海洋栖热菌β-胡萝卜素15,15’双加氧酶原核表达  被引量:2

Prokaryotic expression ofβ-carotene 15,15’-dioxygenase from Oceanithermus sp.

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作  者:欧亚非 赵彩梦 郑亚东 毛多斌 魏涛 OU Yafei;ZHAO Caimeng;ZHENG Yadong;MAO Duobin;WEI Tao(Beijing Work Station,Technology Center,Shanghai Tobacco Group Co.,Ltd.,Beijing 101121,China;School of Food and Biological Engineering,Zhengzhou University of Light Industry,Zhengzhou 450002,China)

机构地区:[1]上海烟草集团技术中心北京工作站,北京101121 [2]郑州轻工业大学食品与生物工程学院,河南郑州450002

出  处:《中国酿造》2020年第3期146-150,共5页China Brewing

基  金:河南省高校科技创新人才项目(18HASTIT040)。

摘  要:以海洋栖热菌(Oceanithermus sp.)DN-1基因组DNA为模板,通过PCR法扩增β-胡萝卜素15,15’双加氧酶BCMOTA基因,构建重组质粒,在大肠杆菌Escherichia coli BL21(DE3)中表达,并采用高效液相色谱(HPLC)法检测酶活性。结果表明,通过镍柱亲和层析和分子筛Sephacryl TM S-100,得到纯化重组β-胡萝卜素15,15’双加氧酶,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析结果表明,该酶分子质量约35 kDa;该酶的最适温度为50℃,最适pH值为8.0。在此优化反应条件下,生成569.3 mg/L全反式视黄醛,转化率达到89.5%。Using the genome DNA of Oceanithermus sp.DN-1 as template,the gene ofβ-carotene 15,15'-dioxygenase BCMOTA was amplified by PCR and the recombinant plasmid was constructed to express the enzyme in Escherichia coli BL21(DE3).The enzyme activity was detected by HPLC.The results showed that the purified recombinantβ-carotene 15,15'-dioxygenase was obtained by Ni-chelating affinity chromatography and molecular sieve Sephacryl TM S-100.The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)showed that the molecular mass of the purified enzyme was approximately 35 kDa.The optimum temperature and pH of the enzyme were 50℃and 8.0,respectively.Under the optimum conditions,569.3 mg/L of all-trans retinal was produced with a conversion rate of 89.5%.

关 键 词:海洋栖热菌DN-1 克隆表达 全反式视黄醛 酶学性质 

分 类 号:TS201.3[轻工技术与工程—食品科学]

 

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