生質柴油是一種可再生且具有環保性質的替代能源，目前利用生物法生產生質柴油，大多是以固定化酵素或全細胞為觸媒，而這些生物觸媒製備繁複、價格昂貴，會增加生產生質柴油的成本和能量消耗，因此需要開發新的具有經濟效益的生物觸媒及生物催化製程。本研究開發了一種同步整合重組脂肪酶製備與原位生質柴油合成的新製程，此製程以畢氏酵母菌Pichia pastoris表達重組疏棉狀嗜熱絲孢菌脂肪酶Thermomyces lanuginosus lipase ( TLL )，直接利用含有胞外酵素及全細胞的培養液作為觸媒催化生質柴油合成，而重組P. pastoris可以利用生質柴油合成過程中所產生的副產物-甘油做為碳源，持續製造分泌重組TLL至培養基中催化生質柴油合成。我們構築了重組質體pGAPZα-TLL，在P. pastoris X33中成功表達具有活性的TLL並分泌到培養基中，分析此重組TLL性質，顯示在37℃，pH 10具有最佳的水解活性，屬於耐鹼性酵素。以此重組P. pastoris培養液為觸媒，大豆油為原料，進行原位生質柴油合成，並採用兩種不同反應流程，即同步轉酯化與酯化反應(concurrent method) 與先水解再酯化逐步反應(stepwise method)，結果顯示這兩種反應流程，油相與水相(即培養液)混合的最佳體積比例相似(皆為1:3)，最佳的甲醇總添加量也相似(皆為32%)，但是所達到的生質柴油轉化率分別為88.9% (concurrent method) 與76.0% (stepwise method) 。我們嘗試在水相中控制添加相同菌量，即油相與培養液混合的體積比例固定為1:1，再以液體培養基調整水相體積，結果發現兩種反應流程的最佳油相與水相混合體積比例皆改變為1:5，最佳甲醇總添加量皆仍為32%，但是在生質柴油轉化率則顯著提升分別為91.4% (concurrent method) 與94.4% (stepwise method)，酵素活性測試亦顯示，控制添加相同菌量的培養液中含有較高的重組脂肪酶活性。本研究結果顯示，整合型生質柴油合成的產率顯著高於非整合型，而且以油相與培養液混合比例1:1，油相與總水相混合比例1:5，並以stepwise method添加32%甲醇，反應96小時後可得最高94.4%的生質柴油轉化率。這種簡化的製程無需酵素製備的過程，故可適用於工業生產生質柴油。

In previous lipase-catalyzed production of biodiesel, a renewable and environmentally friendly alternative liquid fuel, immobilized enzymes or whole cell catalysts have been extensively used. However, enzymes are prepared in an independent process separated from enzymatic biodiesel production. This would increase the cost and energy consumption. Therefore, there is an urgent need to develop novel cost-effective biocatalysts and biocatalytic processes. In this study, a novel and efficient integrated process with coupled lipase production and in situ biodiesel synthesis has been developed. Based on a recombinant Pichia pastoris culture expressing Thermomyces lanuginosus lipase (TLL), the dual biocatalytic system takes advantage of both cell free enzymes and whole cell catalysts. The recombinant P. pastoris utilizes glycerol, the by-product of biodiesel synthesis, as a carbon source and constitutively secretes recombinant TLL into medium to catalyze the synthesis of biodiesel. We had constructed reconstruct pGAPZα-TLL plasmid into P. pastoris X33. The recombinant P. pastoris strain secreted active TLL into culture medium. Biochemical analysis revealed that the recombinant TLL had an optimal temperature at 37℃ and showed alkalophilic property with an optimal pH at 10. Using recombinant P. pastoris liquid culture as biocatalyst and soybean oil as raw material, in-situ biodiesel synthesis was performed through two different reaction processes, namely concurrent and stepwise methods. The results showed that these two methods had a similar optimal oil-water (i.e. oil-culture) ratio of 1:3, and similar optimal total methanol feeding amounts (32%). The biodiesel yields were 88.9% for concurrent method and 76.0% for stepwise method. When adjusting a same amount of yeast cells (i.e. oil-culture ratio is 1:1) in different oil-water ratios, the optimal oil-water ratio became 1:5 between these two methods, and the optimal total methanol feeding amounts did not change (32%). However, the biodiesel yields were significantly increased to 91.4% for concurrent method and 94.4% for stepwise method. Lipase activity assay revealed the culture from adding same amount of cells contained higher amount of lipase than the culture with different amount of cells. This study suggested that the biodiesel yield of the integrated process was higher than the non-integrated process (i.e. using separately prepared catalysts), and the highest 94.4% biodiesel yield can be achieved under 1:1 oil-culture ratio and 1:5 oil-water ratio, and by feeding 32% methanol using stepwise method for 96-hour reaction. This simplified single-step process could represent a significant advance toward achieving economical production of biodiesel at industrial scale.

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