鳳梨皮中鳳梨酵素之分離純化與固定化探討

鳳梨酵素(Bromelain)，屬於半胱胺酸蛋白酶(Cysteine proteinase)，廣泛存在鳳梨全株。本研究利用農業廢棄物的鳳梨皮作為鳳梨酵素的萃取來源，利用超濾濃縮、冷凍乾燥及疏水性層析進行分離純化，得到鳳梨酵素總活性為757 U，比活性為145.11 U/mg，純化倍數為2.55倍，回收率為30.02%。以SDS-PAGE及Zymography對層析樣品進行分析，分子量為24.3 kDa。固定化試驗是將苦茶粕進行前處理，以實驗設計法對固定化因子進行少量實驗組數的排列組合，以Replicate plot、Coefficient plot、Residuals normal probability進行數據優化，得到最適化條件為2.17 mg鳳梨酵素與40 mg載體、4%交聯劑濃度、交聯時間為30分鐘，14C的交聯溫度，其固定化率與相對活性分別為66.81.2%及65.61.1%，鳳梨酵素之單位載體吸附量為29.10.5 g/mg。游離與固定化鳳梨酵素最適反應pH皆為7，游離酵素在pH 6-9可維持75%以上相對活性，固定化酵素則維持43%之相對活性。游離酵素的最適反應溫度為40C，固定化酵素則提升至50C。固定化酵素重複使用5次後相對活性降為44.62.2%；游離酵素在30-40C水浴1小時後可維持70%以上之相對活性，活化能為259.7 kJ/mole。此外，在1 mM Fe3+、Zn2+會抑制鳳梨酵素活性，其他金屬離子(Al3+、Ca2+、Cu2+、Mg2+、Mn2+、Na+)則具有提高活性能力，在10 mM濃度下僅Mg2+、Ca2+有助於提高酵素活性，適合作為酵素的輔因子。鳳梨酵素在高離子強度(1 M)下會被抑制活性，但在1-50 mM NaCl中可維持99.54.6%之相對活性。

關鍵字

鳳梨酵素；苦茶粕；分離純化；酵素固定化；實驗設計法

並列摘要

Bromelain, which belongs to the Cysteine proteinase, is widely present in whole pineapple plants. In this study, pineapple peel from agricultural waste was used as an extraction source of Bromelain. It was separated and purified by ultrafiltration, freeze-drying, and hydrophobic chromatography. The total activity of purified Bromelain was found to be 757 U, and the specific activity was 145.11 U/mg. The purification fold of 2.55 and 30.02% recovery yield were obtained. The chromatographic purified Bromelain was analyzed by SDS-PAGE and Zymography, and the molecular weight was 24.3 kDa. Immobilization was conducted with pretreatment Camellia oleifera seed shell, and the Design of Experiment method was used to array and combine a small number of experimental groups, and the data was optimized using Replicate plot, Coefficient plot, and Residuals normal probability. The optimal immobilization conditions were 1 mL purified Bromelain (2 mg/mL) and 40 mg Camellia oleifera seed shell, 4% cross-linker, cross-linking time 30 minutes, and cross-linking temperature 14C. The Percentage of immobilization and relative activity of Bromelain were 66.81.2% and 65.61.1%, respectively. Adsorption capacity is 29.10.5 g Bromelain/mg of Camellia oleifera seed shell. 　　The optimum pH for free and immobilized Bromelain is centred at 7, and the free Bromelain can maintain 75% relative activity at pH 6-9, whereas immobilized Bromelain maintain its relative activity about 43%. The optimal temperature were 40C and 50C for free and immobilized Bromelain, respectively. The relative activity of the immobilized Bromelain was retain at 44.62.2% after reused 5 cycles; the free Bromelain can maintain the relative activity of more than 70% after 1 hour between 30C and 40C, and the activation energy calculated for enzyme reaction is 259.7 kJ/mole. In addition, the activity of Bromelain were inhibited by Fe3+ and Zn2+ ions at the concentration of 1 mM, and other metal ions (e.g. Al3+, Ca2+, Cu2+, Mg2+ , Mn2+, Na+) have the ability to increase the activity. Only Mg2+ and Ca2+ could increased the Bromelain activity at the concentration of 10 mM, which is suitable as a cofactor for Bromelain. Bromelian activity could be inhibited at high ionic strength (1 M), but the relative activity may maintain at 99.54.6% in 1-50 mM NaCl.