Ng I-Son, Tsai Shau-Wei
Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan 70101, China.
Biotechnol Bioeng. 2005 Jul 5;91(1):106-13. doi: 10.1002/bit.20492.
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 (CPL) was partially purified and employed as the biocatalyst. 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 (DeltaDeltaH) and that of activation entropy (DeltaDeltaS), yet the latter was dominated for most of the reacting systems. The postulated active site model was supported from the variation of DeltaDeltaH and DeltaDeltaS 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.
以水饱和异辛烷中（R，S）-萘普生2,2,2-三氟乙基硫酯的水解拆分作为模型体系，发现当粗制的番木瓜脂肪酶（CPL）经过部分纯化并用作生物催化剂时，其比脂肪酶活性和热稳定性得到了提高。此外，还研究了部分纯化的番木瓜脂肪酶（PCPL）作为一种有效的对映选择性生物催化剂，用于在水饱和有机溶剂中对（R，S）-布洛芬硫酯进行水解拆分。在水饱和异辛烷中的动力学分析表明，酰基供体和酰基受体对脂肪酶活性、E值和对映选择性都有深远影响。对于在酰基部分的2-苯基部分的间位含有庞大取代基的（R，S）-非诺洛芬和（R，S）-酮洛芬硫酯，发现其对映选择性从（S）-硫酯转变为（R）-硫酯。此外，还估算了酰化步骤的动力学常数，以阐明动力学数据并推测活性位点模型。热力学分析表明，对映体识别是由活化焓（ΔΔH）和活化熵（ΔΔS）的差异驱动的，但在大多数反应体系中后者起主导作用。所推测的活性位点模型得到了ΔΔH和ΔΔS随酰基部分变化的支持，其中还说明了良好的线性焓-熵补偿关系。皱褶假丝酵母脂肪酶（CRL）和PCPL性能的比较表明，PCPL在热稳定性更好、对快速反应底物的脂肪酶活性相似或更好、时间进程稳定性以及酶成本较低方面优于CRL。
