Ru M T, Wu K C, Lindsay J P, Dordick J S, Reimer J A, Clark D S
Department of Chemical Engineering, University of California-Berkeley, Berkeley, CA 94720, USA.
Biotechnol Bioeng. 2001 Oct 20;75(2):187-96. doi: 10.1002/bit.1178.
The activation of freeze-dried subtilisin Carlsberg (SC) in hexane has been systematically studied and partially optimized with respect to the freezing method, the addition of inorganic salts and lyoprotectants, the initial concentration and final weight percent of additives, and the amount of water added to the organic solvent. Activity and water content were found to correlate directly with the kosmotropicity of the activating salt (kosmotropic salts bind water molecules strongly relative to the strength of water-water interactions in bulk solution). Combinations of kosmotropic salts with known lyoprotectants such as poly(ethylene glycol) (PEG) and sugars did not yield an appreciably more active catalyst. However, the combination of the kosmotropic sodium acetate with the strongly buffering sodium carbonate activated the enzyme more than the individual additives alone. Enzyme activity was enhanced further by the addition of small amounts of water to the organic solvent. Under optimal conditions, enzyme activity in hexane was improved over 27,000-fold relative to the salt-free enzyme, reaching a catalytic efficiency that was within one order of magnitude of k(cat)/K(m) for hydrolysis of the same substrate in aqueous buffer. Further activation to attain even higher catalytic efficiencies may be possible with additional optimization.
已对冻干的卡尔伯格枯草杆菌蛋白酶（SC）在己烷中的活化进行了系统研究，并在冷冻方法、无机盐和冻干保护剂的添加、添加剂的初始浓度和最终重量百分比以及添加到有机溶剂中的水量方面进行了部分优化。发现活性和含水量与活化盐的促溶能力直接相关（相对于本体溶液中水-水相互作用的强度，促溶盐能强烈结合水分子）。促溶盐与已知的冻干保护剂如聚乙二醇（PEG）和糖类的组合并未产生明显更具活性的催化剂。然而，促溶的醋酸钠与强缓冲剂碳酸钠的组合比单独使用各添加剂更能激活该酶。向有机溶剂中添加少量水可进一步提高酶活性。在最佳条件下，相对于无盐酶，己烷中的酶活性提高了27000倍以上，达到了在水性缓冲液中水解相同底物时k(cat)/K(m)的催化效率范围内。通过进一步优化，可能实现进一步活化以获得更高的催化效率。
