Yun Hyungdon, Cho Byung-Kwan, Kim Byung-Gee
Laboratory of Molecular Biotechnology and Biomaterials, Institute for Molecular Biology and Genetics, Seoul National University, 151-742, Korea.
Biotechnol Bioeng. 2004 Sep 20;87(6):772-8. doi: 10.1002/bit.20186.
The kinetic resolution of (R,S) sec-butylamine with the omega-transaminase (TA) from Vibrio fluvialis JS17 was performed under reduced pressure (e.g., 150 torr) to selectively remove an inhibitory product (2-butanone). The evaporation kinetics of 2-butanone at 150 torr in the buffer solution followed the first-order rate law, and the evaporation rate constant was 2.19 1/h, and independent of pH, while the evaporation kinetics of sec-butylamine is dependent on pH. A simple mathematical model of the evaporation of sec-butylamine allowing the estimation of its concentration in the reaction media was developed. The evaporation rate constant of its free amine form and the protonated amine form were 1.00 1/h, and nearly zero, respectively. Although the optimum pH of the omega-TA activity for sec-butylamine is 9.0, the optimal pH of the enzyme reaction under reduced pressure was pH 7.0, due to the higher evaporation rate of sec-butylamine at higher pH above 7.0. Using the recombinant Escherichia coli BL21 overexpressing omega-TA, 400 mM racemic sec-butylamine was resolved successfully to 98% ee of (R)-sec-butylamine with 53% conversion at 150 torr and pH 7.0.
使用来自河流弧菌JS17的ω-转氨酶（TA）对（R，S）仲丁胺进行动力学拆分是在减压（例如150托）条件下进行的，以选择性地去除抑制性产物（2-丁酮）。2-丁酮在缓冲溶液中150托压力下的蒸发动力学遵循一级速率定律，蒸发速率常数为2.19 1/h，且与pH无关，而仲丁胺的蒸发动力学则取决于pH。建立了一个简单的仲丁胺蒸发数学模型，用于估算其在反应介质中的浓度。其游离胺形式和质子化胺形式的蒸发速率常数分别为1.00 1/h和几乎为零。尽管ω-TA对仲丁胺的最佳活性pH为9.0，但由于仲丁胺在pH高于7.0时蒸发速率较高，减压下酶反应的最佳pH为7.0。使用过表达ω-TA的重组大肠杆菌BL21，在150托和pH 7.0条件下，400 mM外消旋仲丁胺成功拆分为ee值为98%的（R）-仲丁胺，转化率为53%。
