摘要
离子液体双水相提取木瓜蛋白酶有较高的提取率和简便的操作流程,构建合适的离子液体双水相是提取木瓜蛋白酶的研究热点。鉴于此,本实验通过荧光光谱分析、分子对接和离子液体双水相体系分配系数测定等方法,研究离子液体与木瓜蛋白酶相互作用的机制。荧光光谱分析结果表明,在疏水作用力驱动下1-丁基-3-甲基咪唑四氟硼酸盐([C4mim]BF4)和1-丁基氯化吡啶([C4Py]Cl)离子液体可与木瓜蛋白酶发生静态荧光猝灭,结合生成复合物,结合位点数均为1,且[C4Py]Cl离子液体的吉布斯自由能高于[C4mim]BF4离子液体。分子对接结果进一步表明,离子液体的结合部位位于木瓜蛋白酶中间活性区域的疏水凹槽中,并与周围众多的氨基酸残基存在疏水作用、范德华力和π-π相互作用等非共价键力。离子液体双水相体系分配系数测定结果表明,在实际萃取木瓜蛋白酶的过程中,相同条件下[C4Py]Cl离子液体的萃取效果优于[C4mim]BF4离子液体,与荧光光谱和分子对接所得结果相同,即[C4Py]Cl离子液体与木瓜蛋白酶的吉布斯自由能高于[C4mim]BF4离子液体与木瓜蛋白酶的吉布斯自由能,荧光光谱、分子对接、分配系数测定的结果都很好地相互关联。本研究对于筛选合适离子液体构建提取木瓜蛋白酶的双水相萃取体系具有一定的理论指导作用。
Relevant studies have shown that ionic liquid aqueous two-phase extraction of papain gave a high extraction rate through simple operation,and the construction of a suitable ionic liquid aqueous two-phase system is a research hotspot for the extraction of papain.In view of this,this experiment studied the mechanism of the interaction between ionic liquid and papain through fluorescence quenching,molecular docking and the determination of the partition coefficient of papain in ionic liquid aqueous two-phase systems.Fluorescence quenching analysis showed that the ionic liquids[C4mim]BF4 and[C4Py]Cl could bind to papain with one binding site through hydrophobic interaction,forming a complex,and the quenching effect of[C4Py]Cl was higher than that of[C4mim]BF4.Further molecular docking results indicated that the binding site of[C4mim]BF4 was located in the hydrophobic pocket of the active region of papain and interacted non-covalently with many surrounding amino acid residues through hydrophobic interaction,van der Waals force andπ-πinteraction.According to the partition coefficient of papain in ionic liquid aqueous two-phase systems,the extraction efficiency of papain with[C4Py]Cl was better that with[C4mim]BF4 under the same conditions,which was consistent with the results of fluorescence spectroscopy and molecular docking.This study can theoretically guide the selection of suitable ionic liquids for aqueous two-phase extraction of papain.
作者
田康永
杨吕清
胡慧慧
张海德
TIAN Kangyong;YANG Lüqing;HU Huihui;ZHANG Haide(College of Food Science and Engineering,Hainan University,Haikou 570228,China)
出处
《食品科学》
EI
CAS
CSCD
北大核心
2020年第23期15-20,共6页
Food Science
基金
国家自然科学基金地区科学基金项目(31660444)
海南省研究生创新科研课题(Hys2018-198)。
关键词
离子液体双水相萃取
木瓜蛋白酶
荧光光谱
分子对接
ionic liquid aqueous two-phase extraction
papain
fluorescence spectroscopy
molecular docking