异源重组联合酶法环化制备环状Lunasin及其性质研究  

Preparation of Cyclic Lunasin by Heterologous Recombination Combined with Enzymatic Cyclization and Its Properties

作　　者：石帆 侯轶 赵劲松 胡松青[1] SHI Fan;HOU Yi;ZHAO Jinsong;HU Songqing(School of Food Science and Engineering,South China University of Technology,Guangzhou 510640,China;School of Light Industry Science and Engineering,South China University of Technology,Guangzhou 510640,China)

机构地区：[1]华南理工大学食品科学与工程学院,广东广州510640 [2]华南理工大学轻工科学与工程学院,广东广州510640

出　　处：《食品工业科技》2025年第7期133-139,共7页Science and Technology of Food Industry

基　　金：国家自然科学基金(32072157)。

摘　　要：为了实现环状多肽cLunasin的全生物合成,本文设计了合成cLunasin的线性前体aLunasin,构建了含MBP与SUMO双标签的重组表达载体,应用大肠杆菌重组表达aLunasin后经多肽连接酶Butelase 1介导合成cLunasin,进一步通过圆二色光谱、内源荧光光谱和细胞实验探究了环化对Lunasin热稳定性、结构特性与抗结直肠癌活性的影响。结果显示,37℃诱导后重组蛋白成功表达,质谱鉴定结果显示Butelase 1成功环化aLunasin,通过该法每升发酵液可以制备6.36 mg的电泳纯cLunasin。圆二色光谱与内源荧光光谱结果表明,环化在一定程度上提升了Lunasin结构的有序性与致密性。而且,环化改善了Lunasin的热稳定性,在97℃孵育30 min后,多肽保留率从Lunasin的62.3%提高到cLunasin的74.9%。cLunasin对结直肠癌HCT-116细胞的半抑制浓度为15.2μmol/L,显著低于Lunasin的30.3μmol/L,抗结直肠癌活性增强。本文建立了一种cLunasin的绿色生物合成方法,为活性多肽的改造与重组表达提供有益探索。In order to prepare a cyclic polypeptide cLunasin by the method of total biosynthesis,the linear precursor aLunasin was designed for cLunasin synthesis,and a fusion expression vector with the double tags of MBP and SUMO was constructed.After the recombinant expression of aLunasin by Escherichia coli,the cLunasin was catalyzed to synthesize by the polypeptide ligase Butelase 1.The effects of cyclization on the thermal stability,structural properties and anticolorectal cancer activity of the peptide were further investigated by circular dichroism spectroscopy,endogenous fluorescence spectroscopy and cell experiments.The recombinant protein was successfully expressed after induction at 37℃,and aLunasin was successfully cyclized by Butelase 1,which was identified by the mass spectrometry identification.And 6.36 mg of cLunasin could be prepared form 1 liter of fermentation broth by the total biosynthesis.Cyclization could get the peptide structure more ordered and more compact,indicated by circular dichroism spectroscopy and endogenous fluorescence spectroscopy.Morover,the thermal stability of peptide was improved after cyclization.After incubation at 97℃for 30 min,the peptide retention for cLunasin increased to 74.9%,while that of Lunasin was 62.3%.The half maximal inhibitory concentration of cLunasin on colorectal cancer HCT-116 cells was determined to 15.2μmol/L,which was lower than that of for Lunasin 30.3μmol/L,indicating that the anticolorectal cancer activity was enhanced significantly by cyclization.A green biosynthesis method for cLunasin was established,and an useful exploring was provided for the modification and recombinant expression of active peptides.

关 键 词：大豆 Lunasin 重组表达 环化 Butelase 1 稳定性 抗结直肠癌活性 本文网刊: 

分 类 号：TS201.4[轻工技术与工程—食品科学]