丙酮酸钠促进S-腺苷蛋氨酸和谷胱甘肽联合高产及其生理机制
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摘要
为了考察丙酮酸钠对S-腺苷蛋氨酸(SAM)和谷胱甘肽(GSH)联产发酵的影响,本文通过在不同培养时间向摇瓶中添加不同浓度丙酮酸钠,发现0 h添加2 g/L丙酮酸钠最有利于SAM和GSH生物合成。在该条件下进行分批发酵培养,结果表明:SAM和GSH联产量在27 h时达到最大值402.3 mg/L,比不添加丙酮酸钠的对照提高了35.1%。进一步地,对酵母胞内SAM合成酶、γ-谷氨酰半胱氨酸合成酶、己糖激酶、异柠檬酸脱氢酶的活性以及NADH和ATP含量进行测定,结果发现:丙酮酸钠添加不仅提高了SAM和GSH代谢途径中的关键酶活性,还提高了胞内能量代谢物质的水平,最终提升了SAM和GSH的合成能力,实现了SAM和GSH的联合高产。该研究结果为类似耗能合成化合物的高效生产提供了一种可行的思路。
In order to investigate the effect of sodium pyruvate on the biosynthesis of S-adenosylmethionine( SAM) and glutathione( GSH),the addition of different concentrations of sodium pyruvate into medium in flasks during different culture time was carried out. It was found that the addition of 2 g/L sodium pyruvate at 0 h was the optimum condition for the biosynthesis of SAM and GSH.The maximum co-production of SAM and GSH of 402.3 mg/L was obtained at 27 h during batch fermentation under the optimum sodium pyruvate addition strategy,which was increased by 35.1% compared to that of the control without sodium pyruvate addition.Moreover,the assay of methionine adenosyltransferase,γ-glutamylcysteine synthase,hexose kinase and isocitrate dehydrogenase,as well as the determination of intracellular NADH and ATP were conducted.It was indicated that the addition of sodium pyruvate increased not only activities of key enzymes involved in SAM and GSH biosynthesis,but also intracellular levels of energy metabolic substance,which in turn improved the capability of SAM and GSH biosynthesis,and the co-production of SAM and GSH.The results presented in this study would provide one feasible approach for efficient production of analogous chemicals biosynthesized with energy consumption.
In order to investigate the effect of sodium pyruvate on the biosynthesis of S-adenosylmethionine( SAM) and glutathione( GSH),the addition of different concentrations of sodium pyruvate into medium in flasks during different culture time was carried out. It was found that the addition of 2 g/L sodium pyruvate at 0 h was the optimum condition for the biosynthesis of SAM and GSH.The maximum co-production of SAM and GSH of 402.3 mg/L was obtained at 27 h during batch fermentation under the optimum sodium pyruvate addition strategy,which was increased by 35.1% compared to that of the control without sodium pyruvate addition.Moreover,the assay of methionine adenosyltransferase,γ-glutamylcysteine synthase,hexose kinase and isocitrate dehydrogenase,as well as the determination of intracellular NADH and ATP were conducted.It was indicated that the addition of sodium pyruvate increased not only activities of key enzymes involved in SAM and GSH biosynthesis,but also intracellular levels of energy metabolic substance,which in turn improved the capability of SAM and GSH biosynthesis,and the co-production of SAM and GSH.The results presented in this study would provide one feasible approach for efficient production of analogous chemicals biosynthesized with energy consumption.
引文
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[4]陈坚,卫功元,李寅,等.微生物发酵法生产谷胱甘肽[J].无锡轻工大学学报,2004,23(5):104-110.
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[6]Li Y,Wei G,Chen J.Glutathione:a review on biotechnological production[J].Applied Microbiology and Biotechnology,2004,66(3):233-242.
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[8]邵娜,卫功元,葛晓光,等.紫外线γ射线复合诱变筛选S-腺苷甲硫氨酸和谷胱甘肽联产发酵菌株[J].辐射研究与辐射工艺学报,2010,28(2):107-113.
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[11]Hu H,Qian J,Chu J,et al.Optimization of L-methionine feeding strategy for improving S-adenosyl-L-methionine production by methionine adenosyltransferase overexpressed Pichia pastoris[J].Applied Microbiol and Biotechnol,2009,83(6):1105-1114.
[12]Liang G,Liao X,Du G,et al.Elevated glutathione production by adding precursor amino acids coupled with ATP in high cell density cultivation of Candida utilis[J].Journal of Applied Microbiology,2008,105(5):1432-1440.
[13]Zhou J W,Liu L M,Shi Z P,et al.ATP in current biotechnology:regulation,application and perspectives[J].Biotechnology Advances,2009,27(1):94-101.
[14]王大慧,王玉磊,卫功元,等.基于代谢通量分析的SAM和GSH联产溶氧控制策略[J].高校化学工程学报,2013,27(1):102-107.
[15]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.
[16]申凤丹,刘金龙,吴剑荣,等.丙酮酸钠对E.coli CCTCC M208088发酵生产聚唾液酸的影响[J].工业微生物,2011,41(2):37-42.
[17]Miller G L.Use of dinitrosalicylic acid reagent for determination of reducing sugar[J].Analytical Biochemistry,1959,31(3):426-428.
[18]Wang Y,Wang D,Wei G,et al.Enhanced co-production of S-adenosylmethionine and glutathione by an ATP-oriented amino acid addition strategy[J].Bioresource Technology,2012,107(2):19-24.
[19]姚高峰,秦秀林,储炬,等.高效液相色谱法分析胞内S-腺苷L-甲硫氨酸含量及其合成酶活性的优化研究[J].化学与生物工程,2011,28(6):83-86.
[20]Lin A P,Mc Alister-Henn L.Isocitrate binding at two functionally distinct sites in yeast NAD+-specific isocitrate dehydrogenase[J].Journal of Biological Chemistry,2002,277(25):22475-22483.
[21]Hara K Y,Shimodate N,Hirokawa Y,et al.Glutathione production by efficient ATP-regenerating Escherichia coli mutants[J].Fems Microbiology Letters,2009,297(2):217-224.
[22]Hara KY,Kondo A.ATP regulation in bioproduction[J].Microbiol Cell Fact,2015,14(1):198.
[2]杨静,王曼,韦平和.S-腺苷甲硫氨酸的临床及药理研究进展[J].药学进展,2001,25(3):164-167.
[3]Meister A,Anderson M E.Glutathione[J].Annual Review of Biochemistry,1983,52:711-760.
[4]陈坚,卫功元,李寅,等.微生物发酵法生产谷胱甘肽[J].无锡轻工大学学报,2004,23(5):104-110.
[5]Shiozaki S,Shimizu S,Yamada H.Production of S-adenosyl-l-methionine by Saccharomyces sake[J].Journal of Biotechnology,1986,4(6):345-354.
[6]Li Y,Wei G,Chen J.Glutathione:a review on biotechnological production[J].Applied Microbiology and Biotechnology,2004,66(3):233-242.
[7]Brosnan J T,Brosnan M E,Bertolo R,et al.Methionine:a metabolically unique amino acid[J].Livestock Science,2007,112(1-2):2-7.
[8]邵娜,卫功元,葛晓光,等.紫外线γ射线复合诱变筛选S-腺苷甲硫氨酸和谷胱甘肽联产发酵菌株[J].辐射研究与辐射工艺学报,2010,28(2):107-113.
[9]Liu H,Lin J,Cen P,et al.Co-production of S-adenosyl-Lmethionine and glutathione from spent brewer’s yeast cells[J].Process Biochemistry,2004,39(12):1993-1997.
[10]王玉磊,卫功元,邵娜,等.基于能量代谢分析的S-腺苷蛋氨酸和谷胱甘肽联合高产方法[J].化工学报,2012,63(1):223-229.
[11]Hu H,Qian J,Chu J,et al.Optimization of L-methionine feeding strategy for improving S-adenosyl-L-methionine production by methionine adenosyltransferase overexpressed Pichia pastoris[J].Applied Microbiol and Biotechnol,2009,83(6):1105-1114.
[12]Liang G,Liao X,Du G,et al.Elevated glutathione production by adding precursor amino acids coupled with ATP in high cell density cultivation of Candida utilis[J].Journal of Applied Microbiology,2008,105(5):1432-1440.
[13]Zhou J W,Liu L M,Shi Z P,et al.ATP in current biotechnology:regulation,application and perspectives[J].Biotechnology Advances,2009,27(1):94-101.
[14]王大慧,王玉磊,卫功元,等.基于代谢通量分析的SAM和GSH联产溶氧控制策略[J].高校化学工程学报,2013,27(1):102-107.
[15]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.
[16]申凤丹,刘金龙,吴剑荣,等.丙酮酸钠对E.coli CCTCC M208088发酵生产聚唾液酸的影响[J].工业微生物,2011,41(2):37-42.
[17]Miller G L.Use of dinitrosalicylic acid reagent for determination of reducing sugar[J].Analytical Biochemistry,1959,31(3):426-428.
[18]Wang Y,Wang D,Wei G,et al.Enhanced co-production of S-adenosylmethionine and glutathione by an ATP-oriented amino acid addition strategy[J].Bioresource Technology,2012,107(2):19-24.
[19]姚高峰,秦秀林,储炬,等.高效液相色谱法分析胞内S-腺苷L-甲硫氨酸含量及其合成酶活性的优化研究[J].化学与生物工程,2011,28(6):83-86.
[20]Lin A P,Mc Alister-Henn L.Isocitrate binding at two functionally distinct sites in yeast NAD+-specific isocitrate dehydrogenase[J].Journal of Biological Chemistry,2002,277(25):22475-22483.
[21]Hara K Y,Shimodate N,Hirokawa Y,et al.Glutathione production by efficient ATP-regenerating Escherichia coli mutants[J].Fems Microbiology Letters,2009,297(2):217-224.
[22]Hara KY,Kondo A.ATP regulation in bioproduction[J].Microbiol Cell Fact,2015,14(1):198.