摘要
构建植物乳杆菌(Lactobacillus sp.LMY-20)中亚硝酸盐还原酶(nitrite reductase,NiR)的重组大肠杆菌、纯化重组蛋白并对其进行酶学性质分析。将人工合成的密码子优化的亚硝酸盐还原酶基因(nir)亚克隆至载体pET28a(+),构建重组表达载体pET28a(+)-nir并转化到E.coli BL21(DE3)中实现表达。包涵体复性,利用镍柱亲和层析纯化重组亚硝酸盐还原酶。成功构建产亚硝酸盐还原酶的重组大肠杆菌并纯化了重组亚硝酸盐还原酶,纯化后经变性聚丙烯酰氨凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)分析在45 kDa附近出现明显条带。Na 2S 2O 4-MV法测得酶活为2 332.72 U/L,最适pH值为6.5,最适作用温度为37℃,在4~70℃下孵育40 min后可保持超过85%的活力。该研究实现植物乳杆菌来源的NiR在大肠杆菌中的重组表达、纯化及其酶学性质分析,重组NiR具有较好的温度适应性和稳定性,为其在农业、食品及医药等领域应用奠定基础。
This study constructed a recombinant Escherichia coli that expressed nitrite reductase(NiR)from Lactobacillus sp.LMY-20,and the enzymatic properties of the NiR were investigated.A synthetic codon-optimized Lactobacillus plantarum nitrite reductase gene(nir)was cloned into vector pET28a(+)and expressed in Escherichia coli BL21(DE3).The recombinant NiR was purified by nickel column affinity chromatography with a molecular weight of 45 kDa on SDS-PAGE.Moreover,its optimal reaction pH and temperature were 6.5 and 37℃,respectively.Besides,more than 85%of the original enzyme activity was preserved after 40 min incubation at 4-70℃.In conclusion,the recombinant NiR has good temperature adaptability and stability,which lays a foundation for its applications in the fields of agriculture,food,and medicine.
作者
张庆芳
李美玉
王晓辉
胡善松
于爽
迟乃玉
ZHANG Qingfang;LI Meiyu;WANG Xiaohui;HU Shansong;YU Shuang;CHI Naiyu(School of Life Science and Biotechnology,Dalian University,Dalian 116622,China;Liaoning Technology of Marine Microbiological Engineering Research Center,Dalian 116622,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2019年第12期28-34,共7页
Food and Fermentation Industries
基金
国家高技术研究发展计划‘863计划’(No.2018YFC0311100)
国家自然科学基金(No.31500039)
辽宁省自然科学基金项目(No.20170520167、20180550728)
大连市支持高层次人才创新创业项目(No.2017RQ155)
关键词
植物乳杆菌
亚硝酸盐还原酶
重组表达
纯化
酶学性质
Lactobacillus plantarum
nitrite reductase
recombinant expression
purification
enzymatic properties
