Biosynthesis of Cephalexin in PEG400-Ammonium Sulfate and PEG400-Magnesium Sulfate Aqueous Two-Phase Systems

  • Cao, Xuejun (State Key Laboratory of Bioreactor Engineering, Department of Biochemical Engineering, East China University of Science and Technology) ;
  • Zhu, Jianhang (State Key Laboratory of Bioreactor Engineering, Department of Biochemical Engineering, East China University of Science and Technology) ;
  • Wei, Dongzhi (State Key Laboratory of Bioreactor Engineering, Department of Biochemical Engineering, East China University of Science and Technology) ;
  • Hur, Byung-Ki (Department of Biological Engineering, Inha University)
  • Published : 2004.02.01

Abstract

The biosynthesis of cephalexin was carried out in the aqueous two-phase systems using penicillin acylase as a catalyst, and 7-aminodeacetoxicephalosporanic acid (7-ADCA) and phenylglycine methyl ester (PGME) as substrates. 20% PEG400-l7.5% ${(NH_4)}_2SO_4$ containing 0.5 M NaCl and 1.5 M methanol aqueous two-phase systems (ATPS) were selected as reaction medium, and 53% product yield was obtained using immobilized penicillin acylase as a catalyst. 20% PEG400-l5% $MgSO_4$ ATPS was also used for the synthesis of cephalexin, and 60-62% product yield was obtained by using free penicillin acylase as a catalyst. When batch reactions were repeated in the ATPS, the cephalexin yields decreased during the reactions due to deactivation, loss, and product inhibition of penicillin acylase. The effect of different ratio of phenylglycine methyl ester to 7-ADCA on the product yield was investigated, and high cephalexin yield was obtained at a high molar ratio.

Keywords

References

  1. Albertsson, P.-$\AA$., 1986. Partition of Cell Particles and Macromolecules, 3$^rd$ edn. John Wiley, New York, U.S.A
  2. Andersson, E. and H. H. Berbel. 1990. Bioconversions in aqueous two-phase systems. Enzyme Microb. Technol. 12: 242-254
  3. Andersson, E., B. Mattiasson, and H. H. Berbel. 1984. Enzymatic conversion in aqueous two phase systems: deacylation of benzyl penicillin to 6-aminopenicillanic acid with penicillin acylase. Enzyme Microb. Technol. 6: 301- 306
  4. Balasinghan, K., D. Warburton, P. Dunnill, and M. D. Lilly. 1972. The isolation and kinetics penicillin amidase from Escherichia coli. Biochim. Biophs. Acta 276: 250-256
  5. Bermudez., O. and D. Forciniti. 2001. Purification and characterization of crystallins by aqueous two-phase extraction. Biotechnol. Bioprocess. Eng. 6: 395-401
  6. Cao, X. J., J. H. Zhu, D. Z. Wei, and B. K. Hur. 2002. Partition improvement of cephalexin and 7-aminodeacetoxicephalospronic acid in aqueous two-phase systems for cephalexin synthesis. J. Ind. Eng. Chem. 8: 203-211
  7. Cao, X. J., X. A. Meng, Y. M. Liu, and X. Y. Wu. 1994. Study on kinetic behavior of E. coli ATCC 11105 producing penicillin acylase in aqueous two-phase system. Chinese J. Antibiotics 19: 3-5
  8. Cao, X. J., X. Y. Wu, Y. L. Jiang, and Q. Shen. 1994. Penicillin bioconversion in aqueous two-phase systems by recombinant Escherichia coli with intracellular penicillin acylase. Chinese J. Antibiotics 19: 131-133
  9. Guisan, J. M. 1988. Agarose aldehyde gels as supports for protein immobilization-stabilization of enzyme. Enzyme Microb. Technol. 10: 375-382 https://doi.org/10.1016/0141-0229(88)90018-X
  10. Johansson, G. and G. Kopperschläger. 1987. Effects of organic solvents on the partitioning of enzymes in aqueous two-phase systems, J. Chromatog. 388: 295-305
  11. Kim, D. W., S. M. Kang, and K. H. Yoon. 2001. Characterization of glutaryl 7-ACA acylase from Pseudomonas diminuta KAC 1. J. Microbiol. Biotechnol. 11: 452-497
  12. Odette, H. J., F. L. Roberto, T. Marco, and M. J. Guisan. 1998. Use of aqueous two-phase systems for in situ extraction of water-soluble antibiotics during their synthesis by enzymes immobilized on porous supports. Biotechnol. Bioeng. 59: 73-79
  13. Odhankar, S. S. and V. G. Gaikar. 1996. Effect of surface active additives on partitioning of proteins and enzymes in poly(ethylene glycol)/dextran aqueous two-phase systems. J. Chem. Technol. Biotechnol. 73: 251-258
  14. Odhankar, S. S. and V. G. Gaikar. 1996. Effect of surface active additives on partitioning of proteins and enzymes in poly(ethylene glycol)/dextran aqueous two-phase systems. J. Chem. Technol. Biotechnol.73: 251-258
  15. Rito-Palomares, M. 2002. The practical application of aqueous two-phase process for the recovery of biological products. J. Microbiol. Biotechnol. 12: 535-543
  16. Roberto, F. L., C. M. Rosell, and J. M. Guisan. 1991. Enzyme reaction engineering: Synthesis of antibiotic catalyzed by stabilized penicillin G acylase in the presence of organic cosolvents. Enzyme Microb. Technol. 13: 898- 905
  17. Roberto, F. L., C. M. Rosell, and M. J. Guisan. 1998. The presence of methanol exerts a strong and complex modulation of the synthesis of different antibiotics by immobilized penicillin G acylase. Enzyme Microb. Technol. 23: 305-310
  18. Shin, H. J., S. G. Lee, W. S. Lee, and K. H. Yoon. 1996. Enzymatic conversion of glutaryl 7-aminocephalosporanic acid to 7-aminocephosporanic acid with an immobilized glutaryl 7-aminocephalosporanic acid acylase. J. Microbiol. Biotechnol. 6: 336-339
  19. Sun, Y. 1998. Bioseparation Engineering, p. 63. Chemical Industry Publisher, Beijing, China