以果聚糖蔗糖酶催化生產低聚乳果糖之探討

Lactosucrose Production using Levansucrase

低聚乳果糖（O-β-D-galactopyranosyl-（1→2）-β-D-fructofuranoside；lactosucrose）是一種功能性低聚糖，已知具有一系列的生理功能，體內外及動物實驗證實其具保健功效，且安全無毒。此新型營養保健品對腸道保健及癌症預防有很大幫助。開發利用低聚乳果糖具極高之學術與工業價值，在提高人們的健康水準具重要的意義。本研究探討以酵素催化合成系統探討低聚乳果糖之合成，完成探討果聚糖蔗糖酶之製備、分離與純化，並以游離型酵素與固定化酵素合成低聚乳果糖條件探討。果聚糖蔗糖酶（Levansucrase）經沉澱、DEAE-Sepharose及Source 15Q陰離子交換層析後可得到純化之酵素，最後之純化倍數為24.0，回收率為43.7％，比活性達300.1 U／mg。將純化的樣品進行SDS-PAGE電泳分析，樣品之電泳分析呈現單一帶，對照標準品分子量，計算得分子量約為32.5 kDa。以游離型酵素-Levansucrase催化生產低聚乳果糖，結果顯示低聚乳果糖之最佳生產條件為游離型酵素（2.5U）、蔗糖／乳糖（1：1）濃度為100 g／L、溫度27˚C、pH 6的情況，在24h可產出35.8 g／L（生成率 35.8％；1.49 g／L／h）的低聚乳果糖。以固定化酵素-Levansucrase催化生產低聚乳果糖，結果顯示低聚乳果糖之最佳生產條件為固定化酵素2 g（約含150U）、蔗糖／乳糖（1：1）濃度為100 g／L、溫度27˚C、pH 6的情況下，在24h可產出36.9 g／L（生成率 36.9％；1.54 g／L／h）的低聚乳果糖。

Lactosucrose (O-β-D-galactopyranosyl-(1→2)-β-D-fructofuranoside) has a series of physiological functions that are safe and nontoxic and have been proven to be health-promoting in vitro, in vivo, and in animal experiments. This new nutraceutical promotes intestinal health and aids cancer prevention. The development and use of this oligosaccharide has high academic and industrial value and is of considerable significance in improving human health. This study involved the preparation, separation, and purification of levansucrase and its use in free and immobilized forms to synthesize lactosucrose. Precipitation, and DEAE-Sepharose and Source 15Q anion exchange chromatography were used to obtain the purified enzyme. The final purification ratio, recovery, and specific activity were 24.0, 43.7%, and 300.1 U/mg, respectively. The purified sample was subjected to SDS-PAGE analysis, and the estimated molecular weight of the enzyme was approximately 32.5 kDa. For the free enzyme, the optimal production conditions of lactosucrose were free enzyme = 2.5 U, sucrose:lactose (1:1) concentration = 100 g/L, temperature = 27°C, and pH = 6. With the free enzyme, 35.8 g/L of lactosucrose was produced in 24 h (production rate, 35.8%; 1.49 g/L/h). For the immobilized enzyme, the optimal production conditions of lactosucrose were immobilized enzyme = 2 g (approximately 150 U), sucrose:lactose (1:1) concentration = 100 g/L, temperature = 27°C, and pH = 6. With the immobilized enzyme, 36.9 g/L of lactosucrose was produced in 24 h (production rate, 36.9%; 1.54 g/L/h).