用重组大肠杆菌JM109（pKST11）转化苯生产顺-1,2-二羟基-3，5-环己二烯

顺-1，2-二羟基-3，5-环己二烯（简称DHCD）是航天业，电子工业，医药业以及精细化工业上重要的手性化合物，利用重组E.coli JM109（pKST11），采用适时监测发酵过程中全细胞甲苯双加氧酶（Toluene dioxygenase,TDO）活性的方法，研究了发酵生产DHCD工艺中的重要影响因子IPTG以及底物苯的供给方式对DHCD产量的影响，研究结果表明：（1）发酵初期利用IPTG诱导TDO的表达，不利于细胞生长，在对数生长中期（6或8h），采用0.5mmol/L IPTG即可诱导出TDO的最高表达。（2）发酵液中的苯对全细胞甲苯双加氧酶（TDO）的活性有抑制作用，而利用液体石蜡作为缓释剂进行两相法发酵则降低了苯的毒害，明显提高了DHCD的产量。当采用传统的通气供苯方法，DHCD的产量仅有7.5g/L；批式添加液体石蜡与苯的混溶物使DHCD的产量提高到22.6g/L，是通气供苯法的3倍；而采用流加的方式添加液体石蜡与苯的混溶物使DHCD的产量进一步提高到36.8g/L，是通气供苯法的5倍，证明通过发酵工艺的优化可以解决苯的毒害与苯作为反应底物在水相中需要一定浓度之间的矛盾，获得较好的转化结果。

Biotransformation of Benzene to cis-1,2-dihydroxycyclohexa-3,5-diene Using Recombinant Escherichia coli JM109 (pKST11)

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