Samama J P, Lee K M, Biellmann J F
Eur J Biochem. 1987 Mar 16;163(3):609-17. doi: 10.1111/j.1432-1033.1987.tb10910.x.
The activity and the kinetic properties of horse liver alcohol dehydrogenase have been studied in water-in-oil microemulsions containing sodium dodecyl sulfate (SDS) or hexadecyl trimethylammonium bromide (CTAB), 1-butanol or 1-pentanol or 1-hexanol or t-butanol, water and cyclohexane alone or with octane. In the anionic microemulsions (i.e. containing sodium dodecyl sulfate), the enzyme quickly lost its activity, but was efficiently protected by the coenzyme and some adenine nucleotides. In the cationic microemulsions (i.e. containing hexadecyl trimethylammonium bromide), the enzyme activity was more stable and with higher alcohols was stable for at least 20 min. The Michaelis constant of NAD+ calculated with respect to the water content was nearly constant and higher than in water. The maximum velocity in anionic microemulsions depends on the water content whereas in cationic microemulsions, the maximum velocity did not show a clear dependence on the water content and was close to the maximum velocity found in water. The pH dependence of Km and Vmax in these microemulsions was similar to that observed in water. The kinetic data for a hydrophobic substrate, cinnamyl alcohol, showed that this alcohol partitions between the pseudo-phases and thus the apparent Michaelis constant and the concentration at which substrate-excess inhibition appeared were increased. The catalytic properties of the enzyme in microemulsions were illustrated by the preparative reduction of cinnamaldehyde with cofactor recycling. The rate determination of NAD+ reduction and of 1-butanol/cinnamaldehyde redox reaction showed that at low water content (2.8%), the NAD+ reduction rate was close to zero whereas the redox reaction rate was about half of the rate at higher water content. Probably at low water content the coenzyme binding-dissociation rates are reduced much more than the binding-dissociation rates of the substrates and the rates of the ternary complex interconversion. The cationic microemulsions seemed to be very favorable medium for enzyme activity, the tetraalkyl ammonium surfactant causing less denaturation than the anionic detergent dodecyl sulfate.
在含有十二烷基硫酸钠（SDS）或十六烷基三甲基溴化铵（CTAB）、正丁醇或正戊醇或正己醇或叔丁醇、水以及单独的环己烷或与辛烷混合的油包水微乳液中，对马肝醇脱氢酶的活性和动力学性质进行了研究。在阴离子微乳液（即含有十二烷基硫酸钠的微乳液）中，酶迅速失去活性，但辅酶和一些腺嘌呤核苷酸能有效保护它。在阳离子微乳液（即含有十六烷基三甲基溴化铵的微乳液）中，酶活性更稳定，且在存在高级醇的情况下至少20分钟保持稳定。相对于水含量计算得到的NAD⁺的米氏常数几乎恒定且高于在水中的值。阴离子微乳液中的最大反应速度取决于水含量，而在阳离子微乳液中，最大反应速度对水含量没有明显依赖性，且接近在水中发现的最大反应速度。这些微乳液中Km和Vmax对pH的依赖性与在水中观察到的相似。对于疏水底物肉桂醇的动力学数据表明，这种醇在假相之间进行分配，因此表观米氏常数以及出现底物过量抑制的浓度都增加了。通过辅因子循环制备还原肉桂醛说明了酶在微乳液中的催化性质。NAD⁺还原和正丁醇/肉桂醛氧化还原反应的速率测定表明，在低水含量（2.8%）时，NAD⁺还原速率接近零，而氧化还原反应速率约为较高水含量时速率的一半。可能在低水含量时，辅酶的结合 - 解离速率比底物的结合 - 解离速率以及三元复合物相互转化的速率降低得更多。阳离子微乳液似乎是酶活性非常有利的介质，四烷基铵表面活性剂引起的变性比阴离子洗涤剂十二烷基硫酸钠小。
