在本研究中我們首次應用具熱穩定性的Klebsiella oxytoca 水解酵素（SNSM-87）為立體選擇性觸媒，於兩相介質中進行外消旋alpha-醇酯之水解分割，結果顯示結合介質工程（操作條件）、基質工程（醯基和離去醇基）和酵素固定化技術具有可以改善酵素鏡像選擇性或催化活性的效果。
首先以兩相介質中SNSM-87進行外消旋扁桃酸乙酯之水解分割為例，探討溶劑種類、溫度、pH、產物抑制、酵素使用量和基質濃度等各種反應條件對於酵素行為的影響。得到最佳操作條件為：緩衝溶液之pH在7和8之間、反應溫度45oC且酵素量不少於0.5 mg/mL，此時酵素具S選擇性（E = 56）並可在異辛烷相獲得單一鏡像異構R型扁桃酸乙酯。
再以SNSM-87作為觸媒於兩相介質中進一步水解外消旋鄰氯扁桃酸乙酯，所產生的R型鄰氯扁桃酸是合成Clopidogrel的重要中間物。研究各種操作條件對於酵素催化活性和鏡像選擇性的影響，獲得最佳條件為：反應溫度55oC、異辛烷為有機相以及含有2 mg/ml酵素的pH 6緩衝溶液為水相。在此最佳條件下進行動力分析，接著在酵素分割反應中進一步考慮酸抑制和非酵素催化水解，發現實驗和理論的轉化率對時間變化圖十分吻合。
利用外消旋鄰氯扁桃酸酯為基質，探討不同離去醇基對於酵素催化活性和選擇性的影響中，我們採用速率決定步驟為醯化步驟，且利用擴展的Michaelis-Menten機構進行動力分析。發現含有難離去醇基的S型鄰氯扁桃酸酯和含有各種不同的離去醇R型鄰氯扁桃酸酯一樣，兩者速率決定步驟均為四面中間體斷鍵形成acyl-enzyme中間體。然而對於含有易離去醇基的S型鄰氯扁桃酯，形成四面中間體才是速率決定步驟，故導致甲酯有最佳的鏡像選擇性。在兩相介質中也將SNSM-87應用到其他外消旋-醇酯的水解分割，並且發現甲酯擁有最高的選擇性和反應性。在兩相介質中以SNSM-87催化水解分割外消旋alpha-chlorophenyl acetate也得到對於反應快的R型有兩階段Brnsted斜率，但卻對於反應慢的S型酯只有一階段斜率的現象。並更進一步比較將alpha-氫氧基改為alpha-甲基或alpha-氯基時，對於SNSM-87和lipases之酵素催化活性、鏡像選擇性和光學選擇性的影響。
將SNAM-87經由共價鍵結固定在經環氧活化的聚丙烯酸高分子 Eupergit C 250L上，發現溫度、pH和有機溶劑對於固定化酵素催化水解外消旋扁桃酸乙酯的催化活性和鏡像選擇性具有與自由酵素不同的影響。在pH 6、55oC並以異辛烷為有機相的反應條件下，固定化酵素對於S型扁桃酸乙酯維持30%以上的催化活性，並使E值從43.6增加至319。熱力學參數分析顯示，固定化酵素的–ΔΔH從自由酵素的20.1增加至42.0 kJ/mol，而且–ΔΔS也從31.0增加至81.5 J/mol K。動力分析顯示固定化SNSM-87的過渡狀態斷鍵的質子轉移變慢，特別是針對反應慢的R型酯。另外結合基質工程和酵素固定化技術，針對其它alpha-醇酯的分割，發現可以增加酵素鏡像選擇性，而且固定化酵素在兩相系統中可重複使用8次而不損失活性和鏡像選擇性。因此所開發之固定化程序可能是提供SNSM-87工業化應用在兩相介質中水解分割的解決方法。

For the first time, the thermally stable Klebsiella oxytoca hydrolase (SNSM-87) is explored as an enantioselective biocatalyst for the hydrolytic resolution of (R,S)-alpha-hydroxy acid esters in biphasic media. In this study, we show how medium engineering (operating parameters), substrate engineering (acyl part and leaving alcohol) and enzyme immobilization can be combined to improve the enzymatic enantioselectivity or activity.
The hydrolytic resolution of (R,S)-ethyl mandelate via SNSM-87 in biphasic media is first investigated. Effects of various process parameters such as solvent type, temperature, pH, product inhibition, enzyme loading and substrate concentration on the enzyme performance were studied, leading to the high enzyme (S)-enantioselectivity of E = 56 at 45oC for the reaction media consisting of isooctane and pH 7 buffer. The optimal conditions of pH between 7 and 8, temperature 45oC and enzyme loading no less than 0.5 mg/ml were proposed for obtaining optically pure (R)-ethyl mandelate remained in the organic phase.
The hydrolytic resolution of (R,S)-ethyl 2-chloromandelate in biphasic media by using SNSM-87 as a promising biocatalyst is then reported for producing the desired (R)-ethyl 2-chloromandelate as an important intermediate for the synthesis of Clopidogrel. Effects of various operating parameters on the enzyme activity and enantioselectivity were systematically studied, from which the optimal condition of using isooctane as the organic phase, enzyme concentration of 2 mg/mL, temperature of 55oC, and pH 6 of the aqueous phase was selected. The kinetic constants were further estimated from the kinetic analysis at the optimal condition. By further considering the acid product inhibition and non-enzymatic hydrolysis for the enzymatic resolution, good agreements of the time-course conversions from experiments and the theoretical prediction were also obtained.
For studying the effect of leaving alcohol on the enzymatic activity and enantioselectivity using (R,S)-2-chloromandelates as model substrates, an expanded Michaelis-Menten mechanism for the rate-limiting acylation step was adopted for the kinetic analysis. The fast-reacting (S)-2-chloromandelates containing a difficult leaving alcohol moiety, as well as that the slow-reacting (R)-2-chloromandelates in the whole range of leaving alcohol moieties, indicates that the breakdown of tetrahedral intermediates to acyl-enzyme intermediates is rate-limiting. However, the rate-limiting step shifts to the formation of tetrahedral intermediates for the (S)-2-chloromandelates containing an easy leaving alcohol moiety, and leads to an optimal enantioselectivity for the methyl ester substrate. The SNSM-87 is also explored as an enantioselective biocatalyst for the hydrolytic resolution of the other (R,S)-alpha-hydroxy acid esters in biphasic media, where the best methyl esters possessing the highest enantioselectivity and reactivity are selected and elucidated in terms of the structure-enantioselectivity correlations and substrate partitioning in the aqueous phase. Two-step Brnsted slopes for the fast-reacting (R)-ester but only one-step for the slow-reacting (S)-ester are further obtained in the hydrolytic resolution of (R,S)-alpha-chlorophenyl acetate in biphasic media. A replacement of the alpha-hydroxy moiety to alpha-methyl or alpha-chloro group has profound effects on changing the enzymatic activity, enantioselectivity and optical-preference for SNSM-87 and lipases.
The Klebsiella oxytoca hydrolase was immobilized on epoxy-activated acrylic polymers Eupergit C 250L via covalent attachment. The effects of temperature, pH, and organic solvent on the enzymatic activity and enantioselective for the hydrolysis of (R,S)-ethyl mandelate were investigated. The immobilized enzyme retains more than 30% catalytic activity for the (S)-ethyl mandelate and increases the E value from 43.6 to 319 at pH 6 and 55oC with isooctane as the organic phase. An analysis of the thermodynamic parameters indicates that –ΔΔH increased from 20.1 to 42.0 kJ/mol as well as –ΔΔS from 31.0 to 81.5 J/mol K after immobilization when comparing with the free enzyme. The kinetic analysis for the immobilized SNSM-87 shows the slower proton transfer for the breakdown of transition state, especial for the slow reacting (R)-ester. In addition, the combination of substrate engineering and enzyme immobilization for the hydrolytic resolution of other (R,S)-alpha-hydroxy acid esters also results in an increase of enantioselectivity. The immobilized enzyme could be used in the repetitive manner more than 8 times without loss the activity and enantioselectivity in the biphasic system. Therefore, the immobilization procedure developed may provide a promising solution for the industrial application of SNSM-87 in biphasic system.

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