植物乳杆菌素LPL-1的异源表达及理化性质分析  

Heterologous Expression and Physicochemical Properties of Plantaricin LPL-1

作　　者：王玉 王瑶[1] 李平兰[1] WANG Yu;WANG Yao;LI Pinglan(College of Food Science and Nutritional Engineering,China Agricultural University,Beijing 100083,China)

机构地区：[1]中国农业大学食品科学与营养工程学院,北京100083

出　　处：《食品科学》2023年第20期100-106,共7页Food Science

基　　金：国家自然科学基金面上项目(32172172)。

摘　　要：为在大肠杆菌中可溶表达具有生物活性的Ⅱa类细菌素,以植物乳杆菌素LPL-1为研究对象,将植物乳杆菌素LPL-1的表达基因克隆至阿拉伯糖启动子下,C-端与His-tag序列融合表达,以单核细胞性李斯特菌ATCC 54002为指示菌,鉴定重组细菌素的活性。通过CRISPR/Cas9基因编辑技术对大肠杆菌BW25331基因组进行编辑,突变过氧化物酶基因(ahpC),敲除硫氧还蛋白还原酶(trxB)和谷胱甘肽还原酶(gor)基因,成功构建了适宜IIa类细菌素表达的细胞工厂E. coil(ΔtrxB+Δgor+ahpC^(M))。表达产物经亲和层析纯化后,对其理化性质进行分析,证明重组细菌素具有热稳定性(60~100℃)、酸碱稳定性(pH 2~11)、表面活性剂稳定性。本研究实现了植物乳杆菌素LPL-1在大肠杆菌中的活性表达,重组表达的细菌素具有稳定的理化性质,在食品行业具有广泛的应用潜力。The present study aimed to express soluble recombinant class Ⅱa bacteriocin in Escherichia coli.The plantaricin LPL-1 gene was cloned into an arabinose promoter,and the C-terminus was fused with the his-tag sequence for expression.The inhibitory activity of recombinant plantaricin LPL-1 against Listeria monocytogenes 54002 was used as the response variable.A novel cell factory,E.coil(ΔtrxB+Δgor+ahpC^(M)),suitable for class IIa bacteriocin expression was established by CRISPR gene editing technology.It was derived from E.coil BW25331 by deleting the thioredoxin reductase(trxB)gene and glutathione reductase(gor)gene and mutating the gene encoding peroxidase(ahpC).The recombinant plantaricin LPL-1 was purified by affinity chromatography,and its physicochemical properties were analyzed.The purified plantaricin LPL-1 possessed wide pH stability(2–11),high thermal stability(60–100℃),and surfactant stability.The recombinant bacteriocin has wide potential for food industrial applications.

关 键 词：Ⅱa类乳酸菌细菌素 植物乳杆菌素LPL-1 异源表达 包涵体 CRISPR/Cas9 

分 类 号：TS201.3[轻工技术与工程—食品科学]