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Enzymatic synthesis of cefazolin using immobilized recombinant cephalosporin-acid synthetase as the biocatalyst

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Abstract

A method for the synthesis of β-lactam antibiotic cefazolin (CEZ) by enzymatic acylation of 7-amino-3-(5-methyl-l,3,4-thiadiazol-2-yl)thiomethyl-3-cephem-4-carboxylic acid (TDA) using immobilized cephalosporin-acid synthetase (IECASA) from recombinant E. coli strain VKPM B-12316 has been developed. A stepwise pH gradient designed on the basis of investigations on the solubility of components was applied for synthesis. This helped in avoiding the precipitation of TDA in the reaction when its initial concentration was high (150–200 mM). Thus, under optimal conditions a high yield of CEZ (relative to TDA) of 92–95% was obtained. Where the final reaction mixture contained 65–85 mg/mL of CEZ, 4–5 mg/mL of unreacted TDA, and 40–60 mg/mL of the by-product, 1(H)-tetrazolylacetic acid (TzAA). Testing of optimized CEZ synthesis using IECASA in a batch reactor has proved sufficiently high operational stability of the biocatalyst, with its residual activity after the 25th cycle accounting for about 83 ± 2% of its starting value. The half-inactivation period of IECASA was estimated as 85 cycles of CEZ synthesis.

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References

  1. Sklyarenko AV, Eldarov MA, Kurochkina VB, Yarotsky SV (2015) Enzymatic synthesis of cephalosporin-acids. Review Appl Biochem Microbiol 51(6):627–640

    Article  CAS  Google Scholar 

  2. Kurochkina VB, Nys PS (1999) Enzymatic synthesis of beta-lactam antibiotics. I. Cefazolin. Antibiotiki i Chiomiotherapi (Russia) 44(5):12–16

    CAS  Google Scholar 

  3. Kurochkina VB, Nys PS (2002) Kinetic and thermodynamic approach to design the processes for enzymatic synthesis of beta-lactams. Biocatal Biotransform 20:35–41

    Article  CAS  Google Scholar 

  4. Volpato G, Rodriges RC, Fernandez-Lafuente R (2010) Use of enzymes in production of semi-synthetic penicillins and cephalosporins: drawbacks and perspectives. Curr Med Chem 17:3855–3873

    Article  CAS  Google Scholar 

  5. Kurochkina VB, Sklyarenko AV (2008) Enzymatic synthesis of beta-lactam antibiotics. Analytical review. In: Zaikov GE (ed) Biotechnology state of the art and prospects for development, Chap. 20. Nova Science Publishers, New York, pp 175–204

    Google Scholar 

  6. Bruggink A (ed) (2001) Synthesis of β-lactam antibiotics: chemistry, biocatalysis and process integration. Kluver Academic Publishers, Dordrecht

    Google Scholar 

  7. Barber MS, Giesecke UE, Reichert A, Minas W (2004) Industrial enzymatic production of cephalosporin-based β-lactams. Adv Biochem Eng Biotechnol 88:179–215

    CAS  PubMed  Google Scholar 

  8. Nys PS, Kurochkina VB (2000) Methodological approach to development of enzymatic technologies for semisynthetic beta-lactam antibiotic production. Appl Biochem Biotechnol Part A Enzyme Eng Biotechnol 88:221–229

    Article  CAS  Google Scholar 

  9. Patent RU 2210596 (2000) Method of synthesis of cefazolin

  10. Patent RU 24210528 (2011) Method of producing biocatalyst exhibiting cephalosporin acid synthesis activity

  11. Eldarov MA, Sklyarenko AV, Mardanov AV, Beletsky AV,. Dumina MV, Medvedeva NV, Satarova DE, Ravin NV, Yarockii SV (2015) Cephalosporin-acid synthetase of Escherichia coli strain VKPM B-10182: genomic context, gene identification, producer strain production. Appl Biochem Microbiol 51(5):505–510

    Article  CAS  Google Scholar 

  12. Eldarov МА, Sklyarenko AV, Medvedeva NV, Zhgun AA, Satarova DE, Sidorenko AI, Dumina MV, Emperian AS, Sokolov NN, Yarotsky SV (2015) Recombinant cephalosporin-acid synthetase: optimization of expression in E. coli cells, immobilization and application for biocatalytic cefazolin synthesis. Biomed Chimia (Russia) 61(5):646–651

    CAS  Google Scholar 

  13. Park CB, Lee SB, Ryu DD (2000) Penicillin acylase-catalyzed synthesis of cefazolin in water–solvent mixtures: enhancement effect of ethyl acetate and carbon tetrachloride on the synthetic yield. J Mol Catal B Enzym 9(4–6):275–281

    Article  CAS  Google Scholar 

  14. Youshko MI, Moody HM, Bukhanov AL, Boosten WHJ, Svedas VK (2004) Penicillin acylase-catalyzed synthesis of β-lactam antibiotics in highly condensed aqueous systems: beneficial impact of kinetic substrate supersaturation. Biotechnol Bioeng 85(3):323–329

    Article  CAS  Google Scholar 

  15. McDonald MA, Bommarius AS, Rousseau RW (2017) Enzymatic reactive crystallization for improving ampicillin synthesis. Chem Eng Sci 165:81–88

    Article  CAS  Google Scholar 

  16. Kurochkina VB, Sklyarenko AV, Satarova DE, Yarosky SV (2011) Ionization constants and solubility of compounds involved in enzymatic synthesis of aminopenicillins and aminocephalosporins. Bioprocess Biosyst Eng 34(9):1103–1117

    Article  CAS  Google Scholar 

  17. Santana M, Ribeiro M, Leite GA, Giordano RL, Giordano RC, Mattedi S (2010) Solid–liquid equilibrium of substrates and products of the enzymatic synthesis of ampicillin. AIChE J 56:1578–1583

    Article  CAS  Google Scholar 

  18. Muraviev D, Khamizov R (2004) Ion-Exchange isothermal supersaturation: concept, problems, and applications. In: Marcus Y, SenGupta AK (eds) Ion exchange and solvent extraction. A series of advances, V16, Chap. 3. Taylor & Francis, New-York, pp 304–377

    Google Scholar 

  19. Muraviev D, Khamizov R, Tikhonov NA (1998) Ion-Exchange Isothermal Supersaturation. Solvent Extr Ion Exch 16(1):151–221

    Article  CAS  Google Scholar 

  20. Selemenev VF, Kotova DL, Khokhlov VYu O, GYu (2013) The processes of supersaturation and methods for isolation of amino acids using ion-exchange resins. Sorpt Chromatogr Process (Russia) 13(5):623–633

    CAS  Google Scholar 

  21. Patent US 6287799 (2001) Process for preparation of a β-lactam antibiotic

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Funding

The investigation was funded by grants 2017HH0011 of Science and Technology Department of Sichuan Province Project, and 2016-XT00-00023-GX of Science and Technology Bureau, Chengdu Municipal Government Project.

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Correspondence to A. V. Sklyarenko.

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Wang, L., Sklyarenko, A.V., Li, D. et al. Enzymatic synthesis of cefazolin using immobilized recombinant cephalosporin-acid synthetase as the biocatalyst. Bioprocess Biosyst Eng 41, 1851–1867 (2018). https://doi.org/10.1007/s00449-018-2007-z

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  • DOI: https://doi.org/10.1007/s00449-018-2007-z

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