柠檬酸钠对脱氮假单胞杆菌发酵产维生素B_(12)代谢过程的影响
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摘要
以脱氮假单胞杆菌为生产菌,研究了柠檬酸钠对其发酵过程中产维生素B_(12)的代谢调控作用。通过摇瓶培养的方式,在基础培养基中添加不同质量浓度的柠檬酸钠,利用分光光度法和HPLC法分别测定了关键酶活和有机酸等含量。结果表明,添加质量浓度为0.3%的柠檬酸钠最有利于维生素B_(12)的发酵,其产量高达45.73μg/mL。在此基础上,结合有机酸和酶学方法,进一步研究发现,外源添加柠檬酸钠能显著降低菌体柠檬酸和琥珀酸的积累,并且加速苹果酸的消耗,降低EMP途径中丙酮酸激酶和磷酸果糖激酶的活力,提高HMP途径中葡萄糖-6-磷酸脱氢酶的活力,从而避免发酵液pH急剧下降,减少EMP途径的通量,增加HMP途径的通量,为维生素B_(12)的合成提供充足的还原力NADPH,提高维生素B_(12)发酵产量。
In this study,the metabolism of vitamin B_(12) in the process of fermentation was studied by using Pseudomonas aeruginosa as the producing strain.By shake flask method,sodium citrate in the basic culture medium supplemented with different concentrations,the key enzyme activity and organic acid content were determined by spectrophotometry and HPLC respectively.The result showed that 0.3% sodium citrate was more favorable for vitamin B_(12) fermentation,and its yield was as high as 45.73 μg/mL.Based on this fact,combining with organic acids and enzyme methods,further research found that sodium citrate could significantly reduce the citric acid and succinic acid accumulation,accelerate the consumption of malic acid,reduce the EMP pathway of pyruvate kinase and phosphofructokinase activity,and increase the activity of glucose-6-phosphate dehydrogenase in HMP pathway.In order to avoid the rapid decline of pH,the flux of EMP pathway was reduced and the flux of HMP pathway was increased,which provided sufficient reducing power for vitamin B_(12) synthesis.Thus,the fermentation yield of vitamin B_(12) could be improved.
In this study,the metabolism of vitamin B_(12) in the process of fermentation was studied by using Pseudomonas aeruginosa as the producing strain.By shake flask method,sodium citrate in the basic culture medium supplemented with different concentrations,the key enzyme activity and organic acid content were determined by spectrophotometry and HPLC respectively.The result showed that 0.3% sodium citrate was more favorable for vitamin B_(12) fermentation,and its yield was as high as 45.73 μg/mL.Based on this fact,combining with organic acids and enzyme methods,further research found that sodium citrate could significantly reduce the citric acid and succinic acid accumulation,accelerate the consumption of malic acid,reduce the EMP pathway of pyruvate kinase and phosphofructokinase activity,and increase the activity of glucose-6-phosphate dehydrogenase in HMP pathway.In order to avoid the rapid decline of pH,the flux of EMP pathway was reduced and the flux of HMP pathway was increased,which provided sufficient reducing power for vitamin B_(12) synthesis.Thus,the fermentation yield of vitamin B_(12) could be improved.
引文
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[23]Akshay G,Jinwoon L,Michale M,et al.Metabolic fluxes,pool and enzyme measurements suggest a tighter coupling of en.ergeties and biosynthetie reactions associated with reduced pyruvate kinase flux[J].Biotechnol and Bioeng,1999,64(2):129.134.
[24]周佳,程新,彭志远,等.典型性甲基供体对脱氮假单胞杆菌代谢过程的氧调控作用[J].食品工业科技,2013,34(7):162.164.Zhou J,Chen X,Peng Z Y,et al.Regulatory effect of the typical methyl-group donors on the oxygen metabolism in Pseudo.monas denitrificans[J].Science and Technology of Food Industry,2013,34(7):162.164.
[25]彭卫福,陈未,周佳,等.无机氮源对脱氮假单胞杆菌产维生素B12代谢过程的影响[J].食品工业科技,2013,34(18):178.181.Peng W F,Chen W,Zhou J,et al.Effect of inorganic nitrogen sources on the metabolic process of vitamin B12 producing by Pseudomonas denitrificans[J].Science and Technology of Food Industry,2013,34(18):178.181.
[26]Zhou J,Liu L,Chen J.Improved ATP supply enhances acid tolerance of Candida glabrata during pyruvic acid production[J].Applied Microbiology,2011,110(1):44.53.
[2]Eschenmoser A.Organic natural.product synthesis today.vitamin B12 as an example[J].Naturwissenschaften,1974,61:513.525.
[3]Martens H,Barg H,Warren M J,et al.Microbial production of vitamin B12[J].Appl Microbiol Biotechnol,2002,58(3):275.285.
[4]Kang Z,Zhang J,Zhou J,et al.Recent advances in microbial production ofδ.aminolevulinic acid and vitamin B12[J].Bio.technol Adv,2012,30(6):1533.1542.
[5]Debussehe L,Thibaut D,Cameron B,et al.Biosynthesis of the corrin macrocycle of coenzyme B12 in Pseudomonas denitrifi.cans[J].Baeteriol,1993,175(22):7430.7440.
[6]Warren M J,Raux E,Sehubert H L,et al.The biosynthesis of Adenosylcobalamin(vitamin B12)[J].Nat Prod Rep,2002,19(4):390.412.
[7]Moore S J,Lawrence A D,Biedendieck R,et al.Elucidation of the anaerobic pathway for the corrin component of cobalamin(vitamin B12)[J].Proceedings of the National Academy of Sciences,2013,110(37):14906.14911.
[8]Wang Z J,Wang P,Liu Y W,et al.Metabolic flux analysis of the central carbon metabolism of the industrial vitamin B12 pro.ducing strain Pseudomonas denitrificans using 13C.labeled glucose[J].Taiwan Inst Chem Eng,2012,43(2):181.187.
[9]Goel A,Jinwoon L,Domach M M,et al.Metabolic fluxes,pool and enzyme measurements suggest a tighter coupling of en.ergetics and biosynthetic reactions associated with reduced pyruvate kinase flux[J].Biotechnol Bioeng,1999,64(2):129.134.
[10]唐军,蔡水洪,叶勤.克鲁斯假丝酵母分批发酵生产甘油的代谢流分布[J].高校化学工程学报,2002,16(1):58.63.Tang J,Cai S H,Ye Q.Metabolic flux distribution in batch cultivation of Candida krusei for glycerol production[J].Journal of Chemical Engineering of Chinese Universities,2002,16(1):58.63.
[11]刘新星,陈双喜,储炬,等.柠檬酸钠对枯草杆菌生长代谢及肌苷积累的影响[J].微生物学报,2004,44(5):627.630.Liu X X,Chen S X,Chu J,et al.Effect of sodium citrate on the growth metabolism and inosine accumulation by Bacillus sub.tilis[J].Acta Microbiologica Sinica,2004,44(5):627.630.
[12]陈双喜,黄明志,储炬,等.柠檬酸钠对肌苷发酵过程代谢流分布的调节[J].化工学报,2005,56(9):1731.1737.Chen S X,Huang M Z,Chu J,et al.Regulation of sodium citrate on metabolic flux distribution of inosine fermentation process[J].Journal of Chemical Industry and Engineering(China),2005,56(9):1731.1737.
[13]秦永锋,徐庆阳,谢希贤,等.柠檬酸钠对L–缬氨酸发酵及代谢流量分布的影响[J].天津科技大学学报,2010,25(2):9.12.Qin Y F,Xu Q Y,Xie X X,et al.Effects of sodium citrate on metabolic flux distributions of L.Valine fermentation[J].Jour.nal of Tianjin University of Science&Technology,2010,25(2):9.12.
[14]马雷,程立坤,徐庆阳,等.柠檬酸钠对L.异亮氨酸发酵及代谢流量分布的影响[J].天津科技大学学报,2010,25(3):14.18.Ma L,Cheng L K,Xu Q Y,et al.Effects of sodium citrate on metabolic flux distributions of L.isoleucine fermentation[J].Journal of Tianjin University of Science&Technology,2010,25(3):14.18.
[15]王凤,石侃,潘涛,等.柠檬酸钠和生物素对白色链霉菌发酵产ε.聚赖氨酸的影响[J].现代食品科技,2013,29(6):1230.1233.Wang F,Shi K,Pan T,et al.Effect of sodium citrate and biotin onε.poly.lysine fermentation of Streptomyces albulus[J].Modern Food Science and Technology,2013,29(6):1230.1233.
[16]王玉磊,朱健,卫功元,等.柠檬酸钠促进S.腺苷蛋氨酸和谷胱甘肽联合高产[J].中国生物工程杂志,2013,33(8):49.54.Wang Y L,Zhu J,Wei G Y,et al.Increased Co.production of S.adenosylmethionine and glutathione by sodium citrate addi.tion[J].China Biotechnology,2013,33(8):49.54.
[17]Bradford M M.A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein.dye binding[J].Anal Biochem,1976,72(1/2):248.254.
[18]Pestma E,Sehefiers W A,vall Dijken J P.Adaptation of the kinetics of glucose transport to envirmental conditions in the yeast Candida utilis CBS 621 continuous culture study[J].Gen Microbiol,1988,134(5):1109.1116.
[19]Collins L B,Thomas T D.Pyruvate Kinase of Streptococcus lactis[J].Journa of Bacteriology,1974,120(1):52.58.
[20]Avigad G.Inhibition of glucose 6.phosphate dehydrogenase by adenosine 5’.triphosphate[J].Proceedings of the National A.cademy of Sciences,1966,56(5):1543.1547.
[21]Phong W Y,Lin W,Rao S P S,et al.Characterization of phosphofructokinase activity in Mycobacterium tuberculosis reveals that a functional glycolytic carbon flow is necessary to limit the accumulation of toxic metabolic intermediates under hypoxia[J].Plos One,2013,(8)2:1.11.
[22]Fortnagel P,Freese E.Analysis of sporulation mutants.Ⅱ.Mutants blocked in the citric acid cycle[J].Bacteriof,1969,95(4):1434.1438.
[23]Akshay G,Jinwoon L,Michale M,et al.Metabolic fluxes,pool and enzyme measurements suggest a tighter coupling of en.ergeties and biosynthetie reactions associated with reduced pyruvate kinase flux[J].Biotechnol and Bioeng,1999,64(2):129.134.
[24]周佳,程新,彭志远,等.典型性甲基供体对脱氮假单胞杆菌代谢过程的氧调控作用[J].食品工业科技,2013,34(7):162.164.Zhou J,Chen X,Peng Z Y,et al.Regulatory effect of the typical methyl-group donors on the oxygen metabolism in Pseudo.monas denitrificans[J].Science and Technology of Food Industry,2013,34(7):162.164.
[25]彭卫福,陈未,周佳,等.无机氮源对脱氮假单胞杆菌产维生素B12代谢过程的影响[J].食品工业科技,2013,34(18):178.181.Peng W F,Chen W,Zhou J,et al.Effect of inorganic nitrogen sources on the metabolic process of vitamin B12 producing by Pseudomonas denitrificans[J].Science and Technology of Food Industry,2013,34(18):178.181.
[26]Zhou J,Liu L,Chen J.Improved ATP supply enhances acid tolerance of Candida glabrata during pyruvic acid production[J].Applied Microbiology,2011,110(1):44.53.