对自主筛选的不透明红球菌FMME1-41进行发酵营养条件优化，得到最佳发酵培养基：酵母粉6 g/L，大豆蛋白胨2 g/L，(NH4)2SO4 0.8 g/L，葡萄糖25 g/L，KH2PO4 3 g/L，MgSO4 0.6 g/L，MnSO4 0.3 g/L，L-谷氨酸2.5 g/L，在最佳培养基中发酵30 h，LOGX酶活达到6.12 U/mL.在7.5 L发酵罐中进行补料策略优化，使用分批发酵和DO-stat补料相结合的两阶段发酵方法，发酵周期40 h，LOGX酶活达到21.5 U/mL，是分批发酵LOGX酶活的2.8倍. 利用LOGX在2 L体系中转化L-谷氨酸生产α-酮戊二酸（α-KG），α-KG产量达到92.0 g/L，摩尔转化率为92.6%，α-KG生产强度为9.2 g/L/h.
The optimization of fermentation conditions for the independent screening strain Rhodococcus opacus FMME1-41 was carried out. The optimal nutrition conditions was as follows: 6 g/L yeast powder, 2 g/L soya peptone, 0.8 g/L (NH4)2SO4, 25 g/L glucose, 3 g/L KH2PO4, 0.6 g/L MgSO4, 0.3 g/L MnSO4, 2.5 g/L glutamic acid. In addition, fermentation period was 30 h, the LOGX activity reached 6.12 U/mL. Feeding strategy was optimized in 7.5 L fermentation tank. Under the condition of combination of bat. ch fermentation and DO-stat feeding, the fermentation period was 40 h, and the LOGX activity was 21.5 U/mL, which is 2.8 times of the batch fermentation. LGOX was used to produce α-ketoglutaric acid (α-KG) from L-glutamic acid in 2 L systerm. Finally, the maximum titer of α-KG reached 92 g/L, the molar conversion rate was 92.6%, the production intensity of α-KG was 9.2 g/L/h.
[1]郑强, 严卫民, 徐东, 等.谷氨酸氧化酶的来源和特性及其在生化检验中的应用[J].检验医学教育, 2003, 10(2):32-35
[2]Kamei T, Asano K, Nakamura S.Determination of serum glutamate oxaloacetate transaminase and glutamate pyruvate transaminase by L-glutamate oxidase[J].Chem& Pharm Bull, 1986, 34(1):409-412
[3]毕春元, 李玲, 李敬龙.谷氨酸氧化酶的研究进展[J].生命科学, 2012, 24(2):169-173
[4]李青山, 李晓波, 李友荣, 等.用静息细胞培养法研究-谷氨酸氧化酶的生物合成[J].华东理工大学学报, 1994, 20(2):168-172
[5]徐水清, 李友荣.谷氨酸氧化酶产生菌的筛选与产酶条件[J].微生物学报, 1993, 33(4):309-312
[6]牛盼清, 张震宇, 刘立明.酶法转化 -谷氨酸生产α-酮戊二酸[J].生物工程学报, 2014, 30(8):1318-1322
[7] Niu P, Dong X, Wang Y, et al.Enzymatic production of α-ketoglutaric acid from L-glutamic acid via L-glutamate oxidase [J]. J Biotechnol, 2014, 179: 56-62.[J].J Biotechnol, 2014, 176( -):56-62
[8]樊祥臣, 陈瑞东, 刘佳, 等.谷氨酸氧化酶高密度发酵及催化合成α-酮戊二酸[J].过程工程学报, 2016, 16(2):292-297
[9] Fan X, Chen R, Chen L, et al.Enhancement of alpha-ketoglutaric acid production from L-glutamic acid by high-cell-density cultivation[J]. J Mol Catal B-Enzym, 2016, 126:10-17.[J].J Mol Catal B-Enzym, 2016, 126(-):10-17
[10]Wagner B M, Donnarumma F, Wintersteiger R, et al.Simultaneous quantitative determination of alpha-ketoglutaric acid and 5-hydroxymethylfurfural in human plasma by gas chromatography-mass spectrometry[J].Anal and Bioanal Chem, 2010, 396(7):2629-2637
[11]Ogawa J, Yamanaka H, Mano J, et al.Synthesis of 4-hydroxyisoleucine by the aldolase-transaminase coupling reaction and basic characterization of the aldolase from Arthrobacter simplex AKU 626[J].Biosci Biotech and Bioch, 2007, 71(7):1607-1615
[12]Falcioni F, Blank L M, Frick O, et al.Proline availability regulates proline-4-hydroxylase synthesis and substrate uptake in proline-hydroxylating recombinant Escherichia coli[J].Appl Environ Microb, 2013, 79(9):3091-3100
[13]Otto C, Yovkova V, Barth G.Overproduction and Secretion of Alpha-Ketoglutaric Acid by Microorganisms[J].Appl Microbiol Biot, 2011, 92(4):689-695
[14]Zhou J W, Zhou H Y, Du G C, et al.Screening of a thiamine-auxotrophic yeast for α-ketoglutaric acid overproduction[J].Lett Appl Microbiol, 2010, 51(3):264-271
[15]Liu L M, Li Y, Zhu Y, et al.Redistribution of carbon flux in Torulopsis glabrataby altering vitamin and calcium level[J].Metab Eng, 2007, 9(1):21-29
[16]Yin X X, Madzak C, Du G C, et al.Enhanced alpha-ketoglutaric acid production in Yarrowia lipolytica WSH-Z06 by regulation of the pyruvate carboxylation pathway[J].Appl Microbiol Biot, 2012, 96(6):1527-1537
[17]Yovkova V, Otto C, Aurich A, et al.Engineering the alpha-ketoglutarate overproduction from raw glycerol by over of the genes encoding NADP(+)-dependent isocitrate dehydrogenase and pyruvate carboxylase in Yarrowia lipolytica[J].Appl Microbiol Biot, 2014, 98(5):2003-2013
[18]Allain C C, Poon L S, Chan C S, et al.Enzymatic determination of total serum cholesterol[J].Clin Chem, 1974, 20(4):470-475
[19]乔宇, 丁宏标, 闫俊艳, 等.重组大肠杆菌产普鲁兰酶的高密度发酵工艺研究[J].生物技术进展, 2012, 02(3):195-200
[20]占宏德, 李江华, 刘龙, 等.离子交换法纯化α-酮戊二酸[J].食品与生物技术学报, 2013, 32(10):1043-1048
[21]Chen C Y, Wu W T, Huang C J, et al.A common precursor for the three subunits of L-glutamate oxidase encoded by gox gene from StreptomycesplatensisNTU3304[J].Can J Microbiol, 2001, 47(3):269-275
