双肽细菌素Plantaricin EF在乳酸乳球菌中的异源表达及其抑菌活性  

Heterologous Expression of Two-pepride Bacteriocin Plantaricin EF in Lactococcus lactis and the Antibacterial Activity

作　　者：郦萍 王嘉雯 徐栋 顾青 Li Ping;Wang Jiawen;Xu Dong;Gu Qing(Zhejiang Gongshang University, School of Food Science and Biotechnology, Key Laboratory for Food Microbial Technology of Zhejiang Province, Hangzhou 310018)

机构地区：[1]浙江工商大学食品与生物工程学院浙江省食品微生物技术研究重点实验室,杭州310018

出　　处：《中国食品学报》2018年第1期34-41,共8页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金-青年科学基金项目(31601449);浙江省自然科学基金项目(LY16C200002);国家国际科技合作专项(2013DFA32330)

摘　　要：乳酸菌细菌素是乳酸菌在代谢过程中由核糖体合成的具有抗菌作用的多肽或蛋白质,具有抗菌活性强,安全,容易降解,无毒副作用和无耐药性等优点,在食品、医药和农业等领域具有巨大应用潜力。Plantaricin EF(PlnEF)为class IIb类细菌素,抑菌效果好,而在天然乳酸菌中表达水平较低,且难以纯化。本研究拟运用乳酸菌表达系统,进行PlnEF的异源高效表达。通过PCR扩增得到Plantaricin E(PlnE)和Plantaricin F(PlnF)的基因序列,构建重组质粒p NZ8124-pln E和p NZ8124-pln F,并转化乳酸乳球菌NZ9000获得异源表达菌株。运用NICE系统诱导双肽细菌素PlnE和PlnF的表达。建立高效纯化体系,得到的重组蛋白PlnE和PlnF质量浓度分别为2.629 mg/m L和2.397 mg/m L。利用牛津杯双层琼脂拮抗法研究PlnE、PlnF和PlnEF(等物质的量混合)的抑菌活性,结果表明,它们不仅能抑制革兰氏阳性菌,还能抑制革兰氏阴性菌,且PlnEF具有协同作用。Lactic acid bacteria（LAB） bacteriocin is antibacterial peptide or protein synthesized by the ribosome during metabolic process. It has great application potential in the fields of food, medicine and agriculture due to its strong antibacterial activity, safety and other unique characteristics. Plantaricin EF（PlnEF） is a class IIb bacteriocin with great antibacterial effect against food-borne pathogens. However, low expression level in natural LAB and the difficulties of its purification might limit its application. In the present study, we heterologously expressed PlnEF using the Lactobacillus expression system for the first time. The gene sequences of Plantaricin E（PlnE） and Plantaricin F（PlnF） were obtained by PCR amplification and recombinant plasmids p NZ8124-pln E and p NZ8124-pln F were successfully constructed. The recombinant plasmids were introduced into Lactococcus lactis NZ9000. Nisin-Controlled gene expression system（NICE） was used to express PlnE and PlnF. PlnE and PlnF were purified, and the concentrations of recombinant proteins PlnE and PlnF were 2.629 mg/m L and 2.397 mg/m L, respectively. Inhibitory activities of PlnE, PlnF and PlnEF（mole ∶ mole=1 ∶ 1）were tested by oxford cup double agar suspension method. Results showed that both PlnE and PlnF inhibited Gram-positive and Gram-negative bacteria, and PlnEF effect synergistically.

关 键 词：NICE系统 双肽类细菌素Pln EF 异源表达 分离纯化 抑菌活性 

分 类 号：TS201.3[轻工技术与工程—食品科学]