脂肪酶底物结合口袋的分子改造策略及进展

doi:10.13560/j.cnki.biotech.bull.1985.2020-0123

摘要/Abstract

摘要：

脂肪酶作为重要的酶制剂之一,因其对映选择性、立体选择性和广泛的底物特异性等特性被广泛地运用在工业生产中。为了提高脂肪酶的酶学特性和理化性质、迎合更高的工业需求,除了从酶反应的条件上进行优化,还可对脂肪酶进行分子改造,从根源上提高酶的催化效率。一般常采用半理性设计或理性设计的策略,在底物结合口袋或“盖子”结构附近进行酶分子的改造,以达到改变脂肪酶的底物特异性、立体选择性、对映选择性和区域选择性等目的。对近几十年来关于脂肪酶底物结合口袋的分子改造研究进行了综述,以期为今后的相关研究工作提供研究思路。

关键词: 脂肪酶, 底物结合口袋, 半理性设计, 理性设计

Abstract:

Lipase is one of the important enzymes and widely used in industrial processes due to its high enantioselectivity,stereoselectivity and broad substrate specificity. In addition to optimize the reaction conditions of lipases,people can also molecularly modify the lipase and essentially improve the catalytic efficiency in order to improve the enzymatic and physicochemical properties of lipase as well as to meet the high industrial demand. Generally,semi-rational design and rational design are often applied in molecular modification of binding pocket or “lid” domain of lipases for changing their substrate specificities,stereoselectivities,enantioselectivities,regioselectivities,etc. In this article,the molecular modification of lipase substrate binding pocket in recent decades is reviewed,aiming at providing some clues for relevant researches in the future.

Key words: lipase, substrate binding pocket, semi-rational design, rational design

蔡玉贞, 白巧燕, 苏敏, 唐良华. 脂肪酶底物结合口袋的分子改造策略及进展[J]. 生物技术通报, 2020, 36(11): 173-180.

CAI Yu-zhen, BAI Qiao-yan, SU Min, TANG Liang-hua. Strategies and Advances in the Molecular Modification of Substrate Binding Pocket of Lipase[J]. Biotechnology Bulletin, 2020, 36(11): 173-180.

图/表 2

参考文献 22

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	改造策略	脂肪酶	酶性质的改变	突变位点	突变结果	参考文献
理性设计	改变氨基酸侧链大小	Malassezia globosalipase（MgMDL2）	位置选择性	F278 E282	F278A和E282A获得水解三酰甘油的能力	[3]
		Candida Antarcticalipase A（CALA）	底物链长偏好	G237	G237V 和G237Y对pNP-C4：0和pNP-C6：0水解活性显著提高,其中G237Y对pNP-C6：0的水解活性高WT的3倍	[5]
		Lipase from Streptomyces sp. strain W007（MAS1）	底物链长偏好	H108 F153 V233	H108A、F153A和V233A对pNP-C8的k_cat/K_m分别是WT的2.3、2.1、1.4倍;对pNP-C16的比活分别比WT提高了3.0、2.2、2.0倍	[6]
		Candida antarcticalipase B（CALB）	立体选择性	W104	对heptan-4-ol和 nonan-5-ol的k_cat/K_m分别提高了270倍和5 500倍	[8]
	改变底物结合口袋的亲、疏水性	Rhizopus chinensislipase（RCL）	底物链长、不饱和脂肪酸偏好	H284 L285	突变体HQL（H284 和L285之间插入Q）和L285Q对p-NPP（C16）的催化活性分别为WT的2.72倍和1.5倍;HQL水解不饱和脂肪酸的活性是饱和脂肪酸1.45倍,而WT只有1.10倍	[7]
		Geobacillus zalihaelipase（T1 lipase）	对映选择性	Q114	Q114M催化（R,S）-布洛芬与油酸醇的选择性酯化反应中E值为WT的3.2倍	[9]
		Geobacillus zalihae lipase（T1 lipase）	催化活性	Q114	Q114L催化的丁酸薄荷酯的酯化反应的转化率能够达到92％	[10]
	增加π环体系的非经典相互作用	Rhizomucor mieheilipase（RML）	催化活性	P209 L258	对硝基苯酯（C6）的比活提高了3.98倍	[14]
半理性设计	定点饱和诱变	Candida rugosalipase（LIP2）	底物链长偏好	L132	L132A和L132I的底物特异性表现出向中长链的脂肪酸转移	[15]
	组合活性中心饱和突变（CAST）/简并密码子NDT	Candida Antarcticalipase A（CALA）	对映选择性	F233	以2-苯基丙酸对硝基苯基酯为底物的催化反应中,F233G的E值高达259	[16]
	迭代饱和突变（ISM）	Bacillus subtilislipase（Lip A）	热稳定性	R33/D34/K35 K112/ M134 Y139/I157	以4-硝基苯基辛酸酯底物测得两个最佳突变体（M134D/I157M/Y139C/K112D/R33Q/D34N/K35D）和（M134D/I157M/Y139C/K112D /R33G）的T₅₀⁶⁰分别为89℃和93℃,远高于WT（T₅₀⁶⁰为48℃）	[17]
	ISM/简并密码子NDT	Candida antarcticalipase B（CALB）	立体选择性	W104/L144 V149/V154 I189/V190 A281/A282	（V149D/I189V/V190C/A281G/A282V）对2-苯基丙酸对硝基苯酯的k_cat / K_m为WT的近277倍,突变体（W104C/L144Y/V149I/V154I/A281C/A282F）对R-2-苯基丙酸对硝基苯酯的活性比WT高15倍	[18]
	理性聚焦迭代定点诱变（FRISM）	Candida antarcticalipase B（CALB）	对映选择性	W104/ D134 Q157/I189 V190/ A281 A282	突变体（A281G/A282V/V190C）、（Q157L/I189A）、（W104/I189）、（W104A/I189M/V190C/D134L）对各自的最佳对映体的选择性分别高达95%、94%、95%和91%	[19]

	改造策略	脂肪酶	酶性质的改变	突变位点	突变结果	参考文献
理性设计	改变氨基酸侧链大小	Malassezia globosalipase（MgMDL2）	位置选择性	F278 E282	F278A和E282A获得水解三酰甘油的能力	[3]
		Candida Antarcticalipase A（CALA）	底物链长偏好	G237	G237V 和G237Y对pNP-C4：0和pNP-C6：0水解活性显著提高,其中G237Y对pNP-C6：0的水解活性高WT的3倍	[5]
		Lipase from Streptomyces sp. strain W007（MAS1）	底物链长偏好	H108 F153 V233	H108A、F153A和V233A对pNP-C8的k_cat/K_m分别是WT的2.3、2.1、1.4倍;对pNP-C16的比活分别比WT提高了3.0、2.2、2.0倍	[6]
		Candida antarcticalipase B（CALB）	立体选择性	W104	对heptan-4-ol和 nonan-5-ol的k_cat/K_m分别提高了270倍和5 500倍	[8]
	改变底物结合口袋的亲、疏水性	Rhizopus chinensislipase（RCL）	底物链长、不饱和脂肪酸偏好	H284 L285	突变体HQL（H284 和L285之间插入Q）和L285Q对p-NPP（C16）的催化活性分别为WT的2.72倍和1.5倍;HQL水解不饱和脂肪酸的活性是饱和脂肪酸1.45倍,而WT只有1.10倍	[7]
		Geobacillus zalihaelipase（T1 lipase）	对映选择性	Q114	Q114M催化（R,S）-布洛芬与油酸醇的选择性酯化反应中E值为WT的3.2倍	[9]
		Geobacillus zalihae lipase（T1 lipase）	催化活性	Q114	Q114L催化的丁酸薄荷酯的酯化反应的转化率能够达到92％	[10]
	增加π环体系的非经典相互作用	Rhizomucor mieheilipase（RML）	催化活性	P209 L258	对硝基苯酯（C6）的比活提高了3.98倍	[14]
半理性设计	定点饱和诱变	Candida rugosalipase（LIP2）	底物链长偏好	L132	L132A和L132I的底物特异性表现出向中长链的脂肪酸转移	[15]
	组合活性中心饱和突变（CAST）/简并密码子NDT	Candida Antarcticalipase A（CALA）	对映选择性	F233	以2-苯基丙酸对硝基苯基酯为底物的催化反应中,F233G的E值高达259	[16]
	迭代饱和突变（ISM）	Bacillus subtilislipase（Lip A）	热稳定性	R33/D34/K35 K112/ M134 Y139/I157	以4-硝基苯基辛酸酯底物测得两个最佳突变体（M134D/I157M/Y139C/K112D/R33Q/D34N/K35D）和（M134D/I157M/Y139C/K112D /R33G）的T₅₀⁶⁰分别为89℃和93℃,远高于WT（T₅₀⁶⁰为48℃）	[17]
	ISM/简并密码子NDT	Candida antarcticalipase B（CALB）	立体选择性	W104/L144 V149/V154 I189/V190 A281/A282	（V149D/I189V/V190C/A281G/A282V）对2-苯基丙酸对硝基苯酯的k_cat / K_m为WT的近277倍,突变体（W104C/L144Y/V149I/V154I/A281C/A282F）对R-2-苯基丙酸对硝基苯酯的活性比WT高15倍	[18]
	理性聚焦迭代定点诱变（FRISM）	Candida antarcticalipase B（CALB）	对映选择性	W104/ D134 Q157/I189 V190/ A281 A282	突变体（A281G/A282V/V190C）、（Q157L/I189A）、（W104/I189）、（W104A/I189M/V190C/D134L）对各自的最佳对映体的选择性分别高达95%、94%、95%和91%	[19]