中文摘要

木瓜乳汁中所含之木瓜脂肪分解酵素已被發現可應用於對掌性異構酸與其衍生物之動力分割。本論文旨在從篩選和純化酵素、介質工程以及基質工程三個不同的角度切入，探討應用此酵素於(R,S)-naproxen酯類之光學分割的製程改善。
在篩選和純化酵素方面，首先於含有飽和水之有機溶劑中進行水解分割(R,S)-naproxen三氟乙酯時，我們探討溫度及溶劑效應對於此酵素活性及鏡像選擇性(E值)的影響。結果發現以異辛烷為反應溶劑時，在最佳反應溫度60℃時有最大的初始反應速率，且擁有高的E值122。另外，在較親水性的溶劑像是環己烷或是醚類中，酵素的活性和E值都會嚴重下降。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的現象，經偶合動力參數後可得到實驗值與理論值相吻合的結果。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，顯示前者在活性和選擇性上都優於後者，且在60℃、異辛烷為反應溶劑時，前者都有較穩定的表現。
在含有飽和水之有機溶劑中進行反應時，發現利用來自厄瓜多的Carica pentagona Heilborn木瓜脂肪分解酵素(CPHL)亦可得到不錯的結果。另外，與CPL比較，發現兩者對於較親水性的溶劑都有較差的反應結果，且都會被產物(酸或醇)所抑制。當此兩酵素以部份純化的形式進行反應時，在酵素的活性和選擇性上，均可得到不錯的改善。此外，由熱力學分析指出活化焓對於反應選擇性E值有較大的影響，而其中對於利用CPL在(R,S)-fenoprofen三氟乙硫酯的分割上卻是個例外。
在介質工程方面，探討添加有機可溶性的鹼對於利用部份純化木瓜脂肪分解酵素(pCPL)在含有飽和水異辛烷的分割效應時，發現不同種類鹼對酵素的活性和選擇性具有相當不同程度的影響，亦即其可扮演酵素活化劑或酵素抑制劑。當添加120 mM 三乙基胺於反應系統時，可活化pCPL而使反應初始速率有2.24倍增加的效果。在60 mM 的三乙基胺的添加下，對於所有的酵素活性都有改善，但選擇性E值卻有下降的趨勢，不過對以pCPHL為觸媒之反應系統卻有相反的結果。應用Michaelis-Menten不可逆反應機構偶合得到實驗值與理論值吻合的結果，且由動力學分析顯示在酵素進行醯化反應(acylation)時，質子由四面中間體(tetrahedral intermediates)到醯化酵素中間體(acyl-enzyme)的步驟相較於四面中間體的形成有著較顯著受到添加物影響。然而，對於扮演酵素抑制劑的有機鹼而言，並沒有觀察到明顯的相互關係。
在基質工程方面，利用pCPL在含有飽和水的異辛烷酯類水解分割上，發現基質含有不同拉電子能力的離去基對於初始反應速率有著很明顯的影響。利用不含有二甲基胺基質且離去基拉電子能力(inductive number)由0.01增加為0.4時，酵素的活性會有630倍的提升，因而可推論酵素反應決定步驟是在醯化的步驟。另外，當利用inductive number由0.01到0.11的基質時，我們也推測在此反應中，質子由四面中間體裂解成醯化酵素中間體的步驟可能是速率決定步驟。另外，我們也利用了一個新的基質工程的方法-質子轉移設計(proton shuttle device)的概念，顯示由於增加過渡狀態時分子內氫鍵而大大的改善酵素的活性。對於離去基含有相同拉電子能力的兩個基質，應用這種方式可使反應速率增加約60倍，且由動力學分析可得到k2S/KmS有80到90倍的提升。另外，在相同基態假設下，所增加的倍數(相當於11.5~11.9 kJ/mol)也與增加一個氫鍵的能量相符合。對於不同來源脂肪分解酵素的影響方面，此設計也具有同樣增加酵素活性的效果。

ABSTRACT

The papaya lipase stored in the crude papain from spay-dried latex of Carica papaya has been discovered as a versatile enentioselective biocatalyst to obtain chiral acids by using hydrolytic resolution of their racemic esters. The dogma of this dissertation is to improve the lipase-catalyzed hydrolysis of (R,S)-naproxen ester practised by adopting the enzyme screening, purification, medium engineering, and substrate engineering concepts.
In terms of the enzyme screening and purification, we employed the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl ester via a crude Carica papaya lipase in water-saturated organic solvents as the model system, where effects of temperature and solvents on the lipase activity and enantioselectivtiy were studied. The optimal temperature of 60℃ for the maximum initial rate of (S)-ester with a high enantiomeric ratio (E = 122) in water-saturated isooctane is obtainable. Additionally, less hydrophobic solvents of cyclohexane and MTBE than isooctane are vital on decreasing the lipase activity and enentioselectivity. A kinetic analysis is further performed by using a Michaleis-Menten mechanism combined with the product inhibition and lipase deactivation, leading to good agreements of experimental and best-fitted conversions. Comparisons of enzyme performances for Carica papaya and Candida rugosa lipases indicate that the former is more enantioselective, active, and stable for (S)-naproxen ester.
A crude lipase stored in Caraica pentagona Heilborn latex was explored as an effective enantioselective biocatalyst for the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl ester in water-saturated solvents. A comparison of the enzyme performances in terms of activity or enantioselective with those from Carica papaya lipase indicates that both enzymes display low tolerance to the hydrophilic solvent and are inhibited by (S)-naproxen or 2,2,2-trifluoroethanol. Improvements on the lipase activity and enantioselectivity are found when using both lipases in partially purified form as the biocatalysts. By utilizing the thermodynamic analysis, the enantiomeric discrimination is mainly driven by the difference of activation enthalpy for all reaction systems except for the hydrolysis of (R,S)-fenoprofen 2,2,2-trifluoroethyl thioester employing Carica papaya lipases as the biocatalyst.
As for medium engineering, the performance of pCPL for the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl ester in water-saturated isooctane is altered when adding a variety of organo-soluble bases that act as either enzyme activators (i.e. TEA, MP, TOA. DPA, PY, and DMA), or enzyme inhibitors (i.e. PDP, DMAP, and PP). Triethylamine (TEA) was selected as the best enzyme activator as 2.24-fold increase of the initial rate for (S)-naproxen ester was achieved when adding 120 mM of the base. Furthermore, the activity enhancement for all lipases can also be observed, but with a negative effect on enantioselectivity, except for the partially purified lipase from Carica pentagona Heilborn as 60 mM TEA is added. By applying an expanded Michaelis-Menten mechanism for the acylation step, the kinetic analysis indicates that the proton transfer for the breakdown of tetrahedral intermediates to acyl-enzyme intermediates is more affected than that for the formation of tetrahedral intermediates when adding an enzyme activator. However, no correlation for the proton transfer in the acylation step is found when adding the base acting as an enzyme deactivator.
As for substrate engineering, with the hydrolytic resolution of (R,S)-naproxen ester via pCPL in water-saturated isooctane at 45℃ as the model system, the substrate containing a leaving group of different electron-withdraw capability has an extreme influence on the enzyme activity. About 630-fold enhancement of the initial rate for (S)-ester are obtainable when increasing the inductive parameter from 0.01 to 0.4 for the alcohol without containing a dimethylamino group. This implies that the acylation step must be the rate-limiting step. Meanwhile, we also assume that the bond-breaking of tetrahedral intermediate to acyl-enzyme intermediate may be the rate-limiting step for the compounds that alcohol group is hard to leave (e.g. 0.01 to 0.11). On the other hand, a novel means of using substrate-assisted catalysis indicates that the proton shuttle device acts as an effective tool for improving the lipase activity by forming an extra intramolecular hydrogen bond in the transition state. For the substrates with the same inductive parameter of 0.01 or 0.03, about 61-fold enhancement of lipase activity is obtainable. Moreover, about 80 ~ 90-fold increase of k2S/KmS implies the free energy difference of 11.5 ~ 11.9 kJ/mol between the transient states if one assumes the same ground states for both substrates. These values agree well with the stabilization by an additional hydrogen bond. Similar results for the activity enhancement are also found for using other lipases.

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