利瑪原甲藻的黑海綿酸釋放與提純最佳化研究

Abstract

本研究探討黑海綿酸的提純最適化，在研究初期發現藻細胞在培養至穩定期後，釋放黑海綿酸到培養液中的濃度大量提高，不利於從藻細胞內提純黑海綿酸，此時針對培養液中黑海綿酸的提純，較從藻細胞後內提純更簡單，因此本研究以合成吸附樹脂SP825吸附培養液中的黑海綿酸後，再行脫附提純。SP825對黑海綿酸吸附實驗中，發現SP825吸附承載量至少為4.736 mg/g，而SP825對黑海綿酸的脫附率可達96.9 %，整個操作不僅能完全吸附培養基中的黑海綿酸，並可利用甲醇來達到脫附毒素的目的，增加量化生產時濃縮操作的方便性，顯示SP825可用於黑海綿酸吸附萃取；接著以二氯甲烷進行分配萃取，並將二氯甲烷層進行矽膠速分管柱層析，利用正己烷與異戊烷為沖提液，並以梯度方式進行沖提，合併含毒素分液後，以製備型高效液相層析更進一步純化毒素，最後能獲得95%純度的黑海綿酸。 利用SP825茶包與藻細胞培養實驗中，發現SP825茶包組黑海綿酸釋放量遠低於控制組，推測高濃度黑海綿酸對藻細胞是一種生存環境壓力，造成藻細胞本身釋放更多黑海綿酸。另外，本實驗分析17株利瑪原甲藻藻株間的黑海綿酸含量，發現不同藻株之間與藻細胞內外黑海綿酸含量皆有差異，以#1a為黑海綿酸生產之最適藻株，其單位細胞總產毒量47.2 pg/cell為最高。

This research utilize the release of okadaic acid in Prorocentrum lima to probe the best purification method of okadaic acid. When growing to the steady phase in experiment, cells begin to release okadaic acid to media fast which makes better purificaiton than directly from cell. This research utilize SP825 resin absorb okadaic acid in media, and then purify it. In the experiment of okadaic acid content analysis among different Prorocentrum lima , the differences of okadaic acid content are among Prorocentrum lima strains, and cell, and media. The strains #1a, #2 and #15 have relatively high okadaic acid content per cell. And SP825 resin adsorbability of okadaic acid is 4.736 mg / g at least, however SP825 resin deadsorbent rate can be up to 96.9%. To extract okadaic acid using dichloromethane partition SP825 methanol eluent, before running silica gel flash column. The mobile phase are hexane and isopropanol. After combining toxin dichloromethane fraction, it’s reverse phase high performance liquid chromatography purification and then we can get 95% pure okadaic acid. From deadsorption okadaic acid with SP825 to purification, it’s certainly easier procedures and less time and money consuming. . In the experiment of P. lima culture with SP825 teabag, the result shows the okadaic acid SP825 teabag group producing is far less than control group. The author infers that okadaic acid is a key factor as environmental pressure of P. lima. When okadaic acid in media increases, it results in P. lima releasing more okadaic acid instinctively. Besides, in the experiment of okadaic acid content analysis among different Prorocentrum lima , the differences of okadaic acid content are among Prorocentrum lima strains, and cell, and media. The strains #1a, #2 and #15 have relatively high okadaic acid content per cell suiting okadaic acid purification.