基于长链底物亲和的脂肪酶MAS1理性设计改造

唐秀云; 王嘉伟; 张建国; 缪雨露; 赵玥; 高蓓; 张鲁嘉

doi:10.14135/j.cnki.1006-3080.20220328001

基于长链底物亲和的脂肪酶MAS1理性设计改造

doi: 10.14135/j.cnki.1006-3080.20220328001

唐秀云^1,,
王嘉伟²,
张建国²,
缪雨露³,
赵玥³,
高蓓^1, ,,
张鲁嘉^{1, 3, 4, ,}

1.
华东理工大学生物工程学院，生物反应器工程国家重点实验室，上海 200237
2.
上海理工大学健康科学与工程学院，上海 200093
3.
华东师范大学化学与分子工程学院，上海分子治疗与新药创制工程技术研究中心，上海 200241
4.
纽约大学（上海）理论与计算化学联合中心，上海 200122

基金项目: 国家重点研发计划（2020YFA0908400）；国家自然科学基金（31772007, U1805235）；上海市自然科学基金（21ZR1416500）；纽约大学全球种子基金；纽约大学-华东师范大学计算化学中心（ECNU创新001公众平台）

详细信息

作者简介:
唐秀云（1994—），女，安徽安东人，硕士生，主要研究方向：脂肪酶理性设计改造。E-mail：txy13865167321@163.com

通讯作者:
高　蓓，E-mail：gaobei@ecust.edu.cn

张鲁嘉，E-mail：ljzhang@chem.ecnu.edu.cn；ljzhang@nyu.edu

中图分类号: Q55
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- 被引次数: 0
出版历程
- 收稿日期: 2022-03-28
- 网络出版日期: 2022-05-27

Rational Design of Lipase MAS1 for the Long-chain Substrate Affinity

TANG Xiuyun^1
,,
WANG Jiawei²,
ZHANG Jianguo²,
MIAO Yulu³,
ZHAO Yue³,
GAO Bei^{1
, ,},
ZHANG Lujia^{1, 3, 4
, ,}

1.
State Key Laboratory of Bioreactor Engineering, School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China
2.
School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China
3.
Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241, China
4.
NYU-ECNU Center for Computational Chemistry at NYU Shanghai, Shanghai 200122, China

摘要

摘要: 探讨了脂肪酶MAS1对长链底物4-硝基苯酚豆蔻肉酸酯（pNP-C14）催化的最适温度、最适pH、pH稳定性，并通过分子动力学模拟计算（MD得出结合自由能），后根据结合自由能的差值初步筛选出了G145W及T141L两个单点突变体。实验表明，与野生型MAS1相比，结合自由能降低显著的突变体G145W的米氏常数（K_m）值减小了11%，催化常数与K_m(k_cat/K_m值)为野生型MAS1的1.29倍，突变体G1451对长链底物的亲和能力和催化效率均有提高；而结合自由能降低较小的突变体T141L的K_m值增大了22%，k_cat/K_m值为野生型MAS1的0.88倍，说明酶与底物分子结合自由能的降低并非准确的突变筛选标准。通过分子力学/广义波恩表面积（MM/GBSA）残基拆解分析得出：残基T38、F39、L149、F153、V202、V233对脂肪酶活性口袋与长链底物结合的稳定性贡献较其他残基大；残基T38、G40、N41、N45和T237在脂肪酶对提高长链底物的亲和能力中具有重要贡献，预测为热点残基，其结合自由能的降低可以作为突变筛选的参考标准。
- 脂肪酶MAS1 /
- 长链底物 /
- 动力学 /
- MM/GBSA残基拆解 /
- 热点残基
Abstract: In this study, the optimum temperature, optimum pH and pH stability of lipase MAS1 for the catalysis of the long-chain substrate 4-nitrophenol myristate (pNP-C14) were determined. Two single point mutations G145W and T141L were initially screened out according to the decrease of binding free energy by molecular dynamics simulation (Molecular Dynamics, MD). Experiments showed that the Km value of the mutant G145W with a significant decrease in binding free energy was reduced by 11% compared with the wild-type MAS1, and the k_cat/K_m value was 1.29 times that of the wild-type MAS1. Compared with the wild-type MAS1, the Km value of the mutant T141L with a smaller decrease in binding free energy was increased by 22%, and the k_cat/K_m value was 0.88 times that of the wild-type MAS1. Compared with the wild type, the affinity and catalytic efficiency of G145W for long-chain substrates were improved, while those of T141L were lower than those of the wild type. It shows that the absolute zero value of the binding energy difference is not an accurate mutation screening criterion. In-depth analysis was performed by Molecular Mechanics / Generalized Born Surface Area (MM/GBSA) residue disassembly. It was concluded that residues T38, F39, L149, F153, V202 and V233 contributed more to the binding stability of the lipase active pocket and long-chain substrates than other residues; residues T38, G40, N41, N45, and T237 have important contributions to the improvement of lipase affinity for long-chain substrates and are predicted to be hotspot residues whose reduced binding free energy can serve as a reference standard for mutation screening.
- Lipase MAS1 /
- long-chain substrate /
- kinetics /
- MM/GBSA /
- hotspot residues

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图 1 温度对脂肪酶MAS1酶活的影响

Figure 1. Effect of temperature on the lipase MAS1 activity

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图 2 （a）最适反应 pH（b）pH稳定性

Figure 2. (a) Optimal pH curve (b) stability of pH curve

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图 3 脂肪酶MAS1与分子对接分析

Figure 3. Molecular docking Results of lipase MAS1 and pNP-C14

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图 4 MAS1, G145W, T141L双倒数曲线图

Figure 4. Lineweaver-Burk plots of the wild-type MAS1, mutants G145W and T141L.

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表 1 引物序列

Table 1. Primers

Primers	Sequence（5´→3´）	Restriction sites
MAS1-F	ATAGAATTCGATGGCGACCGCG ACCGCGGCC	EcoRI
MAS1-R	ATACTCGAGTCAATGGTGATGGT GATGGTGATGATGG	XhoI
T7-F	TAATACGACTCACTATA	/
T7-R	GCTAGTTATTGCTCAGCG	/

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表 2 定点突变构建引物序列

Table 2. Primers used in sitedirected mutagenes

Primers	Sequence（5´→3´）
T141L-1R	GAGGCCGAGCAGCGTGAGGCCGTGGTTGTCCGG
T141L-2F	CCGGACAACCACGGCCTCACGCTGCTCGGCCTC
G145W-1R	CGGCAGCAGCTTGGTGAGCCAG AGCAGCGTGGTGCCGTG
G145W-2F	CACGGCACCACGCTGCTCTGGC TCACCAAGCTGCTGCCG
MAS1-F	ATAGAATTCGATGGCGACCGCGACCGCGGCC
MAS1-R	ATACTCGAGTCAATGGTGATGGTGATGGTGATGATGG

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表 3 结合自由能计算结果

Table 3. Calculation of total free energy

Lipases	ΔG/(kJ·mol⁻¹)	ΔΔG/(kJ·mol⁻¹)
WT	−159.896	N/A
G145W	−180.611	−20.715
T141L	−165.833	−5.937
A135W	−157.406	2.490
F47K	−159.607	0.289
G76H	−119.533	40.363
V35W	−151.072	8.824
S230K	−151.862	8.034

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表 4 酶动力学参数测定

Table 4. Kinetic parameters of lipases

Lipases	K_m/( mmol·L⁻¹)	k_cat/s⁻¹	k_cat/K_m ( mmol·L⁻¹·s⁻¹)
WT	0.702	8.236×10⁵	1.173×10⁶
G145W	0.625	9.431×10⁵	1.510×10⁶
T141L	0.860	8.852×10⁵	1.030×10⁶

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表 5 MM/GBSA残基分解结果

Table 5. Results of the MM/GBSA residue decomposition

Amino acid residues	ΔG/（kJ·mol⁻¹）
Amino acid residues	WT	T141L	G145W
G37	−4.904	−0.469	−4.820
T38	−9.573	−4.485	−15.573
F39	−12.619	−5.665	−12.142
G40	−3.423	0.134	−5.699
N41	−0.519	0.042	−1.314
N45	−1.130	0.159	−3.506
H75	−0.895	−0.218	−0.243
H108	−3.582	−0.527	−5.657
S109	−5.565	−1.573	−4.577
T141	−4.084	−2.678	−3.197
L146	−6.996	−14.577	−6.058
L149	−11.255	−8.770	−17.782
L150	−3.774	−11.757	−7.841
F153	−15.104	−6.561	−12.460
P154	−5.473	0.126	−2.176
L167	−3.372	−18.769	−5.623
Q170	−4.510	−1.925	−2.962
V202	−8.535	−1.448	−8.393
H232	−1.490	0.0420	−5.724
V233	−15.138	−1.423	−14.544
A234	−4.619	−0.0330	−0.527
T237	−2.753	0.0170	−5.079

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