木瓜乳汁中之木瓜脂肪分解酵素首次被發現可應用於對掌性異構物之分割上。本論文主旨在於應用此酵素於2芳香取代基丙酸硫酯之動力分割及動態動力分割製程開發。

　　在以(R,S)-naproxen三氟乙硫酯於含有飽和水之有機溶液之分割系統中，我們探討溫度變化及溶劑效應對於脂肪分解酵素之活性及其鏡像選擇性(E值)的影響。結果發現最佳溫度在60oC時酵素具有最大的初始反應速率，而且反應溫度於45oC、溶劑為異辛烷時E值仍大於100。進一步進行動力學分析，應用Michaelis-Menten不可逆反應機構並考慮產物抑制及酵素失活現象，經偶合動力參數後得到實驗值與理論值相吻合。比較粗Carica papaya脂肪分解酵素(CPL)與Candida rugosa脂肪分解酵素(CRL)之表現，在以(R,S)-naproxen硫酯或氧酯為基質的反應中，顯示以異辛烷或環己烷為溶劑時均具有相似的反應初始速率、E值及熱力學參數ΔΔH、ΔΔS等。

　　部分純化木瓜脂肪分解酵素(pCPL)能夠提昇對(R,S)-naproxen三氟乙硫酯之動力分割效果。再利用極性溶劑、pH 8之緩衝溶液或含methyl-b-cyclodextrin之水溶液對部分純化木瓜脂肪分解酵素進行前處理，發現均可以改善酵素活性及鏡像選擇性。其中以異丙醚處理之效果最佳，但以氯化鈉或環冠醚處理則效果不彰。繼續以pCPL應用在各種(R,S)-芳香取代基丙酸硫酯分割中，探討乙醯提供者及乙醯受質對酵素活性及E值之影響時，發現在間位含有巨大取代基之fenoprofen及ketoprofen，其酵素鏡像選擇性會有反轉現象，配合動力分析結果，本文提出此酵素活性中心結構假說。此外，由熱力學分析指出pCPL或CRL進行分割時活化焓與活化熵之差值呈現良好的線性關係（稱為焓與熵的互補效應）。到目前階段為止，發現pCPL在熱穩定性、活反應性、隨時間之穩定性及價格都有優於CRL。

　　動態動力分割探討中，鹼觸媒三辛基胺於各類(R,S)-芳香取代基丙酸硫酯反應中，無論是消旋或非酵素之自我水解反應行為均符合Hammett關係。經由鹼觸媒之添加，除了提昇光學產品純度及轉化率，還可以減少產物酸之抑制，而且三辛基胺尚可活化酵素對於naproxen， fenoprofen及flurbiprofen三氟乙硫酯之反應性。

　　因為番木瓜乳汁成分受地區之氣候及培養方式有顯著差異，本文企圖以橄欖油為水解反應基質結合profen分割，篩選出更佳之酵素來源，其中由印尼地區生產之木瓜脂肪分解酵素(pCPL-Indo)在水相反應中，以pH 8.5為最佳反應pH值，而且在7到10之pH範圍仍具有相當高的活性。於動力分析與熱力分析之結果，顯示pCPL-Indo在60oC時其酵素構形有明顯改變，又以動力參數k2S及k2R之結果指出反應屬於乙醯化步驟為主要主導鏡像選擇之因素。此外，配合產物抑制及酵素失活之模式，偶合參數後得理論與實驗值吻合之結果。

　For the first time, the crude papain as the spary-dried latex from Carica papaya (EC 3.4.22.2) is discovered as a versatile enantioselective biocatalyst for obtaining chiral acids from their racemic thioesters. The dogma of this thesis is to develop the lipase-catalyzed kinetic resolution and dynamic kinetic resolution processes for obtaining the chiral profen from the corresponding (R,S)-profen thioesters.

　The hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl thioester via a crude Carica papaya lipase in water-saturated organic solvents is first employed as the model system, in which effects of temperature and solvents on the lipase activity and enantioselectivity are first investigated. An optimal temperature of 60oC for the initial rate of (S)-thioester and a high enantiomeric ratio greater than 100 at 45oC in isooctane are obtained. A kinetic analysis by considering product inhibition and lipase deactivation is furthermore performed in water-saturated isooctane at 45oC, leading agreements of experimental and best-fit time-course conversions. A detailed comparison of the kinetic and thermodynamic behaviors of Carica papaya and Candida rugosa lipases in isooctane and cyclohexane indicates that both lipases are very similar in terms of the thermodynamic parameters of ΔΔH and ΔΔS, E value and initial rate for the (S)-substrate when either (R,S)-naproxen thioester or ester is the substrate.

　With the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl thioesters in water-saturated isooctane as a model system, improvements of the specific lipase activity and thermal stability were found when a crude Carica papaya lipase was partially purified and employed as the biocatalyst. Improvements of lipase activity and enantioselectivity were obtained when polar organic solvents, pH 8 buffer or aqueous solution containing methyl-b-cyclodextrin, but not NaCl or 18-crown-6, were employed in the pretreatment. The lipase pretreated with isopropyl ether was selected as the best lipase and employed for the resolution of (R,S)-profen 2,2,2-trifluoroethyl thioesters. The partially purified Carica papaya lipase (pCPL) was furthermore explored as an effective enantioselective biocatalyst for the hydrolytic resolution of (R,S)-profen thioesters in water-saturated organic solvents. The kinetic analysis in water-saturated isooctane indicated that both acyl donor and acyl acceptor have profound influences on the lipase activity, E value and enantioselectivity. Inversion of the enantioselectivity from (S)- to (R)-thioester was found for (R,S)-fenoprofen and (R,S)-ketoprofen thioesters that contained a bulky substituent at the meta position of 2-phenyl moiety of the acyl part. Kinetic constants for the acylation step were furthermore estimated for elucidating the kinetic data and postulating an active site model. The thermodynamic analysis indicated that the enantiomer discrimination was driven by the difference of activation enthalpy (ΔΔH) and that of activation entropy (ΔΔS), yet the former was dominated for most of the reacting systems. The postulated active site model was supported from the variation of ΔΔH and ΔΔS with the acyl moiety, in which a good linear enthalpy-entropy compensation relationship was also illustrated. A comparison of the performances between Candida rugosa lipase (CRL) and pCPL indicated that pCPL was superior to CRL in terms of the better thermal stability, similar or better lipase activity for the fast-reacting substrate, time-course-stability and lower enzyme cost.

Both racemization and non-enzymatic hydrolysis of (R,S)-profen thioesters with trioctylamine as the catalyst follow the Hammett relationships. The dynamic kinetic resolution of the substrate by adding trioctylamine as the racemization catalyst and enzyme activator was furthermore performed, giving improvements on the yield and stereo-purity for the (S)-naproxen product. Similar enzyme behaviors for pCPL were demonstrated when 2,2,2-trifluoroethyl thioesters of (R,S)-fenoprofen and (R,S)-flurbiprofen were replaced as the substrate.
With hydrolysis of olive oil in aqueous solutions and hydrolytic resolution of (R,S)-profen 2,2,2-trifluoroethyl thioesters in water-saturated isooctane as the model systems, the lipolysis and enantioselective hydrolysis activities of four partially purified Carica papaya lipases of different plant variety and geography location of cultures were compared and selected pCPL-Indo from 　Indonesia as the best lipase preparation. For lipolysis, an optimal pH of 8.5 for all lipase preparations was found. Yet, pCPL-Indo possessed the highest activity at pH ranged from 7 to 10. For the kinetic resolution, the thermodynamic analysis implied that pCPL-Indo has changed the conformation at 60 oC and the enantiomer discrimination was dominated by ΔΔH. The kinetic analysis also indicated that the enantiomeric discrimination was mainly due to the difference of k2S and k2R in the acylation step. Agreements between the experimental time-course conversions XS and best-fitted results were illustrated by considering product inhibition and enzyme deactivation effects.

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