乙酸与钾离子强度对聚唾液酸合成及过程分子质量变化的影响
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摘要
聚唾液酸是由大肠杆菌K235合成的一种Group Ⅱ型胞外多糖。发酵液中有机酸积累、pH控制方式和K_2HPO_4对聚唾液酸合成和分子质量有较大影响。该研究发现,代谢累积有机酸及外源乙酸胁迫使得聚唾液酸产量和分子量降低;相比摇瓶所需100 mmol/L的K_2HPO_4,在发酵罐控制pH发酵时,20 mmol/L左右的K_2HPO_4可以获得较高的聚唾液酸分子量和产量;在发酵16 h后把氨水变成KOH流加来控制pH,并补入适量胰蛋白胨作为有机氮源,聚唾液酸产量有所提高,而且在发酵结束前较长时间内聚唾液酸的产量和分子质量维持稳定状态。
Polysialic acid( PSA) belongs to group II extracellular polysaccharide produced by Escherichia coli K235. Previous studies have shown that the accumulation of organic acids in the fermentation broth,pH control mode and K_2HPO_4 have a great influence on the biosynthesis and molecular weight of polysialic acid. In this study,we found that the accumulation of organic acids and exogenous acetic acid addition reduced the production and molecular weight of polysialic acid. Compared with the 100 mmol/L K_2HPO_4 required for shake flask cultivation,20 mmol/L K_2HPO_4 could obtain higher molecular weight and yield of polysialic acid in fermenter. After 16 h of fermentation,the ammoniawater was switched to KOH feeding to control the pH,and the appropriate amount of tryptone was added as the organic nitrogen source. As a result,the production of polysialic acid was increased and the molecular weight and yield of PSA remained stable. Results from this study can provide a good hint for industrial scale production of PSA with stable molecular weight.
Polysialic acid( PSA) belongs to group II extracellular polysaccharide produced by Escherichia coli K235. Previous studies have shown that the accumulation of organic acids in the fermentation broth,pH control mode and K_2HPO_4 have a great influence on the biosynthesis and molecular weight of polysialic acid. In this study,we found that the accumulation of organic acids and exogenous acetic acid addition reduced the production and molecular weight of polysialic acid. Compared with the 100 mmol/L K_2HPO_4 required for shake flask cultivation,20 mmol/L K_2HPO_4 could obtain higher molecular weight and yield of polysialic acid in fermenter. After 16 h of fermentation,the ammoniawater was switched to KOH feeding to control the pH,and the appropriate amount of tryptone was added as the organic nitrogen source. As a result,the production of polysialic acid was increased and the molecular weight and yield of PSA remained stable. Results from this study can provide a good hint for industrial scale production of PSA with stable molecular weight.
引文
[1]FERRERO M,APARICIO L R.Biosynthesis and production of polysialic acids in bacteria[J].Appl Microbiol Biotechnol,2010,86(6):1 621-1 635.
[2]KISS J Z N,ROUGONT G.Cell biology of polysialic acid[J].Current Opinion in Neurobiology,1997,7(5):640-646.
[3]王欣,关锋.多聚唾液酸(PSA)及其修饰的神经粘附分子(PSA-NCAM)对肿瘤及细胞信号通路的影响[J].遗传,2014,36(8):739-746.
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[5]GREGORIADIS G,MCCORMACK B,Wang Z,et al.Polysialic acids:potential in drug delivery[J].FEBS Letters,1993,315(3):271-276.
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[7]卢绍曾,吴剑荣,朱莉等.聚乙二醇聚唾液酸嵌段聚合修饰尿酸酶[J].生物加工过程,2015(2):86-92.
[8]付浩,冯昌雨,詹晓北等.聚唾液酸-透明质酸接枝聚合物的合成及其在药物缓释载体中的应用[J].合成化学,2016(7):570-575.
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[11]闫霞,吴剑荣,郑志永等.大肠杆菌K235能量代谢扰动影响聚唾液酸合成的研究[J].生物技术通报,2015,31(9):209-217.
[12]MOHLENHOFF M,ECKHARDT M,GERARDY-SCHAHN R.Polysialic acid:three-dimensional structure,biosynthesis[J].Current Opinion in Structural Biology,1998,8(5):558-564.
[13]SVENNERHOLM L.Quantitative estimation of silalic acid:a colorimetric rescorcinol-hydrochloruc acid[J].Biochimica Et Biophysica Acta,1957,24(3):604-611.
[14]MARKELY L R A,ONG B T,HOI K M,et al.A highthroughput method for quantification of glycoprotein sialylation[J].Analytical Biochemistry,2010,407(1):128-133.
[15]张佳程,骆承庠,张丽娟.水杨酸钠-次氯酸钠比色法测定食品中总凯氏氮的研究[J].中国农业科学,1999(1):85-88.
[16]姜岷,雷丹,陈可泉等.纤维素水解液厌氧发酵产丁二酸发酵液中有机酸和混合单糖的测定[J].分析化学,2009,37(4):605-608.
[17]RODE B,ENDRES C,RAN C,et al.Large-scale production and homogenous purification of long chain polysialic acids from E.coli K1[J].Journal of Biotechnology,2008,135(2):202-209.
[18]MANZI A E,HIGAS H H,DIAZ S,et al.Intramolecular self-cleavage of polysialic acid[J].The Journal of Biowgical Chemistry,1994,38(269):23 617-23 624.
[19]LULI G,WILLIAMR S.Comparison of growth,acetate production,and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations[J].Applied and Environmental Microbiology,1990,4(56):1 004-1 011.
[20]ZHANG Y,LEEY C.Acid-catalyzed lactonization ofα-2,8-linked oligo/polysialic acids studied by high performance anion-exchange chromatography[J].The Journal of Biological Chemistry,1999,10(274):6 183-6 189.
[21]李国顺,郑志永,李丹,等.端基法测定聚唾液酸平均聚合度[J].工业微生物,2012,42(5):19-23.
[22]叶进富.蛋白质zeta电位与离子交换层析分离的相关性研究[D].杭州:浙江大学,2006.
[23]刘金龙.微生物发酵法制备聚唾液酸的研究[D].无锡:江南大学,2013.
[24]STEENBERGEN S.M,VIMR E.R.Mechanism of polysialic acid chain elongation in Escherichia coli K1[J].Molecular Microbiology,1990,4(4):603-611.
[2]KISS J Z N,ROUGONT G.Cell biology of polysialic acid[J].Current Opinion in Neurobiology,1997,7(5):640-646.
[3]王欣,关锋.多聚唾液酸(PSA)及其修饰的神经粘附分子(PSA-NCAM)对肿瘤及细胞信号通路的影响[J].遗传,2014,36(8):739-746.
[4]CIESLEWICZ M,VIMR E.Reduced polysialic acid capsule expression in Escheichia coli K1 mutants with chromosomal defects in Kps F[J].Molecular Microbiology,1997,2(26):237-249.
[5]GREGORIADIS G,MCCORMACK B,Wang Z,et al.Polysialic acids:potential in drug delivery[J].FEBS Letters,1993,315(3):271-276.
[6]FERNANDES A I,GREGORIADIS G.Polysialylated asparaginase:preparation,activity and pharmacokinetics[J].Biochimica et Biophysica Acta(BBA)-Protein Structure and Molecular Enzymology,1997,1 341(1):26-34.
[7]卢绍曾,吴剑荣,朱莉等.聚乙二醇聚唾液酸嵌段聚合修饰尿酸酶[J].生物加工过程,2015(2):86-92.
[8]付浩,冯昌雨,詹晓北等.聚唾液酸-透明质酸接枝聚合物的合成及其在药物缓释载体中的应用[J].合成化学,2016(7):570-575.
[9]ZHENG Zhi-yong,WANG Shun-zhi,LI Guo-shun,et al.A new polysialic acid production process based on dualstage p H control and fed-batch p H control and fed-batch fermentation for higher yield and resulting high molecular weight product[J].Appl Microbiol Biotechnol,2013,97(6):2 405-2 412.
[10]CHEN Fang,TAO Yong,JIN Cheng,et al.Enhanced production of polysialic acid by metabolic engineering of Escherichia coli[J].Biotechnological Products and Process Engineering,2015,99(6):2 603-2 611.
[11]闫霞,吴剑荣,郑志永等.大肠杆菌K235能量代谢扰动影响聚唾液酸合成的研究[J].生物技术通报,2015,31(9):209-217.
[12]MOHLENHOFF M,ECKHARDT M,GERARDY-SCHAHN R.Polysialic acid:three-dimensional structure,biosynthesis[J].Current Opinion in Structural Biology,1998,8(5):558-564.
[13]SVENNERHOLM L.Quantitative estimation of silalic acid:a colorimetric rescorcinol-hydrochloruc acid[J].Biochimica Et Biophysica Acta,1957,24(3):604-611.
[14]MARKELY L R A,ONG B T,HOI K M,et al.A highthroughput method for quantification of glycoprotein sialylation[J].Analytical Biochemistry,2010,407(1):128-133.
[15]张佳程,骆承庠,张丽娟.水杨酸钠-次氯酸钠比色法测定食品中总凯氏氮的研究[J].中国农业科学,1999(1):85-88.
[16]姜岷,雷丹,陈可泉等.纤维素水解液厌氧发酵产丁二酸发酵液中有机酸和混合单糖的测定[J].分析化学,2009,37(4):605-608.
[17]RODE B,ENDRES C,RAN C,et al.Large-scale production and homogenous purification of long chain polysialic acids from E.coli K1[J].Journal of Biotechnology,2008,135(2):202-209.
[18]MANZI A E,HIGAS H H,DIAZ S,et al.Intramolecular self-cleavage of polysialic acid[J].The Journal of Biowgical Chemistry,1994,38(269):23 617-23 624.
[19]LULI G,WILLIAMR S.Comparison of growth,acetate production,and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations[J].Applied and Environmental Microbiology,1990,4(56):1 004-1 011.
[20]ZHANG Y,LEEY C.Acid-catalyzed lactonization ofα-2,8-linked oligo/polysialic acids studied by high performance anion-exchange chromatography[J].The Journal of Biological Chemistry,1999,10(274):6 183-6 189.
[21]李国顺,郑志永,李丹,等.端基法测定聚唾液酸平均聚合度[J].工业微生物,2012,42(5):19-23.
[22]叶进富.蛋白质zeta电位与离子交换层析分离的相关性研究[D].杭州:浙江大学,2006.
[23]刘金龙.微生物发酵法制备聚唾液酸的研究[D].无锡:江南大学,2013.
[24]STEENBERGEN S.M,VIMR E.R.Mechanism of polysialic acid chain elongation in Escherichia coli K1[J].Molecular Microbiology,1990,4(4):603-611.