生物技术通报 ›› 2023, Vol. 39 ›› Issue (4): 1-9.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1305
• 酶工程专题 • 下一篇
收稿日期:
2022-10-25
出版日期:
2023-04-26
发布日期:
2023-05-16
通讯作者:
曲戈,男,博士,副研究员,研究方向:酶理性设计与工程生物学;E-mail: qug@tib.cas.cn;基金资助:
Received:
2022-10-25
Published:
2023-04-26
Online:
2023-05-16
摘要:
作为生物催化剂,酶蛋白介导的生化反应具有条件温和、绿色环保等优点。然而相比化学催化剂,天然酶功能的局限性制约了它在生物制造领域的广泛应用。前期研究表明,酶蛋白除了催化专一性外,同时还展现出混杂性的一面,可在特定条件下催化非天然模式反应。这一特性为酶分子功能重塑提供了新思路,可用来指导人工酶设计,拓展天然酶的催化边界,实现新颖酶促反应类型,以扩大酶催化应用场景。本文从酶催化功能混杂性背后可能的进化机制入手,综述了当前诱导酶催化功能混杂性的常用策略,如定向进化、构象动力学、反应条件诱导及祖先酶重构等技术,并从催化机制、构效关系及适应性进化等多个角度,结合近年来相关研究实例,探讨了催化功能混杂性背后的分子机制,为突破天然酶促反应局限性、创制催化非天然反应的高效人工酶元件提供参考。
曲戈, 孙周通. 催化混杂性驱动的酶功能重塑[J]. 生物技术通报, 2023, 39(4): 1-9.
QU Ge, SUN Zhou-tong. Catalytic Promiscuity-driven Redesign of Enzyme Functions[J]. Biotechnology Bulletin, 2023, 39(4): 1-9.
图2 人工Kemp消除酶设计 a:Kemp消除反应方程式;b:基于不同模板获得的KE07、KE59及KE70,其中KE59以KE59 R1 7/10H突变体结构展示;c:基于木聚糖酶设计的HG3
Fig. 2 Design of artificial Kemp elimination enzymes a: Formula of Kemp elimination reaction. b: KE07, KE59 and KE70 designed from three distinct templates. KE59 is depicted using the crystallographic structure of its mutant R1 7/10H. c: HG3 designed from a xylanase
Template | Enzyme | kcat(s-1) | Km(mM-1) | kcat/Km(M-1s-1) | Reference |
---|---|---|---|---|---|
裂合酶等 | KE07 | 0.018 | 1.4 | 12.2 | [ |
KE59 | 0.29 | 1.8 | 163 | [ | |
KE70 | 0.16 | 2.1 | 78.3 | [ | |
KE07.7 | 1.37 | 0.54 | 2 590 | [ | |
KE59.13 | 9.53 | 0.16 | 60 430 | [ | |
KE70.6 | 5.0 | 0.088 | 57 300 | [ | |
木聚糖酶 | HG3 | 0.68 | 1.6 | 425.0 | [ |
HG3.17 | 700 | 3.0 | 230 000 | [ | |
P450 BM3 | P450-BM3 | 1.5 | 6 | 240 | [ |
A82F | 8.4 | 0.27 | 31 000 | [ |
表1 人工Kemp消除酶相关代表性突变体列表
Table 1 Representative mutants related to artificial Kemp elimination enzymes
Template | Enzyme | kcat(s-1) | Km(mM-1) | kcat/Km(M-1s-1) | Reference |
---|---|---|---|---|---|
裂合酶等 | KE07 | 0.018 | 1.4 | 12.2 | [ |
KE59 | 0.29 | 1.8 | 163 | [ | |
KE70 | 0.16 | 2.1 | 78.3 | [ | |
KE07.7 | 1.37 | 0.54 | 2 590 | [ | |
KE59.13 | 9.53 | 0.16 | 60 430 | [ | |
KE70.6 | 5.0 | 0.088 | 57 300 | [ | |
木聚糖酶 | HG3 | 0.68 | 1.6 | 425.0 | [ |
HG3.17 | 700 | 3.0 | 230 000 | [ | |
P450 BM3 | P450-BM3 | 1.5 | 6 | 240 | [ |
A82F | 8.4 | 0.27 | 31 000 | [ |
图3 构象动力学指导的TbSADH酶改造 a:催化反应示意图;b:底物结合口袋关键位点的运动性分析;c-d:A85及I86两位点突变后(c)与突变前(d)所在loop区域柔性分析;e:关键位点P84的空间位置;f-h:野生型(f)与两个最优突变体ΔP84/A85G(g)及P84S/I86L(h)的构象自由能路径分布
Fig. 3 Conformational dynamics-guided modification of enzyme TbSADH a: Schematic representation of catalyzed reaction. b: Dynamics analysis of the substrate binding pocket. c-d: Flexibility exploration of the loop region that accommodates residues A85 and I86 after(c)and before mutagenesis(d). e: Position of residue P84. f-h: Free energy landscapes(kcal/mol)of wild type(f), ΔP84/A85G(g)and P84S/I86L(h)
图4 羧酸还原酶介导的混杂性催化反应类型 a:氧化还原反应;b:分子间酰胺化反应;c:分子间酯化反应;d:分子内酰胺化反应
Fig. 4 Promiscuous reactions mediated by carboxylic acid reductase a: Redox reaction; b: intermolecular amidation; c: intermolecular esterification; d: intramolecular lactamization
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