Lindsay John P, Clark Douglas S, Dordick Jonathan S
Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
Biotechnol Bioeng. 2004 Mar 5;85(5):553-60. doi: 10.1002/bit.20002.
A combinatorial experimental technique was used to identify salts and salt mixtures capable of activating penicillin amidase in organic solvents for the transesterification of phenoxyacetate methyl ester with 1-propanol. Penicillin amidase was lyophilized in the presence of various chloride and acetate salts within 96-deep-well plates and catalytic rates measured to determine lead candidates for highly salt-activated preparations. The kinetics of the most active formulations were then further evaluated. These studies revealed that a formulation consisting of 98% (w/w) of a 1:1 KAc:CsCl salt mixture, 1% (w/w) enzyme, and 1% (w/w) potassium phosphate buffer was approximately 35,000-fold more active than the salt-free formulation in hexane, as reflected in values of V(max)/K(m). This extraordinary activation could be extended to more polar solvents, including tert-amyl alcohol, and to formulations with lower total salt contents. A correlation was found between the kosmotropic/chaotropic behavior of the salts (as measured by the Jones-Dole B coefficients) and the observed activation. Strongly chaotropic cations combined with strongly kosmotropic anions yielded the greatest activation, and this is likely due to the influence of the ions on protein-water and protein-salt interactions.
采用一种组合实验技术来鉴定能够在有机溶剂中激活青霉素酰胺酶以实现苯氧乙酸甲酯与1-丙醇酯交换反应的盐及盐混合物。在96孔深孔板中，于各种氯化物和醋酸盐存在的情况下对青霉素酰胺酶进行冻干，并测定催化速率以确定高盐激活制剂的潜在候选物。然后进一步评估最具活性制剂的动力学。这些研究表明，由98%（w/w）的1:1 KAc:CsCl盐混合物、1%（w/w）的酶和1%（w/w）的磷酸钾缓冲液组成的制剂，在己烷中其活性比无盐制剂高约35000倍，这在V(max)/K(m)值中得到体现。这种非凡的激活作用可扩展到更多极性溶剂，包括叔戊醇，以及总盐含量较低的制剂。发现盐的促溶剂/离液剂行为（通过琼斯 - 多尔B系数测量）与观察到的激活作用之间存在相关性。强离液剂阳离子与强促溶剂阴离子结合产生了最大的激活作用，这可能是由于离子对蛋白质 - 水和蛋白质 - 盐相互作用的影响。
