Qu Xiang-Hua, Chen Jin-Chun, Ma Qi-Xiang, Sun Shi-Yao, Chen Guo-Qiang
College of Biological Science and Biotechnology, Xinjiang University, 830046, China.
Sheng Wu Gong Cheng Xue Bao. 2003 Jan;19(1):74-80.
Cis-1,2-dihydroxycyclohexa-3,5-diene (DHCD) can be used as a valuable chiral intermediates for applications in pharmaceuticals, aerospace, electrical and fine chemical industries. By on-line detection of toluene dioxygenase (TDO) activity in whole recombinant Escherichia coli JM109 (pKST11) cells that harbored TDO gene under a tac promoter, effects of IPTG and various benzene addition strategies on bioransformation of benzene to DHCD were investigated. When IPTG was used at the beginning of fermentation, the growth of cells was inhibited and TDO activity only maintained for 4 hours while same experiments with addition of IPTG at 6h or 8h generated TDO activity for 18 hours. Suitable induction time for IPTG was in the cell logarithmic growth phase and 0.5 mmol/L IPTG was sufficient for inducing maximum TDO activities. Benzene strongly inhibited the activity of TDO which catalyses the conversion of benzene to DHCD. It was found that both cell growth and TDO activity was remarkably inhibited by feeding of benzene vapor, only 7.5 g/L DHCD was obtained. While the benzene inhibition effect was ameliorated by two-liquid phase culture fermentation in which liquid paraffin was used as second phase in the broth. Using different initial ratios of paraffin to benzene in fed-batch culture, DHCD contents were increased to 22.6 g/L, which was 3-fold more compared with that in benzene vapor culture. A further improvement of DHCD production was achieved when the mixture of liquid paraffin and benzene was added continuously by peristaltic pump, the DHCD contents were increased to a final concentration of 36.8 g/L. It was proven that the key to improving DHCD production by recombinants is to prolong TDO activity in cells, which can be achieved by using suitable addition benzene strategies.
顺式-1,2-二羟基环己-3,5-二烯（DHCD）可作为一种有价值的手性中间体，应用于制药、航空航天、电子和精细化工等行业。通过在线检测在tac启动子下携带甲苯双加氧酶（TDO）基因的重组大肠杆菌JM109（pKST11）全细胞中的TDO活性，研究了异丙基-β-D-硫代半乳糖苷（IPTG）和各种苯添加策略对苯生物转化为DHCD的影响。当在发酵开始时使用IPTG时，细胞生长受到抑制，TDO活性仅维持4小时，而在6小时或8小时添加IPTG的相同实验中，TDO活性维持了18小时。IPTG的合适诱导时间是在细胞对数生长期，0.5 mmol/L的IPTG足以诱导最大TDO活性。苯强烈抑制催化苯转化为DHCD的TDO活性。发现通过通入苯蒸气，细胞生长和TDO活性均受到显著抑制，仅获得7.5 g/L的DHCD。而在双液相培养发酵中，以液体石蜡作为发酵液中的第二相，苯的抑制作用得到改善。在分批补料培养中使用不同的石蜡与苯初始比例，DHCD含量增加到22.6 g/L，比苯蒸气培养中的含量增加了3倍。当通过蠕动泵连续添加液体石蜡和苯的混合物时，DHCD产量进一步提高，DHCD含量增加到最终浓度36.8 g/L。事实证明，提高重组体生产DHCD的关键是延长细胞中的TDO活性，这可以通过使用合适的苯添加策略来实现。
