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

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.