chance to share information between sectors as diverse as food and agriculture, pharmaceuticals, biotechnology, materials, chemicals, environmental science and safety. As well as organising educational events, SCI awards a number of prestigious honours and scholarships each year, publishes peer-reviewed journals, and provides Members with news from their sectors in the respected magazine, Chemistry & Industry . Originally established in London in 1881 and in New York in 1894, SCI is a registered charity with Members in over 70 countries.