頭髮菜絲狀體R型藻藍蛋白之提純與性質分析及光質對其生長與色素含量的影響

Purification and Characterization of R-phycocyanin from Bangia atropurpurea Conchocelis and the Effects of Different Light Qualities on the Growth and Pigment Contents

林書吟(Shu-Yin Lin)

指導教授 ： 周宏農

國立臺灣大學/生命科學院/漁業科學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.01745

摘要

R型藻藍蛋白 (R-phycocyanin, R-PC) 與R型藻紅蛋白 (R-Phycoerythrin, R-PE)、異藻藍蛋白 (Allophycocyanin, APC) 同為紅藻中光合作用的輔助色素，可作為螢光標誌應用於醫事檢驗試劑或天然染料應用於食品、化妝品色素。由於最近發現到其在醫療上的活性，擬對此蛋白質特徵進一步研究。藉由頭髮菜B. atropurpurea絲狀體的培養，冷凍乾燥藻體磨粉後以磷酸緩衝液萃取，經梯度飽和硫酸銨沉澱後，再經膠濾製備層析排除大量R-PE後，以羥基磷酸石灰管柱層析配合逐漸提高磷酸緩衝液濃度的方式沖提，純化出R-PC，其在618 nm與280 nm波長吸收的比值 (A618/A280) 為4.11，R-PC吸收光譜有兩個吸收高峰，分別位於553 nm及618 nm，使用618 nm波長激發，可在640 nm處獲得最大螢光強度。R-PC的SDS-PAGE呈現兩明顯之色帶：17.40 kDa與21.72 kDa，顯示其與文獻相符之α和β次單元體，這些證據顯示所設計的分離操作可以獲得高純度的PC，其回收率約為22.0%。在純化R型藻藍蛋白的過程中，同時成功純化了A565/A280為6.14之R型藻紅蛋白與A650/A280為4.18之異藻藍蛋白，R型藻紅蛋白有明顯的三個吸收峰，位於497 nm、543 nm及565 nm，並釋出波長在574 nm之螢光，異藻藍蛋白的最大吸收值位於650 nm，於618 nm附近有一肩峰，於664 nm處得到最大釋放螢光強度，皆與文獻相符。利用純質藻膽蛋白溶液於565 nm、618 nm及650 nm之吸收，求其於此三波長處之消光係數，建立從藻膽蛋白粗萃混合液中估算各藻膽蛋白含量之運算公式，並將其試用於比較不同光質對於色素含量之影響。從不同光質 (白光、藍光、綠光和紅光) 對頭髮菜絲狀體生長與色素含量的實驗結果中發現，以白光、藍光及綠光培養之頭髮菜絲狀體生長情形較以紅光培養者為佳，在培養22天後，不同光質對於頭髮菜絲狀體R型藻紅蛋白含量之影響沒有顯著差異，藍光及紅光會促進其R型藻藍蛋白與異藻藍蛋白的合成，綠光則是增加其葉綠素a的含量。

關鍵字

R型藻藍蛋白 ； 頭髮菜 ； 絲狀體 ； 藻膽蛋白 ； 純化 ； 光質

並列摘要

R-phycocyanin, R-PC, R-phycoerythrin, R-PE and allophycocyanin, APC are the photosynthetic accessory pigments of the red algae. As good fluorescent pigments and natural dyes, they have been applied in food, cosmetics and medical diagnostic reagents. Recently we found the immunological modulation bioactivity of R-PC, so as, tried to isolate more of this protein for further characterization studies. In this study, the Con-chocelis phase of Bangia atropurpurea was used for phycobiliprotein extraction. Am-monium sulfate salting-out and gel filtration chromatography, alter a series of R-PC was isolated and subject to a chromatographic purification by a hydroxylapatite column with a two-step gradient elution of K-phosphate buffers. Pure R-PC obtained had an absorb-ance ratio A618/A280 of 4.11. The purple fluorescent protein, R-PC was identified by its two major absorption peaks at 553 and 618 nm. The emission spectra of R-PC obtained had a maximal peak at 640 nm under excitation at 618 nm. From SDS-PAGE, R-PC was determined to consist of α and β subunits of molecular weights of 17.40 and 21.72 kDa, respectively. Both gel electrophoresis and spectroscopic data showed the purity of R-PC through the designed operation. In the same process, fractions, obtained alter gel filtration (A565/A280=6.14), showed the typical spectroscopic characters of R-PE, maxi-mal absorptions were detected at 497, 543 and 565 nm and emission at 574 nm. Allo-phycocyanin was also obtained with fractions (A650/A280=4.18) after R-PC in separation and determined the maximal absorption at 650 nm and emission 660 nm. In order to know the effect of light quality on the growth and comparison of biliproteins, B. atro-purpurea Conchocelis cultures were exposed to white, blue, green and red light in a 22-days experiment. Red light resulted in the lowest production of algal biomass and blue and green light illumination had no different in growth from the white light. R-phycoerythrin content was not affected by light quality, whereas R-phycocyanin and allophycocyanin content were enhanced by blue or red light. Green light increased the content of chlorophyll a.

並列關鍵字

R-phycocyanin ； Bangia atropurpurea ； Conchocelis phase ； phycobiliprotein ； purification ； light quality

參考文獻

Chiang Y.M., 1962. Marine algae of northern Taiwan (Rhodophyta). Taiwania 8, 143–165.

Chiang Y.M., 1972. Keys to the genera of marine algae of Taiwan. Taiwania 17, 8–13.

Airth R.L. and Blinks L.R., 1956. A new phycoerythrin from Porphyra naiadum. Biol. Bull. 3, 321–327.

Baker N.R., 1991. A possible role for photosystem II in environmental perturbations of photosynthesis. Physiol. Plant. 81, 563–570.

Bhat V.B., Gaikwad W.N. and Madyastha K.M., 1998. Hepatoprotective effect of C- phycocyanin for carbon tetrachloride and R-(+)-pulegone-mediated hepatotoxicity in rats. Biochem. Bioph. Res. Co. 249, 428–431.