摘要
【目的】从凡纳滨对虾(Litopenaeus vannamei)中分离纯化多酚氧化酶(Polyphenol Oxidase,PPO),进行质谱鉴定,分析其生物学信息。【方法】采用硫酸铵沉淀结合柱层析法从虾头胸部中提取纯化PPO,电泳法确定其分子质量,采用液相质谱联用法分析氨基酸序列结构,根据分析结果,同源建模并进行评估。【结果与结论】获得了高纯度的PPO,其纯化倍数为65.7,酶比活力达650.1 U/mg蛋白,得率为29.5%。PPO分子质量约为72 ku。经质谱检测分析和basic local alignment search tool(BLAST)序列比对,纯化蛋白与凡纳滨对虾酚氧化酶原的氨基酸序列相似性为65%,匹配度分值为11 321,确认为PPO。PPO是一种亲水性大于疏水性的两性蛋白,二级结构以α-螺旋和无规则卷曲为主,建立的PPO三维结构模型可靠性较高。
【Objective】To isolate and purify the PPO from Litopenaeus vannamei,the biological information was identified and analyzed by using mass spectrometry.【Methods】PPO was isolated and purified from the shrimp cephalothorax by using ammonium sulfate precipitation and column chromatography.The molecular weight was determined by electrophoresis,and the amino acid sequence was determined by liquid chromatography-electrospray ionization-tandem mass spectrometry(LC-ESIMS/MS).Based on these results,homology modeling was constructed and the evaluations were performed.【Results and Conclusion】Purified PPO was obtained with a purification of 65.7 folds,the specific activity of PPO was 650.1 U/mg protein and with a yield of 29.5%.The molecular weight of PPO was about 72 ku.The similarity of amino acid sequence between the purified protein and Propolyphenoloxidase of L.vannamei was 65%with a matching score of 11321 points by identification and analysis of mass spectrometry and basic local alignment search tool(blast)sequence comparison.The results have confirmed that the purified protein was PPO.PPO is an amphoteric protein and it has a higher hydrophilicity than hydrophobicity.The secondary structure of PPO mainly consists ofα-helix and random coil.In conclusion,the predicted three-dimensional structure of PPO had a high reliability.
作者
魏帅
刘媛
刘蒙娜
孙钦秀
刘书成
吉宏武
郝记明
邓楚津
毛伟杰
WEI Shuai;LIU Yuan;LIU Meng-na;SUN Qin-xiu;LIU Shu-cheng;JI Hong-wu;HAO Ji-ming;DENG Chu-jin;MAO Wei-jie(College of Food Science and Technology,Guangdong Ocean University,Guangdong Provincial Key Laboratory of Aquatic Products Processing and Safety,Guangdong Province Engineering Laboratory for Marine Biological Products,Guangdong Provincial Engineering Technology Research Center of Marine Food,Key Laboratory of Advanced Processing of Aquatic Product of Guangdong Higher Education Institution,Zhanjiang,524088,China;Collaborative Innovation Center of Seafood Deep Processing,Dalian Polytechnic University,Liaoning 116034,China;Southern Marine Science and Engineering Guangdong Laboratory(Zhanjiang),Zhanjiang 524088,china)
出处
《广东海洋大学学报》
CAS
2020年第4期100-108,共9页
Journal of Guangdong Ocean University
基金
广东省区域联合基金-青年基金项目(2019A1515110419)
国家重点研发计划资助(2019YFD0902003)
国家自然科学基金面上项目(31771997)
南方海洋科学与工程广东省实验室(湛江)资助(ZJW-2019-06)
广东海洋大学创新强校重大培育项目(GDOU2017052603)
国家虾蟹产业技术体系(G-48)
广东省普通高校青年创新人才项目(2019KQNCX040)
广东普通高等学校海洋食品绿色加工技术研究团队(2019KCXTD011)。
关键词
凡纳滨对虾
多酚氧化酶
质谱
鉴定
生物信息学
Litopenaeus vannamei
PPO
mass spectrometry
identification
bioinformatics
作者简介
第一作者:魏帅(1986-),男,博士,讲师,研究方向为海洋食品加工新技术。E-mail:weishuaiws@126.com;通信作者:孙钦秀(1986-),女,博士,讲师,研究方向为海洋食品加工新技术。E-mail:sunqinxiugo@163.com;通信作者:刘书成(1977-),男,教授,研究方向为海洋食品加工新技术。E-mail:Lsc771017@163.com。
