Noritomi H, Almarsson O, Barletta G L, Klibanov A M
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
Biotechnol Bioeng. 1996 Jul 5;51(1):95-9. doi: 10.1002/(SICI)1097-0290(19960705)51:1<95::AID-BIT11>3.0.CO;2-3.
The enantioselectivities of subtilisin Carlsberg and Rhizomucor miehei lipase in organic solvents are found to strongly depend on the method by which the enzymes are prepared. For the transesterification between sec-phenethyl alcohol and vinyl butyrate in dioxane at 7 degrees C, the enantioselectivity of subtilisin precipitated with isopropanol is more than twice that of the enzyme prepared by lyophilization from aqueous buffer. Furthermore, the temperature dependence of the enantioselectivity is influenced by the mode of enzyme preparation. For example, in the aforementioned process the enantioselectivities of subtilisin lyophilized from aqueous buffer and crosslinked subtilisin crystals increase when the temperature is raised from 7 to 45 degrees C. In contrast, the enantioselectivities decrease with temperature for the enzyme precipitated from aqueous solution with acetone or isopropanol and for the enzymatic hydrolysis in water. The temperature dependence of the enantioselectivity of subtilisin lyophilized from buffer is markedly affected by the solvent: In acetonitrile and nitromethane the enzyme is more enantioselective at higher temperatures, while negligible temperature effects have been found in tetrahydrofuran and pyridine. Lyophilized lipase exhibits striking temperature dependencies of its enantioselectivity in dioxane, acetonitrile, and nitromethane, while showing almost none in pyridine, triethylamine, and tetrahydrofuran. The results underscore the importance of the mode of enzyme recovery on enantioselectivity and its temperature dependence in enzymatic reactions in organic solvents (in contrast to those in water). (c) 1996 John Wiley & Sons, Inc.
人们发现，枯草杆菌蛋白酶卡尔伯格和米黑根毛霉脂肪酶在有机溶剂中的对映体选择性强烈依赖于酶的制备方法。对于在7℃下二氧六环中仲苯乙醇与丁酸乙烯酯之间的酯交换反应，用异丙醇沉淀的枯草杆菌蛋白酶的对映体选择性是由水性缓冲液冻干制备的酶的两倍多。此外，对映体选择性的温度依赖性受酶制备方式的影响。例如，在上述过程中，当温度从7℃升高到45℃时，从水性缓冲液冻干的枯草杆菌蛋白酶和交联枯草杆菌蛋白酶晶体的对映体选择性增加。相反，用丙酮或异丙醇从水溶液中沉淀的酶以及在水中的酶促水解反应中，对映体选择性随温度降低。从缓冲液冻干的枯草杆菌蛋白酶的对映体选择性的温度依赖性受溶剂的显著影响：在乙腈和硝基甲烷中，酶在较高温度下对映体选择性更高，而在四氢呋喃和吡啶中发现温度影响可忽略不计。冻干的脂肪酶在二氧六环、乙腈和硝基甲烷中其对映体选择性表现出显著的温度依赖性，而在吡啶、三乙胺和四氢呋喃中几乎没有。结果强调了酶回收方式对有机溶剂中酶促反应（与水中的反应相反）的对映体选择性及其温度依赖性的重要性。(c) 1996约翰威立父子公司
