Ru M T, Dordick J S, Reimer J A, Clark D S
Department of Chemical Engineering, University of California, Berkeley, California 94720, USA.
Biotechnol Bioeng. 1999 Apr 20;63(2):233-41. doi: 10.1002/(sici)1097-0290(19990420)63:2<233::aid-bit12>3.0.co;2-s.
The addition of simple inorganic salts to aqueous enzyme solutions prior to lyophilization results in a dramatic activation of the dried powder in organic media relative to enzyme with no added salt. Activation of both the serine protease subtilisin Carlsberg and lipase from Mucor javanicus resulting from lyophilization in the presence of KCl was highly sensitive to the lyophilization time and water content of the sample. Specifically, for a preparation containing 98% (w/w) KCl, 1% (w/w) phosphate buffer, and 1% (w/w) enzyme, varying the lyophilization time showed a direct correlation between water content and activity up to an optimum, beyond which the activity decreased with increasing lyophilization time. The catalytic efficiency in hexane varied as much as 13-fold for subtilisin Carlsberg and 11-fold for lipase depending on the lyophilization time. This dependence was apparently a consequence of including the salt, as a similar result was not observed for the enzyme freeze-dried without KCl. In the case of subtilisin Carlsberg, the salt-induced optimum value of kcat/Km for transesterification in hexane was over 20,000-fold higher than that for salt-free enzyme, a substantial improvement over the previously reported enhancement of 3750-fold (Khmelnitsky, 1994). As was found previously for pure enzyme, the salt-activated enzyme exhibited greatest activity when lyophilized from a solution of pH equal to the pH for optimal activity in water. The active-site content of the lyophilized enzyme samples also depended upon lyophilization time and inclusion of salt, with opposite trends in this dependence observed for the solvents hexane and tetrahydrofuran. Finally, substrate selectivity experiments suggested that mechanism(s) other than selective partitioning of substrate into the enzyme-salt matrix are responsible for salt-induced activation of enzymes in organic solvents.
在冻干之前向酶水溶液中添加简单的无机盐，相对于未添加盐的酶，可使干燥后的粉末在有机介质中显著活化。在KCl存在下冻干导致的卡尔伯格枯草杆菌蛋白酶和爪哇毛霉脂肪酶的活化对冻干时间和样品的水含量高度敏感。具体而言，对于含有98%（w/w）KCl、1%（w/w）磷酸盐缓冲液和1%（w/w）酶的制剂，改变冻干时间表明，在达到最佳值之前，水含量与活性之间存在直接相关性，超过该最佳值后，活性随冻干时间的增加而降低。根据冻干时间的不同，卡尔伯格枯草杆菌蛋白酶在己烷中的催化效率变化高达13倍，脂肪酶变化高达11倍。这种依赖性显然是包含盐的结果，因为对于未添加KCl冻干的酶未观察到类似结果。就卡尔伯格枯草杆菌蛋白酶而言，盐诱导的己烷中酯交换反应的kcat/Km最佳值比无盐酶高20000多倍，比先前报道的3750倍的增强有了显著提高（赫梅利茨基，1994年）。如先前在纯酶中发现的那样，盐活化的酶在从pH值等于其在水中最佳活性的pH值的溶液中冻干时表现出最大活性。冻干酶样品的活性位点含量也取决于冻干时间和盐的加入，在己烷和四氢呋喃这两种溶剂中观察到这种依赖性的相反趋势。最后，底物选择性实验表明，除了底物选择性分配到酶-盐基质之外的其他机制是有机溶剂中盐诱导酶活化的原因。
