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標題: 植酸酵素phyA基因在Pichia pastoris X33重組菌株之表現與應用
Expression and application of recombinant Pichia pastoris X33 constructed by phytase gene phyA
作者: 張明暉
Chang, Ming-Hui
關鍵字: 植酸酵素;phytase;Pichia pastoris X33;pPICZαA;pGAPZαA;SDS-PAGE;磷;米糠;大豆粕;油菜籽粕;葵花籽粕;Pichia pastoris X33;pPICZαA;pGAPZαA;SDS-PAGE;phosphorous;rice bran;soybean meal;rapeseed meal;sunflower meal
出版社: 土壤環境科學系所
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Attempts were made to enhance the availability of phytate-phosphate, poorly metabolized by monogastric animals, such as human, swine, poultry, and plants. The Aspergillus ficuum phytase gene phyA was cloned and constructed by pPICZαA and pGAPZαA, expressed in Pichia pastoris X33 (pPICZαA) and Pichia pastoris X33 (pPGAPZαA). The crude phytase extracts were applied to recover phosphorus from the fermented liquids of rice bran and soybean meal, rapeseed meal and sunflower meal, separately. The recombinant P. pastoris X33 were confirmed by a screening medium containing 100 μg mL-1 Zeocin, PCR and restriction enzyme. In addition, through electrophoresis the size of major fragment coincided with phyA.
The phytase was overexpressed in P. pastoris X33 (pPICZαA) after 2 days incubation with BMGY medium containing 1% (v v-1) glycerol then replaced with fresh BMMY medium containing 1% methanol (on daily basis) as carbon sources. The phytase activity increased evidently with time and reached 200 U mL-1 after 9 days induction. Also a practical way of releasing phosphorus from rice bran was found when 0.1 M sodium acetate buffer replaced with de-ionised water (pH 5.5 ± 0.1). Phosphorus recovery increased with time and reached 1.31% after 24 h incubation contributing to 81% inorganic P released from rice bran phytate. Hydrolysis of rice bran phytate adding phytase of the recombinant yeast showed no significant effect on hydrolysis rates at different activities of 200, 100, 50 U mL-1. However, hydrolysis rates differed significantly with 20, 5, 0 U mL-1. The dry weight of lettuce shoot after harvest treated with phytase was slightly higher than that treated with inactived phytase.
The phytase was successfully expressed in P. pastoris X33 (pPGAPZαA) without methanol induction and replacing culture medium over incubation period. The activity of extracellular crude phytase extract reached 16.5 U mL-1 after 4 days incubation at 30℃. Notably, all the non-recombinant P. pastoris X33 showed no enzyme activities. The SDS-PAGE analyses also presented the same result. The enzyme characteristics indicated two pH (5.5 and 2.5), and 50℃ were optimal for phytase. The thermostabilities of phytase appeared higher than a commercial enzyme (from A. niger). The Km 0.115 mM and Vmax 1.4 μmole min-1 from enzyme kinetics indicated the affinity for phytate of the phytase was higher than A. niger phytase, but lower than P. pastoris Pholp. P. pastoris X33 (pPGAPZαA) was able to metabolize glucose, fructose and glycerol, but unable to effectively metabolize xylose, disaccharides (sucrose, maltose, lactose) and polysaccharides (starch, cellulose). After 6 days incubating with various glucose concentration in YPD liquid media, the phytase activity was 6% > 4% > 3% > 2% > 1%. Moreover the molasses with 2% glucose could substitute glucose in YPD liquid media to produce phytase for reducing cost. Comparing phosphorus releasing effect of phytase, it was the highest when applied to sunflower meal with 7 fold increased of the initial concentration, then followed by appling to rapeseed meal and soybean meal with 5 fold and 3 fold increased, respectively.

為增進人類、豬及家禽等單胃的動物和植物難以吸收、代謝之植酸磷的有效性,本研究乃自可分泌植酸酵素之Aspergillus ficuum選殖出植酸酵素基因phyA,分別構築於pPICZαA和pGAPZαA表現載體,建構P. pastoris X33(pPICZαA)和Pichia pastoris X33(pGAPZαA)兩株重組菌株,產出胞外植酸酵素。並分別應用於水解米糠和大豆粕、油菜籽粕及葵花籽粕植酸磷以釋出磷素。構築之重組菌株皆可生長於含有 100 μg mL-1 Zeocin抗生素之YPDS(Yeast Extract Peptone Dextrose Sorbitol)篩選培養基,且重組載體和染色體DNA經由PCR與限制酵素確認,另主要核酸片段大小之電泳分析均與phyA符合。
P. pastoris X33(pPICZαA)重組菌株以含1%(v v-1)甘油之BMGY生長培養基培養2日之後,再以含1% 甲醇之新鮮BMMY培養基置換 BMGY,並以1% 甲醇追加、誘導培養至第9日,酵素活性可達200 U mL-1 。另pH 5.5 ± 0.1之去離子水可以替代pH 5.5的0.1 M醋酸鈉緩衝液作為粗酵素液水解米糠之反應溶液,且反應24小時之後磷濃度達1.31%,相當於81% 無機磷由米糠之植酸磷水解釋出。以不同稀釋濃度之粗酵素液分別與米糠反應24小時,結果顯示粗酵素液水解米糠釋出水溶性磷的濃度與100、50 U mL-1 無顯著差異,但與20、5及0 U mL-1 則差異顯著;萵苣盆栽試驗的結果顯示採收後地上部乾重,施用含有植酸酵素活性與米糠反應後濾液之Hoagland solution皆略高於酵素先失活之對照處理,且稀釋倍數較高之處理差異比較明顯。
P. pastoris X33(pGAPZαA)重組菌株不需甲醇誘導與培養基置換,即可分泌胞外植酸酵素,以YPD液體培養基於30℃與200 rpm條件下培養4日後,粗酵素液之植酸酵素活性達16.5 U mL-1,而非重組菌株P. pastoris X33則明顯無活性表現。SDS-PAGE 分析亦顯現重組菌株明顯有酵素活性表現而非重組菌株則無。酵素特性分析顯示該酵素具有兩個最適化pH值,分別為pH 5.5和pH 2.5,最適化溫度為50℃。熱穩定性比源自A. niger之商品化植酸酵素高。由酵素動力學分析Km = 0.115 mM和Vmax = 1.4 μmole min-1,顯示該植酸酵素與植酸之間的親和力較A. niger弱,但比P. pastoris Pholp強。P. pastoris X33(pGAPZαA)重組菌株可以代謝利用葡萄糖、果糖和甘油,但無法有效利用木糖和雙糖(蔗糖、麥芽糖、乳糖)以及多醣(澱粉、纖維素)。含有不同濃度葡萄糖之YPD液體培養基培養6日後之酵素活性依序為 6% > 4% > 3% > 2% > 1% ,而且含有2% 葡萄糖之糖蜜可以取代對照YPD液體培養基中之等濃度葡萄糖,降低植酸酵素生產成本;粗酵素液與大豆粕、油籽粕及葵花籽粕於pH 5.5之0.1 M 醋酸鈉緩衝液中,在37℃反應24小時,皆明顯有無機磷釋出,尤其以葵花籽粕最為明顯,比初始無機磷濃度約增加7倍,而油菜籽粕與大豆粕亦可分別約增加5倍與3倍。
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