Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/52115
標題: 利用高蝦紅素生產變異株Xanthophyllomyces dendrorhous同步生產蝦紅素與果寡糖生產酵素之可行性探討
Feasibility of simultaneous production of astaxanthin and transfructosylating enzyme from an astaxanthin-hyperproducing mutant Xanthophyllomyces dendrorhous
作者: Lee, Shu-Chih
李書志
關鍵字: astaxanthin;蝦紅素;fructooligosaccharides;果寡糖
出版社: 食品暨應用生物科技學系所
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摘要: 
In this study, the wild strains of X. dendrorhous CBS 6938 and P. rhodozyma BCRC 21346 were treated with mutagenic agent N-methyl-N''-nitro-N-nitroso-guanidine (NTG) and plated on yeast malt (YM) agar containing b-ionone as a selective medium. One of mutant isolate (X. dendrorhous NCHU-FS701) was found to contain more than threefold of astaxanthin content compared with the parental strain X. dendrorhous CBS 6938. The total carotenoids content of the mutant NCHU-FS701 was 1,508 ug g-1 yeast, and 87% of the total carotenoids was astaxanthin. The mutant NCHU-FS701 could produce higher astaxanthin concentration, but it grew slower than parental strain CBS 6938 in YM broth.
The cost-down medium including molasses, urea, and CaCl2 were selected and optimized by using Box-Behnken design under shaker flask cultivation of X. dendrorhous NCHU-FS701. Maximum astaxanthin production of 11 g ml-1 was obtained with the medium composition of 43.4 g L-1sugar as glucose equivalent in molasses, 2.49 g L-1 urea, and 0.73 g L-1 CaCl2. Based on the media composition, cell growth and astaxanthin production were enhanced when scale up of astaxanthin production was conducted in a 5-L stirred tank reactor with continuous air supply and pH control.
Cell disruption was needed because the astaxanthin and transfructosylating enzyme are intracellular. The optimal setting for 30 g-DCW L-1 yeast cell disruption was found to consist of 0.5-mm-diameter glass bead at 10C by using a bead beater for 20 min, in which more than 97% yeast disruption could be observed under microscopy. The disrupted cell culture was then extracted with hexane/ethyl acetate, and 72% total carotenoids in the organic solvent phase and 446 U mg-1 protein of transfructosylating enzyme activity in the water phase was obtained, indicating a simple step for simultaneously obtaining astaxanthin and transfructosylating enzyme.
For the production of fructooligosaccharides (FOS), the transfructosylating enzyme was immobilized onto chitosan using Tris (hydroxymethyl) phosphine (THP) as coupling agents. The optimal pH was 5.0 and the optimal temperature was 60oC for both free and immobilized enzyme. The THP-immobilized enzyme had better thermal stability, with 70% residual activity even after 10 repeated runs at 60oC. Metal ions, such as K+ and Fe3+ had positive effect on the enzyme activity.

本研究利用致突變劑N-methyl-N''-nitro-N-nitroso-guanidine (NTG)誘導X. dendrorhous CBS 6938及P. rhodozyma BCRC 21346等野生菌株發生突變,進而用含b-ionone的YM培養基篩選出蝦紅素高產量的菌株,高蝦紅素生產變異株X. dendrorhous NCHU-FS701之總類胡蘿蔔素產量為1,508 ug g-1 yeast,其中色素的87%為蝦紅素,其產量較X. dendrorhous CBS 6938或P. rhodozyma BCRC 21346皆高出3倍以上,變異株NCHU-FS701在YM 培養基的培養條件與原始菌株CBS 6938並無差異,然其生長速率較為緩慢。
利用Box-Behnken design的實驗設計,探討廉價碳源、氮源與金屬離子之最適化蝦紅素生產條件;結果顯示於三角瓶規模中,當以糖蜜43.4 g L-1、尿素2.49 g L-1及氯化鈣0.73 g L-1為培養基時有最大蝦紅素之生產(10.97 mg L-1)。於相同培養基且在固定溶氧及pH 6.5調控下的5 L發酵槽中培養120 hr,則可獲得13.58 mg L-1蝦紅素;若碳源提升2倍時,蝦紅素的生產可進一步提升至25.61 mg L-1。
X. dendrorhous NCHU-FS701所生產的果寡糖以neokestose為主,1-kestose次之,其果寡糖生產酵素屬胞內酵素。本研究利用珠磨式組織研磨機進行菌體破碎,以0.5 mm的玻璃珠處理菌液(30 g-DCW L-1)20分鐘能獲得97%的菌體破碎,再以有機溶劑充分混合破碎細胞懸浮液,於有機層中可回收72%的蝦紅素,水層中則可回收果寡糖生成酵素(446 U mg-1 protein),此一步驟可同步獲得蝦紅素及果寡糖生成酵素。
X. dendrorhous NCHU-FS701所萃取的果寡糖生成酵素在60oC與pH 5.0時有最大比活性(446 U mg-1 protein),而金屬離子如K+與Fe3+的添加能提升酵素活性,鎂離子則能提升neokestose於所生產總果寡糖中之比例。當果寡糖生成酵素之粗酵素液以Tris (hydroxymethyl) phosphine (THP)為交聯劑,與幾丁聚醣共交聯進行酵素固定化後,可提升果寡糖生成酵素的熱穩定性,且固定化的酵素經10次的重覆利用後,仍保有70%的酵素活性。
URI: http://hdl.handle.net/11455/52115
Appears in Collections:食品暨應用生物科技學系

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