Synthesis of Peptide Amides using Sol-Gel Immobilized Alcalase in Batch and Continuous Reaction System
Two commercial proteases from Bacillus
licheniformis (Alcalase 2.4 L FG and Alcalase 2.5 L, Type DX) were
screened for the production of Z-Ala-Phe-NH2 in batch reaction.
Alcalase 2.4 L FG was the most efficient enzyme for the C-terminal
amidation of Z-Ala-Phe-OMe using ammonium carbamate as
ammonium source. Immobilization of protease has been achieved by
the sol-gel method, using dimethyldimethoxysilane (DMDMOS) and
tetramethoxysilane (TMOS) as precursors (unpublished results). In
batch production, about 95% of Z-Ala-Phe-NH2 was obtained at
30°C after 24 hours of incubation. Reproducibility of different
batches of commercial Alcalase 2.4 L FG preparations was also
investigated by evaluating the amidation activity and the entrapment
yields in the case of immobilization. A packed-bed reactor (0.68 cm
ID, 15.0 cm long) was operated successfully for the continuous
synthesis of peptide amides. The immobilized enzyme retained the
initial activity over 10 cycles of repeated use in continuous reactor at
ambient temperature. At 0.75 mL/min flow rate of the substrate
mixture, the total conversion of Z-Ala-Phe-OMe was achieved after 5
hours of substrate recycling. The product contained about 90%
peptide amide and 10% hydrolysis byproduct.
[1] D. J. Merkler, "C-Terminal amidated peptides: Production by the in vitro
enzymatic amidation of glycine-extended peptides and the importance of
the amide to bioactivity," Enzyme Microb. Technol., vo1. 16, pp. 450-
456, 1994.
[2] D. I. Chan, E. J. Prenner, H. J. Vogel, "Tryptophan- and arginine-rich
antimicrobial peptides: Structures and mechanisms of action,"
Biochimica et Biophysica Acta, vol. 1758, pp. 1184-1202, 2006.
[3] J. James, B. K. Simpson, "Application of enzymes in food processing,"
Crit. Rev. Food Sci. Nutr., vol. 36, no. 5, pp. 437-463, 1996.
[4] T. Yoshimaru, K. Matsumoto, Y. Kuramoto, K. Yamada, M. Sugano,
"Preparation of Microcapsulated Enzymes for Lowering the Allergenic
Activity of Foods," J. Agric. Food Chem., vol. 45, no. 10, pp. 4178-
4182, 1997.
[5] K. Sangeetha, V. B. Morris, T. E. Abraham, "Stability and catalytic
properties of encapsulated subtilisin in xerogels of alkoxisilanes,"
Applied Catalysis A: General, vol. 341, no. 1-2, pp. 168-173, 2008.
[6] K. S. Bisht, L. A. Henderson, R. A. Gross, D. L. Kaplan, G. Swift,
"Enzyme-catalyzed ring-opening polymerization of ¶Çêª-
pentadecalactone," Macromolecules, vol. 30, pp. 2705-2710, 1997.
[7] M. T. Reetz, P. Tielmann, W. Wiesenhöfer, W. Könen, A. Zonta,
"Second generation sol-gel encapsulted lipases: robust heterogeneous
biocatalysts, " Adv. Synth. Catal. Vol. 345, pp. 717-728, 2003.
[8] S. Ota, S. Myyazaki, H. Matsuoka, K. Morisato, Y. Shintani, K.
Nakanishi, "High-throughput protein digestion by trypsin-immobilized
monolithic silica with pipette-tip formula," J. Biochem. Biophys.
Methods, vol. 70, pp. 57-62, 2007.
[9] S. Xie, F. Svec, J. M. J. Frechet, "Design of reactive porous polymer
supports for high throughput bioreactors: poly(2-vinyl-4,4-
dimethylazlactone -co-acrylamide-co-ethylene dimethacrylate)
monoliths," Biotechnol. Bioeng., vol. 62, pp. 30-35, 1999.
[10] L. Ferreira, M. A. Ramos, J. S. Dordick, M. H. Gil, "Influence of
different silica derivatives in the immobilization and stabilization of a
Bacillus licheniformis protease (Subtilisin Carlsberg)," Journal of
Molecular Catalysis B: Enzymatic, vol. 21, pp. 189-199, 2003.
[11] F. Peter, L. Poppe, C. Kiss, E. Sz¶Çäÿcs-B├¡ro, G. Preda, C. Zarcula, A.
Olteanu, "Influence of precursors and additives on microbial lipases
stabilized by sol-gel entrapment," Biocatal.Biotransform., vol. 23, pp.
251-260, 2005.
[12] C. G. Boeriu, A. E. Frissen, E. Boer, K. van Kekem, D- J. van Zoelen, I.
F. Eggen, "Optimized enzymatic synthesis of C-terminal peptide amides
using subtilisin A from Bacillus licheniformis," Journal of Molecular
Catalysis B: Enzymatic, vol. 66, pp. 33-42, 2010.
[13] C. G. Boeriu, I. F. Eggen, D- J. van Zoelen, G. H. Bours, "Selective
enzymatic hydrolysis of C-terminal tert-butyl esters of peptides," in
Peptides for Youth: The Proceedings of the 20th APS Symposium,
Montreal, pp. 115-116, 2009,
[14] C. G. Boeriu, I. F. Eggen, "Selective enzymatic hydrolysis of C-terminal
tert-butyl esters of peptides," WO 2007/082890 A1.
[15] M. M. Bradford, "Rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding," Anal. Biochem., vol. 72, pp. 248-254, 1976.
[16] J. E. Celis, N. Carter, T. Hunter, K. Simons, J. V. Small, D. Shotton, Cell
Biology: A Laboratory Handbook, 3th Edition, San Diego, Elsevier
Academic Press, 2006.
[17] L. Betancor, H. R. Luckarift, "Bioinspired enzyme encapsulation for
biocatalysis," Trends Biotechnol., vol. 26, no. 10, pp. 566-572, 2008.
[18] I. F. Eggen, C. G. Boeriu, "Process for the conversion of C-terminal
peptide esters or acids to amides employing subtilisin in the present of
ammonium salts," WO 2009/000814.
[1] D. J. Merkler, "C-Terminal amidated peptides: Production by the in vitro
enzymatic amidation of glycine-extended peptides and the importance of
the amide to bioactivity," Enzyme Microb. Technol., vo1. 16, pp. 450-
456, 1994.
[2] D. I. Chan, E. J. Prenner, H. J. Vogel, "Tryptophan- and arginine-rich
antimicrobial peptides: Structures and mechanisms of action,"
Biochimica et Biophysica Acta, vol. 1758, pp. 1184-1202, 2006.
[3] J. James, B. K. Simpson, "Application of enzymes in food processing,"
Crit. Rev. Food Sci. Nutr., vol. 36, no. 5, pp. 437-463, 1996.
[4] T. Yoshimaru, K. Matsumoto, Y. Kuramoto, K. Yamada, M. Sugano,
"Preparation of Microcapsulated Enzymes for Lowering the Allergenic
Activity of Foods," J. Agric. Food Chem., vol. 45, no. 10, pp. 4178-
4182, 1997.
[5] K. Sangeetha, V. B. Morris, T. E. Abraham, "Stability and catalytic
properties of encapsulated subtilisin in xerogels of alkoxisilanes,"
Applied Catalysis A: General, vol. 341, no. 1-2, pp. 168-173, 2008.
[6] K. S. Bisht, L. A. Henderson, R. A. Gross, D. L. Kaplan, G. Swift,
"Enzyme-catalyzed ring-opening polymerization of ¶Çêª-
pentadecalactone," Macromolecules, vol. 30, pp. 2705-2710, 1997.
[7] M. T. Reetz, P. Tielmann, W. Wiesenhöfer, W. Könen, A. Zonta,
"Second generation sol-gel encapsulted lipases: robust heterogeneous
biocatalysts, " Adv. Synth. Catal. Vol. 345, pp. 717-728, 2003.
[8] S. Ota, S. Myyazaki, H. Matsuoka, K. Morisato, Y. Shintani, K.
Nakanishi, "High-throughput protein digestion by trypsin-immobilized
monolithic silica with pipette-tip formula," J. Biochem. Biophys.
Methods, vol. 70, pp. 57-62, 2007.
[9] S. Xie, F. Svec, J. M. J. Frechet, "Design of reactive porous polymer
supports for high throughput bioreactors: poly(2-vinyl-4,4-
dimethylazlactone -co-acrylamide-co-ethylene dimethacrylate)
monoliths," Biotechnol. Bioeng., vol. 62, pp. 30-35, 1999.
[10] L. Ferreira, M. A. Ramos, J. S. Dordick, M. H. Gil, "Influence of
different silica derivatives in the immobilization and stabilization of a
Bacillus licheniformis protease (Subtilisin Carlsberg)," Journal of
Molecular Catalysis B: Enzymatic, vol. 21, pp. 189-199, 2003.
[11] F. Peter, L. Poppe, C. Kiss, E. Sz¶Çäÿcs-B├¡ro, G. Preda, C. Zarcula, A.
Olteanu, "Influence of precursors and additives on microbial lipases
stabilized by sol-gel entrapment," Biocatal.Biotransform., vol. 23, pp.
251-260, 2005.
[12] C. G. Boeriu, A. E. Frissen, E. Boer, K. van Kekem, D- J. van Zoelen, I.
F. Eggen, "Optimized enzymatic synthesis of C-terminal peptide amides
using subtilisin A from Bacillus licheniformis," Journal of Molecular
Catalysis B: Enzymatic, vol. 66, pp. 33-42, 2010.
[13] C. G. Boeriu, I. F. Eggen, D- J. van Zoelen, G. H. Bours, "Selective
enzymatic hydrolysis of C-terminal tert-butyl esters of peptides," in
Peptides for Youth: The Proceedings of the 20th APS Symposium,
Montreal, pp. 115-116, 2009,
[14] C. G. Boeriu, I. F. Eggen, "Selective enzymatic hydrolysis of C-terminal
tert-butyl esters of peptides," WO 2007/082890 A1.
[15] M. M. Bradford, "Rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding," Anal. Biochem., vol. 72, pp. 248-254, 1976.
[16] J. E. Celis, N. Carter, T. Hunter, K. Simons, J. V. Small, D. Shotton, Cell
Biology: A Laboratory Handbook, 3th Edition, San Diego, Elsevier
Academic Press, 2006.
[17] L. Betancor, H. R. Luckarift, "Bioinspired enzyme encapsulation for
biocatalysis," Trends Biotechnol., vol. 26, no. 10, pp. 566-572, 2008.
[18] I. F. Eggen, C. G. Boeriu, "Process for the conversion of C-terminal
peptide esters or acids to amides employing subtilisin in the present of
ammonium salts," WO 2009/000814.
@article{"International Journal of Chemical, Materials and Biomolecular Sciences:60714", author = "L. N. Corîci and A. E. Frissen and D -J. Van Zoelen and I. F. Eggen and F. Peter and C. M. Davidescu and C. G. Boeriu", title = "Synthesis of Peptide Amides using Sol-Gel Immobilized Alcalase in Batch and Continuous Reaction System", abstract = "Two commercial proteases from Bacillus
licheniformis (Alcalase 2.4 L FG and Alcalase 2.5 L, Type DX) were
screened for the production of Z-Ala-Phe-NH2 in batch reaction.
Alcalase 2.4 L FG was the most efficient enzyme for the C-terminal
amidation of Z-Ala-Phe-OMe using ammonium carbamate as
ammonium source. Immobilization of protease has been achieved by
the sol-gel method, using dimethyldimethoxysilane (DMDMOS) and
tetramethoxysilane (TMOS) as precursors (unpublished results). In
batch production, about 95% of Z-Ala-Phe-NH2 was obtained at
30°C after 24 hours of incubation. Reproducibility of different
batches of commercial Alcalase 2.4 L FG preparations was also
investigated by evaluating the amidation activity and the entrapment
yields in the case of immobilization. A packed-bed reactor (0.68 cm
ID, 15.0 cm long) was operated successfully for the continuous
synthesis of peptide amides. The immobilized enzyme retained the
initial activity over 10 cycles of repeated use in continuous reactor at
ambient temperature. At 0.75 mL/min flow rate of the substrate
mixture, the total conversion of Z-Ala-Phe-OMe was achieved after 5
hours of substrate recycling. The product contained about 90%
peptide amide and 10% hydrolysis byproduct.", keywords = "packed-bed reactor, peptide amide, protease, sol-gel immobilization.", volume = "5", number = "4", pages = "369-6", }
