条斑紫菜高纯度藻红蛋白规模制备及光学性质研究  

Pilot Preparation and Optical Properties of Highly Purified Phycoerythrin from Porphyra yezoensis

作　　者：郭子叶[1] 蔡春尔[1,2] 李春霞[1] 耿中雷[1] 贾睿[1,2] 何培民[1,2] 

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]上海海洋大学海洋科学研究院,上海201306

出　　处：《生物技术通报》2013年第3期192-198,共7页Biotechnology Bulletin

基　　金：国家科技支撑计划课题(2012BAC07B03);上海市教委重点学科项目(S30701)

摘　　要：旨在大量制备条斑紫菜高纯度藻红蛋白并研究其光学性质。以江苏吕泗条斑紫菜为原料,采用"溶胀+组织捣碎"法破碎2 kg冻干条斑紫菜叶状体细胞,经多次硫酸铵盐析和一次羟基磷灰石层析后获得纯度>3.20的藻红蛋白的产率为0.184%,再经一次同样层析可获得纯度>4.50的藻红蛋白的产率为0.109%。蛋白纯度已通过吸收/荧光激发光谱,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)/非变性聚丙烯酰胺凝胶电泳(Native-PAGE)的考马斯亮蓝/醋酸锌双染验证。该蛋白摩尔消光系数经Folin-酚法和Bradford法测定分别为1.79 mL/(mg.cm)和1.73 mL/(mg.cm),20℃时在505 nm处的荧光量子产率为0.90,在日光灯、发光二极管、碘钨灯和激光4种光源下的光漂白效果以激光最显著,240μg/mL藻红蛋白10 min可接近碘钨灯辐照240min的光漂白率。Pilot preparation were tried to obtain highly purified phyeoerythrin （ PE ） in abundance from P. yezoensis in Lvsi city, Jiangsu Province and to study its optical properties. 2 kg freeze-dried P. yezoensis thallus was broken using ＂swelling ＆ smash＂ method. Further purification was performed by means of ammonium sulphate co-precipitation （ ASCP ） and hydroxyapatite （ HA ） column chromatogram and the purity of PE achieved 1.67 （ A565/A280 ） , corresponding to yield of 0.184%, The purity of PE could be improved beyond 4.5 with a yield of 0.109% if HAP chromatography repeated. PE was validated by absorption/fluorescence spectra and SDS-PAGE/Native-PAGE stained with coomassie brilliant blue/zinc acetate for its purity. Molar extinction coefficient （ MEC ） of PE were 1.79mL/ （ mg· cm ） and 1.73 mL/ （ mg· cm ） determined by folin-phenol method and Bradford method, respectively. Fluorescence quantum yield of PE was 0.90 at 505 nm wavelength at 20℃ . The PE effect of photo bleaching was the most significant under irradiating of laser among daylight lamp, light emitting diode （ LED ） , iodine-tungsten lamp and laser. For example, the photo bleaching rate of 240 μg/mL PE after 10 minutes＇ irradiating under laser was close to the value using iodine-tungsten lamp of 240 minutes＇ irradiating.

关 键 词：条斑紫菜 藻红蛋白 纯化 摩尔消光系数 荧光量子产率 光漂白 

分 类 号：S968.431[农业科学—水产养殖]