Monti Daniela, Ferrandi Erica Elisa, Righi Monica, Romano Diego, Molinari Francesco
Istituto di Chimica del Riconoscimento Molecolare, CNR, Via Mario Bianco 9, 20131 Milano, Italy.
J Biotechnol. 2008 Jan 1;133(1):65-72. doi: 10.1016/j.jbiotec.2007.09.004.
An intracellular esterase from the yeast Kluyveromyces marxianus CBS 1553 with interesting enantioselective hydrolytic activity towards racemic esters of 1,2-O-isopropylidene glycerol (IPG) was purified and characterized. Optimal culture conditions for the obtainment of the enantioselective esterase on a 5 l-fermentation scale were investigated. Two esterase activities (EST1 and EST2) in the crude cell extract were identified by native PAGE with specific activity staining and separated from each other by anion-exchange chromatography. EST1 showed higher activity and enantioselectivity than EST2 in the resolution of racemic IPG acetate and was further purified by hydrophobic interaction chromatography and preparative electrophoresis (final specific activity approximately = 300 U mg(-1), showing a main protein band with a molecular mass of 29 kDa. EST1 showed optimal activity between pH 8.0 and 10.0 and was stable in the pH range 7.0-10.0. Moreover, it was rather thermostable and active up to 80 degrees C, and retained most of its activity in the presence of 15% (v/v) of various organic solvents. The enzyme showed similar Vmax in the hydrolysis of the acetate esters of IPG, whereas the Km value towards (S)-IPG acetate was significantly lower than the one towards the (R)-enantiomer (5.3 and 70 microM, respectively). Finally, comparison of EST1 activity in the presence of different glycerol esters and synthetic substrates with different chain lengths showed a strong preference of this biocatalyst for short-chain substrates.
对来自马克斯克鲁维酵母CBS 1553的一种细胞内酯酶进行了纯化和表征，该酶对1,2-O-异亚丙基甘油（IPG）的外消旋酯具有有趣的对映选择性水解活性。研究了在5升发酵规模下获得对映选择性酯酶的最佳培养条件。通过天然聚丙烯酰胺凝胶电泳（PAGE）和特异性活性染色鉴定了粗细胞提取物中的两种酯酶活性（EST1和EST2），并通过阴离子交换色谱将它们彼此分离。在拆分外消旋IPG乙酸酯时，EST1比EST2表现出更高的活性和对映选择性，并通过疏水相互作用色谱和制备电泳进一步纯化（最终比活性约为300 U mg(-1)，显示出一条分子量为29 kDa的主要蛋白带）。EST1在pH 8.0至10.0之间表现出最佳活性，在pH 7.0 - 至10.0范围内稳定。此外，它具有相当高的热稳定性，在高达80℃时仍有活性，并且在15%（v/v）的各种有机溶剂存在下保留了大部分活性。该酶在水解IPG的乙酸酯时显示出相似的Vmax，而对(S)-IPG乙酸酯的Km值明显低于对(R)-对映体的Km值（分别为5.3和70 microM）。最后，比较EST1在不同甘油酯和不同链长的合成底物存在下的活性，表明这种生物催化剂对短链底物有强烈偏好。
