Research Progress on the Mechanisms of Decreasing Catalytic Activity of Lipase in the Production of Structured Lipids Synthesized by Enzymatic Method
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摘要: 结构脂质是一类将具有特殊生理功能的脂肪酸结合到甘油三酯骨架上特定位置重新组合生成的新型脂质。酶法催化合成结构脂质具有反应条件温和，能耗低，分离纯化工艺简单，以及可以合成特定酰基位置上的功能性脂肪酸而受到重视。工业上随着酯酶使用次数的增加，酯酶催化活力显著下降，导致目标结构脂质产量降低。本文综述了近年来国内外关于酯酶催化合成结构脂质酯酶结构改变、活性变化和催化活性下降机理，酶活性下降主要是因为分子间强相互作用或弱相互作用破坏酶催化活性中心，本研究可为解决酶法合成结构脂质面临的酯酶活力降低的技术瓶颈提供参考。Abstract: Structured lipids are a new class of lipids recombined by combining fatty acids with special physiological functions at specific positions on the triglyceride backbone. Enzymatic catalyzed synthesis of structured lipids has attracted attention due to its mild reaction conditions, low energy consumption, simple separation and purification process, and the ability to synthesize functional fatty acids at specific acyl positions. However, with the increase of use times of lipases in industry, the catalytic activity of lipase decreases significantly, resulting in a decrease in the production of target structural lipids. This paper reviews the structure change, activity change and decreasing catalytic activity mechanisms of lipase-catalyzed synthesis of structured lipids in China and outside China in recent years. It found that the decrease of enzyme activity is mainly due to the destruction of the catalytic active center by strong or weak intermolecular interaction. This study provides a reference for solving the technical bottleneck of reducing lipase activity in the enzymatic synthesis of structured lipids.
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图 1 脂肪酶合成结构脂质的催化机理
Figure 1. The reaction mechanism of structured lipid synthesis catalyzed by lipase
图 2 有机溶剂分子对脂肪酶结构的影响
Figure 2. Influence of organic solvent molecules on the structure of lipase
注：酶蛋白分子来源南极假丝酵母脂肪酶B（CALB，ID:1LBS，http://www.rcsb.org/pdb/home/home.do）。
图 3 常见离子液体阴阳离子结构示意图
Figure 3. Schematic diagram of the anion and cation structures of common ionic liquids
图 4 外界环境和脂肪酶活性降低的关联分析
Figure 4. Correlation analysis between the external environment and the reduction of lipase activity
表 1 有机溶剂对脂肪酶结构的影响及催化活性降低相关机制
Table 1. The effects of organic solvents on the structure of lipase and the related mechanism of catalytic activity reduction
脂肪酶 有机试剂 方法 结构变化 活性变化 机制 参考文献 Candida antarctica
lipase A乙腈、丙酮、
乙醇、乙酯，
正庚烷傅里叶红外
光谱用乙腈或丙酮处理CALA，α-螺旋含量增加，无规线圈含量减少。然而，当用乙酯处理时，发现α-螺旋含量减少，β-折叠含量增加 α-螺旋含量的降低可能会
影响CALA的活性位点，
从而对其活性产生
重要影响β-折叠物含量的增加可归因于水分子与蛋白质表面之间氢键相互作用的丧失，从而导致处理后的CALA具有更不稳定的结构 Yang等[30] Penicillium chrysogenum lipase 极性和非极性有机溶剂 圆二色性和
本征荧光光谱天然脂肪酶由一个主要的α-螺旋结构组成，该结构在极性和非极性溶剂中均保持不变，丁酸乙酯除外，丁酸乙酯的
活性降低，结构被破坏在己烷存在下（log P=3.5），螺旋含量增加（~85% α-螺旋），表明脂肪酶的
结构稳定性增强在二氯甲烷存在下观察到显著的荧光猝灭，可能是由于表面色氨酸残基埋藏在蛋白的疏水核心中。丁醇和乙醇导致脂肪酶三级结构发生显著变化，并伴随红移 Sadaf等[31] Candida antarctica
lipase B非极性溶剂如氯仿、环戊烷、己烷；极性溶剂如甲醇、丙酮、四氢呋喃 分子动力学
模拟和量子
力学模拟在有机溶剂中，CALB的整体构象是稳定的。在非极性溶剂中，活性中心的构象保持
稳定氯仿、环戊烷、己烷等非极性溶剂保持活性中心的稳定性，而在极性溶剂中，溶剂分子进入活性中心并与活性中心发生强烈的相互作用 甲醇、丙酮、四氢呋喃等极性溶剂通过氢键与活性中心相互作用，从而破坏活性中心。活性位点区域的构象变化是影响CALB活性的主要因素 Li等[32] PML （Proteus mirabilis） and
Lip S （Pseudomonas mandelii）极性溶剂如二甲基亚砜、甲醇和乙醇 荧光光谱 在PML和Lip S之间，最大酶活性所需的构象灵活性不同，表明PML和Lip S在每种有机溶剂中的构象可获得最大酶活性 极性有机溶剂如DMSO甲醇和乙醇，会导致PML和Lip S的构象变化，从而增加构象灵活性；由于有机溶剂中构象的灵活性增加，PML和Lip S的结构变得不稳定 PML和Lip S的构象变化是由于表面不同氨基酸排列不同，在DMSO存在下构象相似，但在甲醇和乙醇中构象不同 Dachuri等[33] Y. lipolytica lipase 甲醇和己烷 分子动力学
模拟脂肪酶在甲醇中的构象
变化较大Ser274-Asn288和Thr106-His126等区域与lid区域相互作用 Y. lipolytica脂肪酶的闭合机制是在甲醇中的“盖子”运动 Jiang等[34] Double mutant porcine pancreatic lipase 乙醇、甲苯、
辛醇分子动力学
模拟Asp250Val和Glu254Leu突变体在较高温度下表现出“盖子”打开 Asp250Val和Glu254Leu在保持“盖子”处于闭合状态中的重要作用 在辛醇中酯酶“盖子”打开的动力学比在水中快，非极性溶剂有利于“盖子”的开放构造 Haque等[35] chimeric C. rugosa LIP1/lid3 氯乙基-2-羟基己酸酯、甲醇、醋酸乙烯酯与6-甲基-5-庚烯-2-醇 醇解反应 与野生型相比，嵌合C. rugosa LIP1/lid3的活性和对映选择性更低 在有机溶剂中，与野生型相比，嵌合体C. rugosa LIP1/lid3对氯乙基-2-羟基己酸酯与甲醇的醇解反应和醋酸乙烯酯与6-甲基-5-庚烯-2-醇的醇解反应的活性和对映选择性都较低 活性的降低可能是由于嵌合体酶具有较低比例的开放形式的酶分子，阻碍了对酶活性位点的接触 Secundo等[36] Burkholderia cepacia lipase （BCL） 叔戊醇、叔丁醇、石油醚、正己烷和异辛烷 傅里叶红外
光谱，CD在选择性有机溶剂和RTIL中，BCL的二级结构发生了一些改变 BCLα螺旋含量的降低可能
影响脂肪酶活性位点α-螺旋含量越低，活性位点的“开放”构象越高，从而更容易接近底物 Liu等[37] Geobacillus zalihae T1 lipase 甲醇、乙醇、丙醇、丁醇和戊醇）和水的混合物 分子动力学
模拟骨架原子结构偏差阐明水/有机溶剂混合物在降低溶剂极性时对蛋白质平衡状态的动态影响 有机溶剂Mt-OH、Et-OH和Pr-OH中形成更多氢键，蛋白质内氢键的增加最终与蛋白质的稳定性相关 在有机溶剂体系中，活性口袋四面体中间填料的距离变化不守恒，这可能导致催化氢键网络的弱点，很可能导致催化活性下降 Maiangwa等[38] 
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表 2 离子液体对脂肪酶结构的影响及催化活性降低相关机制
Table 2. The effects of ionic liquids on the structure of lipase and the related mechanism of catalytic activity reduction
脂肪酶 离子液体 方法 结构变化 活性变化 机制 参考文献 Burkholderia cepacia lipase （BCL） [OMIM][Cl]，[EMIM][TfO]，[BMIM][Cl]，[OMIM][BF4]，
[BMIM][CH3SO3]，
[EMIM][Cl]，[NMP][CH3SO3]，
[HMIM][TfO]，
[HMIM][CH3SO3]，
[BMIM][PF6]，
[BMIM][OH]，[EMIM][PF6]， [HMIM][Cl]，[BMIM][Tf2N]，
[BMIM][BF4]，[OMIM][PF6]，
[EMIM][BF4]，[HMIM][PF6] and [OmPy][BF4]傅立叶红外
光谱室温离子液体中的BCL二级结构发生部分改变 阴离子对酯交换活性的影响远大于阳离子 [EMIM][Tf2N]会压缩酶RTIL的天然结构，从而阻止其展开 Liu等[37] Candida antarctica
lipase B[BMIM][NO3]，[BMIM][lactate]，
[EMIM][Et SO4]
and [Et NH3][NO3]傅立叶红外
光谱结构相似的离子液体对酶的行为有很大不同 分子大、空间要求高的离子不容易穿透蛋白质基质，需要许多分子间氢键解离来产生一些新的氢键，维持活性 分子大、空间要求高的离子不容易穿透蛋白质基质，需要许多分子间氢键的解离来产生部分新的氢键，维持酶活性 Lau等[40] Bacillus amyloliquefaciens and Bacillus lichiniformis [BMIM][Cl]，[HMIM][Cl] 差示扫描量热法和荧光 与中温酶相比，嗜热酶更稳定 随着离子液体浓度的变化，酶的活性和稳定性都会降低 根据解淀粉芽孢杆菌和地衣芽孢杆菌热展开过程中获得的热谱图，[HMIM][Cl]的抗聚集能力强 Dabirmanesh等[41] Rhizopus oryzae NRRL 3562 1-Hexadecyl-3-methylimidazolium bromide （（C16 MIM）Br） 紫外可见光谱和圆二色谱 在低表面活性剂浓度下，导致β-折叠物含量增加和α-螺旋降低的结构变化 高浓度下的失活与光谱所描述的更大结构变化相关。等温滴定量热研究表明，这种结合在本质上是自发的，涉及非共价相互作用 高的熵负值表示疏水域的暴露和结构刚性的增加，与表面活性剂存在时活性位点更易接近和刚性相关 Adak等[42] Candida rugosa, Chromobacterium viscosum and Thermomyces lanuginosa Water-in-ionic liquid （W/IL） microemulsions/1-butyl-3-methylimidazolium hexafluorophosphate （[BMIM]PF6） 傅立叶红外光谱和圆二色谱 与迄今为止用于各种生物催化反应的其他微多相介质相比，在新体系中，来自C. rugosa、C. viscosum和T. lanuginosa的脂肪酶表现出更高的催化性能和操作稳定性 与传统有机溶剂相比，W/IL微乳液中酶的稳定性增强，易于从反应体系中分离产物，酶的可重复使用性好，以及离子液体独特的溶剂性质 将酶分子包埋在W/IL微乳液中形成的水微滴中，表明基于IL的反应系统为酶提供保护环境 Pavlidis等[43] Candida antarctica
lipase Btert-Butyl alcohol，[BMIM][BF4]，
[BMIM][PF6]，
[BMIM][lactate]，
[BMIM][NO3]，
[Et3Me N][Me SO4]傅立叶红外光谱 BMIM[dca]中α-螺旋和β-折叠含量的损失 在含有二氰酰亚胺、烷基硫酸盐、硝酸盐和乳酸阴离子的离子液体中溶解CALB，反应至少比[BMIM][BF4]慢十倍。[Et3Me N][Me SO4]是例外，溶解的CALB在该溶剂中保持其活性 在[BMIM][dca]中，CALB的交联酶聚集体的活性是叔丁醇中的两倍，而游离酶在叔丁醇离子液体中不可逆地失活。形成强氢键的离子液体，尤其是阴离子，可能会解离维持α-螺旋和β-折叠结构完整性的氢键，导致蛋白质全部或部分展开 Pavlidis等[43] Candida antarctica
lipase Bwater-immiscible ionic liquids （ILs），1-ethyl-3-methylimidizolium bis
（trifluoromethylsulfonyl）-
imide and butyltrimethylammonium bis（trifluoromethylsulfonyl）imide圆二色谱和荧光光谱 在50 °C的水和己烷培养基中培养4 d后，CALB的剩余活性损失超过75%，这与α-螺旋和-链二级结构的巨大变化有关 在所研究的两个ILs中观察到的CALB的稳定与维持50%的初始α-螺旋含量和增强-链有关。此外，内在荧光研究清楚地表明，在水和己烷介质中，经典酶的去折叠是如何随时间发生 酶在两种ILs中的培养相关的结构变化可能归因于紧凑而活跃的酶构象，从而提高在非水环境中的稳定性 De等[44] Candida antarctica （Novozyme 435） [BMIM][BF4]，[HMIM][BF4]，[OMIM][BF4]，[BMIM][PF6]，[HMIM][PF6]，[OMIM][PF6]，[EMIM][Tf2N]，[BMIM][Tf2N]，[HMIM][Tf2N]，
[OMIM][Tf2N]，
[BMMIM][Tf2N]，
[HMMIM][Tf2N]，
[OMMIM][Tf2N]，[BMIM][N（CN）2]，[HMIM][N（CN）2]，[BMIM][OAc]傅立叶红外
光谱随着浸泡时间增加，α-螺旋含量降低，β-折叠含量增加 酶435在[BF4]、[PF6]和[TF2N]基离子液体中浸泡24 h后，保留约5%、40%
和100%的空白样品活性，揭示离子液体性质的初始酶活性与疏水性（log P）、极性（Et）、氢键碱性（B）和粘度有关。初始酶活性随log P值增加而增加，随ENT值降低而降低初始酶活性在较窄的范围内（0.24~0.26）随b值的增加而增加，然后随b值的增加而持续下降。对于基于[Tf2N]的离子液体，初始酶活性随粘度增加而增加，而在基于[BF4]
和[PF6]的离子液体中发现了不利关系。诺维信435α-螺旋含量的降低可能会影响脂肪酶活性位点。α-螺旋含量越低，底物越容易接近脂肪酶的活性部位Qin等[45] Candida antarctica lipase B （CALB） [HOOCMMIM][Cl]，
[HOOCEMIM][Cl]，
[HOOCBMIM][Cl]，
[HOOCBMIM][H2PO4]，
[HOOCMMIM][PF6]傅立叶红外光谱，圆二色谱和衰减总反射FTIR 与天然酶相比，所有修饰酶的α-螺旋含量均较高 改性在水相中获得更高的活性和催化效率，而含有更多潮向阳离子的ILs改性导致了更高的改性程度和活性 修饰增强脂肪酶的稳定性，尤其是带有疏水性阳离子的离子液体，而接枝到脂肪酶上的疏水性阴离子有助于提高脂肪酶的热稳定性和对有机溶剂的耐受性 Ru等[46] Aspergillus niger lipase [C4MIM]Cl，[C6MIM]Cl，[C8MIM]Cl，[C10MIM]Cl
and [C12MIM]Cl荧光和圆二色光谱 在0.3%（v/v）的[C10MIM]Cl和[C12MIM]Cl水溶液中暴露脂肪酶，不受IL浓度的影响，会导致脂肪酶二级结构发生变化，表明其椭圆度降低 [CnMIM]基于Cl的ILs可以对脂肪酶的活性（增强、维持甚至抑制）和结构构象施加不同的行为，取决于阳离子烷基链长度及其相对浓度 具有自聚集特性的ILs引起蛋白质结构的不可逆变化，导致蛋白质展开 Nascimento
等[47]
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表 3 超临界二氧化碳对脂肪酶结构的影响及催化活性降低相关机制
Table 3. The effect of supercritical carbon dioxide on the structure of lipase and the related mechanism of catalytic activity reduction
脂肪酶 超临界流体 条件/方法 结构变化 活性变化 机制 参考文献 Pseudomonas fluorescens，Rhizopus javanicus，Rhizopus niveus，porcine pancreas and Candida rugosa 超临界二氧化碳
（SC-CO2）100 bar，40 oC 后期结构稳定性降低 活性下降 SC-CO2分子和酶之间的相互作用；SC-CO2插入到酶和水分子之间 Habulin[50] Candida rugosa lipase（CRL） 亚临界或超临界
二氧化碳傅立叶变换红外光谱和荧光发射光谱 α-螺旋含量改变 在大多数高压处理中，脂肪酶活性显著增强 超临界流体对其酶刚性结构的保护 Chen[51] Lipozyme IM20 SC-CO2，加压/减压循环、减压速率和暴露 酶催化活性 - Lipozyme IM20在重复暴露于SC-CO2和低降压率后表现出最佳活性 提取固定化基质中的抑制成分 Aucoin[52] Palatase 20000 L，Lipozyme CALB L，Lipozyme RM IM and Lipozyme 435 SC-CO2 荧光发射光谱，扫描电子显微照片，红外光谱 蛋白结构发生改变，
形态改变高温度和强压力以及最长的暴露时间导致活性损失 SC-CO2处理可以通过构象变化和结构改变来影响酶的活性 Melgosa[53]
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