甜菜碱类离子液体化学修饰猪胰脂肪酶提升其酶学性能

doi:10.16085/j.issn.1000-6613.2022-0210

摘要/Abstract

摘要：

设计并合成了不同链长、不同阴离子的甜菜碱离子液体对猪胰脂肪酶（porcine pancreatic lipase，PPL）进行化学修饰。所有修饰酶的各项酶学性能都有了明显提高，修饰剂的链长和阴离子对修饰效果有一定的影响；经[BetaineC₁₆][H₂PO₄]修饰的PPL表现出最佳的酶学性能，其酶活是原酶的3.4倍，在60℃下保存45min后的酶活是原酶的8.7倍，在强极性非质子溶剂二甲基亚砜中储存2h后稳定性提高了1.7倍，催化α-苯乙醇转酯化反应的对映体选择性提高了2.0倍。动力学测试结果表明修饰酶较原酶有更高的底物亲和力。荧光光谱表征表明，随着修饰度的增加，荧光强度逐渐降低。圆二色谱表征表明离子液体修饰剂的引入引起了PPL二级结构的变化，修饰后酶的α-螺旋含量降低，β-折叠含量上升，使酶的活性和稳定性得以提升。证实以甜菜碱类离子液体作为修饰剂可以同时有效提升脂肪酶的各项酶学性能，为酶的化学修饰拓展了一种新型、高效的修饰剂。

关键词: 脂肪酶, 化学修饰, 离子液体, 蛋白质稳定性, 动力学, 生物催化

Abstract:

The structure of porcine pancreatic lipase (PPL) was selected as the research object, and betaine ionic liquids with different chain lengths and different anions were designed and synthesized as modifiers to chemically modify PPL. The protein concentration was determined by the BCA method, the degree of modification was determined by the trinitrobenzene sulfonic (TNBS) method, and the enzymatic activity was determined by the p-nitrophenol palmitate (p-NPP) method. The changes in enzymatic properties such as the optimum temperature, optimum pH, thermal stability, organic solvent tolerance and the kinetics of the native and modified PPLs were tested. Results showed that the chain length and anion of modifiers had a certain influence on the modification effect. The PPL modified by [BetaineC₁₆][H₂PO₄] presented the best enzymatic performance, the enzymatic activity of which was 3.4 times that of the native PPL, the enzymatic activity after stored at 60℃ for 45min was 8.7 times that of the native PPL, the stability was increased by 1.7 times after stored in strongly polar aprotic solvent dimethyl sulfoxide for 2h, and the selectivity of enantiomers in the catalytic reaction of α‍-phenyl ethanol was increased by 2.0 times. In addition, the temperature tolerance and pH tolerance of modified PPLs had been significantly improved. The kinetic test results also showed that modified PPLs had a higher substrate affinity so that it had higher activity. The results of fluorescence spectroscopy showed that the introduction of modifiers had a certain impact on the fluorescent group of PPLs. Fluorescence intensity decreased as the modification degree increased. The results of circular dichroism showed that the α-helix content of the modified PPLs decreased while the β-sheet content increased, which improved the activity and stability of PPLs. This experiment confirmed that betaine ionic liquids as modifiers could simultaneously improve various enzymatic properties of lipase, and expand a new type of modifier with good modification effects for chemical modification of enzymes.

Key words: lipase, chemical modification, ionic liquid, protein stability, kinetics, biocatalysis

中图分类号:

Q814

鲁泽平, 裴新华, 薛誉, 张晓光, 胡燚. 甜菜碱类离子液体化学修饰猪胰脂肪酶提升其酶学性能[J]. 化工进展, 2022, 41(11): 6045-6052.

LU Zeping, PEI Xinhua, XUE Yu, ZHANG Xiaoguang, HU Yi. Chemical modification of porcine pancreatic lipase with betaine ionic liquid to improve its enzymatic properties[J]. Chemical Industry and Engineering Progress, 2022, 41(11): 6045-6052.

图/表 11

参考文献 34

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酶	修饰度/%	酶活/U·mg^-1	相对酶活/%
PPL		8.75	100
［BetaineC₄］‍［Cl］-PPL	49.85	19.23	219.77
［BetaineC₈］［Cl］-PPL	49.39	20.34	232.46
［BetaineC₁₂］［Cl］- PPL	45.92	21.52	245.94
［BetaineC₁₆］［Cl］- PPL	40.81	29.23	334.06
［BetaineC₁₆］［BF₄］- PPL	40.74	28.71	328.11
［BetaineC₁₆］［H₂PO₄］- PPL	41.36	30.12	344.23
SarcosineC₁₆- PPL	40.87	10.27	117.37

酶	修饰度/%	酶活/U·mg^-1	相对酶活/%
PPL		8.75	100
［BetaineC₄］‍［Cl］-PPL	49.85	19.23	219.77
［BetaineC₈］［Cl］-PPL	49.39	20.34	232.46
［BetaineC₁₂］［Cl］- PPL	45.92	21.52	245.94
［BetaineC₁₆］［Cl］- PPL	40.81	29.23	334.06
［BetaineC₁₆］［BF₄］- PPL	40.74	28.71	328.11
［BetaineC₁₆］［H₂PO₄］- PPL	41.36	30.12	344.23
SarcosineC₁₆- PPL	40.87	10.27	117.37

酶	K_m/mg·mL^-1	V_max/U·mg^-1
PPL	20.32	0.459
［BetaineC₄］［Cl］-PPL	19.11	0.501
［BetaineC₈］［Cl］-PPL	18.62	0.611
［BetaineC₁₂］［Cl］-PPL	15.62	0.736
［BetaineC₁₆］［Cl］-PPL	12.95	0.869
［BetaineC₁₆］［BF₄］-PPL	12.36	0.860
［BetaineC₁₆］［H₂PO₄］-PPL	13.31	0.874

酶	K_m/mg·mL^-1	V_max/U·mg^-1
PPL	20.32	0.459
［BetaineC₄］［Cl］-PPL	19.11	0.501
［BetaineC₈］［Cl］-PPL	18.62	0.611
［BetaineC₁₂］［Cl］-PPL	15.62	0.736
［BetaineC₁₆］［Cl］-PPL	12.95	0.869
［BetaineC₁₆］［BF₄］-PPL	12.36	0.860
［BetaineC₁₆］［H₂PO₄］-PPL	13.31	0.874

酶	C_总/%	C_R/%	C_S/%	ee_S/%	ee_R/%	E
PPL	29.4	49.2	15.8	24.1	62.2	5.5
［BetaineC₄］［Cl］-PPL	36.2	50.5	13.2	28.9	61.7	5.9
［BetaineC₈］［Cl］-PPL	43.5	39.5	11.25	29.2	60.3	6.3
［BetaineC₁₂］［Cl］-PPL	55.7	53.2	12.9	26.9	62.9	10.3
［BetaineC₁₆］［Cl］-PPL	62.0	44.6	12.7	30.4	59.6	15.8
［BetaineC₁₆］［BF₄］-PPL	60.7	43.9	11.5	28.7	61.9	15.4
［BetaineC₁₆］［H₂PO₄］-PPL	64.5	44.3	12.5	36.4	54.5	16.3