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CN 34-1054/N

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Separation and purification to prepare N-acetylneuraminic acid after oxalic acid hydrolysis of polysialic acid

  • 1.

    Institute of plasma physics, Chinese Academy of Sciences(ASIPP), Hefei 230031,China

  • 2.

    Huainan new energy research center, Hefei 232001,China

  • 3.

    University of Science and Technology of China, Hefei, 230026,China

Cite this:
https://doi.org/10.3969/j.issn.0253-2778.2020.05.008
  • Received Date: 16 April 2019
  • Accepted Date: 25 November 2019
  • Rev Recd Date: 25 November 2019
  • Publish Date: 31 May 2020
    Abstract

  • Abstract

    The polysialic acid was obtained from fermentation of escherichia coli, and hydrolyzed to separate, purify and prepare the N-acetylneuraminic acid. In order to study the process conditions of separation and purification to prepare high purity of n-acetylneuraminic acid from the polysialic acid which was hydrolyzed by the oxalic acid, so as to simplify the process and reduce the production cost, the authors investigated emphatically the hydrolysis of the polysialic acid by oxalic acid , contrasted firstly the effects on hydrolysis of polysialic acids between oxalic acids as other conventional hydrolytic acids and determined oxalic acids as hydrolytic acids. The best operating parameters of the oxalic acid hydrolysis of the polysialic acid was determined by the orthogonal experiment: hydrolysis temperature 90 ℃, hydrolysis time 3.5 h, hydrolysis acid concentration 0.1 mol/L and diluted multiples of polysialic acid 5 times. In these conditions, the hydrolyze rate of the polysialic acid was more than 94 %, and the hydrolyze yield was more than 97 %. The separation and purification conditions were optimized through experiments, and the separation and purification process for the preparation of high purity N-acetylneuraminic acid was obtained. In these process conditions, the analytic results of the infrared spectrum and HPLC showed that the purity of the N-acetylneuraminic acid product was 98.5 %, and the yield was 58.4 %.

    Abstract

    The polysialic acid was obtained from fermentation of escherichia coli, and hydrolyzed to separate, purify and prepare the N-acetylneuraminic acid. In order to study the process conditions of separation and purification to prepare high purity of n-acetylneuraminic acid from the polysialic acid which was hydrolyzed by the oxalic acid, so as to simplify the process and reduce the production cost, the authors investigated emphatically the hydrolysis of the polysialic acid by oxalic acid , contrasted firstly the effects on hydrolysis of polysialic acids between oxalic acids as other conventional hydrolytic acids and determined oxalic acids as hydrolytic acids. The best operating parameters of the oxalic acid hydrolysis of the polysialic acid was determined by the orthogonal experiment: hydrolysis temperature 90 ℃, hydrolysis time 3.5 h, hydrolysis acid concentration 0.1 mol/L and diluted multiples of polysialic acid 5 times. In these conditions, the hydrolyze rate of the polysialic acid was more than 94 %, and the hydrolyze yield was more than 97 %. The separation and purification conditions were optimized through experiments, and the separation and purification process for the preparation of high purity N-acetylneuraminic acid was obtained. In these process conditions, the analytic results of the infrared spectrum and HPLC showed that the purity of the N-acetylneuraminic acid product was 98.5 %, and the yield was 58.4 %.
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    • References(15)
  • [1]
    REVILLA-NUIN B, REGLERO A, FERRERO M A, et al. Uptake of N-acetyl-D-mannosamine: an essential intermediate in polysialic acid biosynthesis by Escherichia coli K92[J]. FEBS Letters, 1999, 449: 183-186.
    [2]
    BRUSS J L, RUTISHAUSER U. Roles, regulation, and mechanism of acid function during neural development[J]. Biochimie, 2001, 83: 635-643.
    [3]
    BLIX F G, GOTTSCHALK A, KLENK E. Proposed nomenclature in the field of neuraminic and sialic acid[J]. Nature, 1957, 179:1088.
    [4]
    SHEN G J, DATTA A K, IZUMI M, et al. Expression of α2, 8/2, 9-Polysialyltransferase from Escherichia coli K92: Characterization of the enzyme and its reaction products[J]. Journal of Biological Chemistry, 1999, 274(49):35139-35146.
    [5]
    刘景, 任婧, 刘翠平, 等. 动物乳及婴幼儿配方奶中唾液酸的研究及应用进展[J]. 食品研究与开发, 2013, 34(11): 105-108.
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    WITCZAK Z J, KARL A N. Carbohydrate in Drugs Design[M]. New York: Marcel Dekker, 1997.
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    高剑峰, 冯万祥. 血液中唾液酸的提取和结晶纯化[J]. 中国医药工业杂志, 1996, 27(6): 246-247.
    [8]
    JUNEJA L R, KOKETSU M, NISHIMOTO K, et al. Large-scale preparation of sialic acid from chalaza and egg-yolk membrane[J]. Carbohydrate Research, 1991, 214:179-186.
    [9]
    WHITEHOUSE M W, ZILLIKEN F. Isolation and determination of neuraminic(sialic) acids[J]. Methods of Biochemical Analysis, 1960, 8: 199-218.
    [10]
    MASAHARU S, YUKIO U, ICHIROU M, et al. Process for manufacturing sialic acids-containing composition: US5270462[P/OL]. 1993-12-14[2019-03-15]. https://europepmc.org/article/PAT/US5270462.
    [11]
    赵慧, 詹晓北, 朱一晖， 等. 聚唾液酸的水解与唾液酸的纯化[J]. 无锡轻工业大学学报, 2004, 23(4): 16-19.
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    李文强. 唾液酸发酵控制及其分离纯化的研究[D]. 无锡：江南大学，2005.
    [13]
    郁丹凤. 聚唾液酸和唾液酸提取工艺的研究[D]. 无锡：江南大学，2008.
    [14]
    吴金勇, 陈祥松， 袁丽霞， 等. 磁场条件下Fe2+对产聚唾液酸大肠杆菌的诱变选育[J]. 河南师范大学学报, 2018, 46(6): 80-85.
    [15]
    SVENNERHOLM L. Quantitative estimation of sialic acids：II. A colorimetric resorcinol-hydrochloric acid method[J]. Biochimica et Biophysica Acta, 1957, 24(4): 604-611.
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    [1]
    REVILLA-NUIN B, REGLERO A, FERRERO M A, et al. Uptake of N-acetyl-D-mannosamine: an essential intermediate in polysialic acid biosynthesis by Escherichia coli K92[J]. FEBS Letters, 1999, 449: 183-186.
    [2]
    BRUSS J L, RUTISHAUSER U. Roles, regulation, and mechanism of acid function during neural development[J]. Biochimie, 2001, 83: 635-643.
    [3]
    BLIX F G, GOTTSCHALK A, KLENK E. Proposed nomenclature in the field of neuraminic and sialic acid[J]. Nature, 1957, 179:1088.
    [4]
    SHEN G J, DATTA A K, IZUMI M, et al. Expression of α2, 8/2, 9-Polysialyltransferase from Escherichia coli K92: Characterization of the enzyme and its reaction products[J]. Journal of Biological Chemistry, 1999, 274(49):35139-35146.
    [5]
    刘景, 任婧, 刘翠平, 等. 动物乳及婴幼儿配方奶中唾液酸的研究及应用进展[J]. 食品研究与开发, 2013, 34(11): 105-108.
    [6]
    WITCZAK Z J, KARL A N. Carbohydrate in Drugs Design[M]. New York: Marcel Dekker, 1997.
    [7]
    高剑峰, 冯万祥. 血液中唾液酸的提取和结晶纯化[J]. 中国医药工业杂志, 1996, 27(6): 246-247.
    [8]
    JUNEJA L R, KOKETSU M, NISHIMOTO K, et al. Large-scale preparation of sialic acid from chalaza and egg-yolk membrane[J]. Carbohydrate Research, 1991, 214:179-186.
    [9]
    WHITEHOUSE M W, ZILLIKEN F. Isolation and determination of neuraminic(sialic) acids[J]. Methods of Biochemical Analysis, 1960, 8: 199-218.
    [10]
    MASAHARU S, YUKIO U, ICHIROU M, et al. Process for manufacturing sialic acids-containing composition: US5270462[P/OL]. 1993-12-14[2019-03-15]. https://europepmc.org/article/PAT/US5270462.
    [11]
    赵慧, 詹晓北, 朱一晖， 等. 聚唾液酸的水解与唾液酸的纯化[J]. 无锡轻工业大学学报, 2004, 23(4): 16-19.
    [12]
    李文强. 唾液酸发酵控制及其分离纯化的研究[D]. 无锡：江南大学，2005.
    [13]
    郁丹凤. 聚唾液酸和唾液酸提取工艺的研究[D]. 无锡：江南大学，2008.
    [14]
    吴金勇, 陈祥松， 袁丽霞， 等. 磁场条件下Fe2+对产聚唾液酸大肠杆菌的诱变选育[J]. 河南师范大学学报, 2018, 46(6): 80-85.
    [15]
    SVENNERHOLM L. Quantitative estimation of sialic acids：II. A colorimetric resorcinol-hydrochloric acid method[J]. Biochimica et Biophysica Acta, 1957, 24(4): 604-611.
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