克雷伯杆菌生产1,3-丙二醇关键酶发酵条件研究

研究了克雷伯杆菌生产1,3-丙二醇关键酶(甘油脱氢酶、1,3-丙二醇氧化还原酶、甘油脱水酶)的发酵条件。研究各种碳源、无机氮源、有机氮源及无机盐对产酶的影响,应用均匀设计、神经网络和遗传算法优化发酵培养基组成,结果为(gL-1):甘油30、KCl 1.6、NH4Cl 6.7、CaCl2 0.28、酵母浸膏2.8。在温度37C、初始pH 7.0、摇床转速200rmin-1和接种量5%条件下,发酵24h,无细胞抽提液中甘油脱氢酶、1,3-丙二醇氧化还原酶和甘油脱水酶活力(U·mg-1)分别为9.16、18.72、0.48,发酵34h,发酵液中1,3-丙二醇(1,3-PD)最大浓度为7.96gL-1。代谢过程中关键酶酶活与1,3-PD峰值出现时间不一致,且提早于1,3-PD峰值的出现。此外,结果表明优化后的培养基去除了多种微量元素,克雷伯杆菌生产1,3-丙二醇关键酶可以在微氧条件下进行。

Studies on Fermentation Conditions for Key Enzymes in1,3-Propanediol Production with Klebsiella Pneumoniae

Fermentation conditions for key enzymes of 1,3-propanediol(1,3-PD) production (glycerol dehydrogenase GDH, 1,3-propanediol oxidoreductase PDOR, glycerol dehydratase GDHt) using Klebsiella Pneumoniae were studied. Several carbon sources, organic nutrients, nitrogen sources and salts were examined for their effects on key enzymes formation. The concentrations of medium components were optimized by the methods of uniform design, artificial neural networks and genetic algorithms. The obtained optimal medium ingredients are (gL-1): glycerol 30, KCl 1.6, NH4Cl 6.7, CaCl2 0.28 and yeast extract 2.8. Meanwhile, the optimization of fermentation conditions was conducted. When the strain grew in the medium under optimal conditions such as 37C, initial pH 7.0, shaker speed 200 rmin-1 as well as 5% inoculum, the GDH, PDOR and GDHt activities(Umg-1) in cell-free extract were 9.16, 18.72, 0.48 after 24h cultivation and the maximum concentration of 1,3-PD was 7.96gL-1 after 34h cultivation. This suggests that appearance of maximum activities of key enzymes is earlier than that of maximum concentration of 1,3-PD. Moreover, results show that the trace elements in medium can be saved and productions of GDH, PDOR and GDHt with Klebsiella Pneumoniae can be carried out under microaerobic condition.