摘要：靈芝為白腐菌類，比其他微生物擁有更多酵素種類，而各種靈芝中成功可為人工栽培的靈芝Ganoderma lucidum，其生長所製造的多醣除常用在健康食品外，特殊醣類生物轉換特性也被用在植物皂苷的轉換，所產生的特定醣基型式皂苷在維持健康及疾病輔助治療上有顯著效果，也用於食品風味調整。然而靈芝生物轉換過程受到許多因子影響難以穩定控制，除導致轉換目標物含量不一難以品管外，連帶影響生物轉換的應用性。此外，雖然已知酵素具有穩定生產的特性，但因多數靈芝研究只集中在其多醣功效，造成具有高度應用價值的靈芝來源酵素只有少數被鑑定出，關於能轉換皂苷醣基的酵素研究，至今仍停留於生物轉換層次而不知實際運作酵素且未被清楚探討，影響實際特定皂苷的生產。為了解決以上問題，探討靈芝酵素轉換特定醣基型式皂苷之作用機制與應用顯得重要且刻不容緩，一來可專一性生產特定醣基型式皂苷促進品管，另一方面可較有效獲得標準品有利於後續針對純物質之生理活性研究。本研究將應用目前已知能透過靈芝生物轉換並產生特殊生理活性成分的原料為素材，包括羅漢果皂苷(mogrosides)、芝麻木酚素(sesame lignans)、絞股藍皂苷(gypenosides)及茶葉醣苷前驅物，篩選靈芝中參與特定皂苷醣基轉換的酵素。三年期研究計畫將先以剛發現之靈芝特定培養條件下所產特殊酵素能力之結果為基礎，分離酵素混合物後詳細分析其對各類皂苷醣基之轉換活性、轉化能力與轉換機制等；另外也設計加入其他來源酵素，包括β-1,2醣苷鍵水解酵素、酵母菌醣基外切酵素與植物專門酵素對皂苷進行修飾，補足靈芝酵素轉換上的限制。第二年，針對特定培養條件下靈芝所產酵素進行鑑定，透過結合各種分子生物學方法、蛋白質體學、轉錄體定序分析，找出主要作用之關鍵候選酵素基因，再針對這些酵素之基因進行選殖及表現。第三年，將表現酵素純化並探討最適反應酸鹼值與溫度、酵素動力、基質專一性等多種特性；隨後也利用靜電紡絲酵素固定化技術在材質選用上的廣用特性，再配合不同固定化材質之化學官能基修飾，研製高度酵素相容性、抗菌性、抗垢性、磁吸性、酸鹼反應性與混入半導體之即時酵素活性監控特點之多功能奈米酵素纖維膜。最後使用微量滴定板系統對產製酵素薄膜最佳使用條件進行測試，也套用數學模型評估膜體通透性與質量傳輸特性，提供未來放大製程所需參數之依據。
Abstract: Lingzhi or Ganoderma spp. is a white-rot fungus with more kinds of extracellular enzymes than other microorganisms. Among various Ganoderma spp., G. lucidum is the only species that has been successfully cultivated for the bioconversion process of plant materials and are used as flavor enhancers in food and as an adjuvant in the treatment of diseases. However, the bioconversion processes of G. lucidum are difficult to control. Enzymes are known to be a better choice in the production of many products compared to the bioconversion methods in terms of stability, while most of the researches on G. lucidim still focus on its physiological effect of their polysaccharides, and few of the G. lucidim-derived enzymes with high application value have been identified. All the current researches still deal with the characterization of biotransformation attributes without knowing the actual enzymes involved, affecting the development of specific saponins production. To address these challenges, it is important to explore the mechanism and the application potential of G. lucidum’s enzymes in converting specific glycosyl saponins. Through enzymatic investigation along with new enzyme discovery, better quality controlled products would be produced due to the steady production of target compounds. In addition, more pure compounds would be obtained and facilitating the subsequent researches. In this proposal, we will use materials already known for special physiologically activities after the biotransformation process of G. lucidum to screen special enzymes, including mogrosides, sesame lignans, gypenosides, and tea glycoside precursors. This three-year research proposal will first be built up based on the preliminary data of special enzymes produced under the specific culture conditions of G. lucidum. After the enzyme mixture is isolated, its conversion activity, capacity, and mechanism for various saponin glycosyl groups will be analyzed. In addition, other sources of enzymes will also be investigated (i.e. β-1,2 glucosidase, yeast glycosyl modification enzymes and other plant-derived enzymes) to compensate G. lucidum’s restricted enzymatic functions and to assist the modification of saponins for specific saponin production. In the second year, the enzymes produced by G. lucidum under specific culture conditions will be identified. Our research strategy is using the combination of various molecular biological methods, the proteomics approach, and the transcript sequencing analysis, all of which will be performed to find the key enzymes contributing to the special saponin conversion properties. In the third year, the characteristics of the enzymes will be explored under various reaction conditions, such as pH and temperature, enzyme dynamics, and substrate specificity. Subsequently, with the versatility of material choices for enzyme immobilization during electrospinning, we will perform chemical modifications in materials for fiber synthesis. The functionalized fiber membranes will present high enzyme compatibility, antibacterial property, anti-fouling activity, magnetic attraction property, acid-base reactivity, and real-time enzyme activity monitoring characteristics. Finally, the microtiter plate system will be used to test the optimal conditions for operating the enzyme film. A mathematical model will also be used to evaluate the permeability and mass transfer properties of the membrane in order to provide the parameters needed for scale-up production in the future.