Synthesis Of Peptide Amides Using Sol-Gel Immobilized Alcalase In Batch And Continuous Reaction System

Autor: Corîci, L. N., Frissen, A. E., Zoelen, D. -J, Eggen, I. F., Francisc Peter, Davidescu, C. M., Boeriu, C. G.
Jazyk: angličtina
Rok vydání: 2011
Předmět:
Zdroj: Scopus-Elsevier
World Academy of Science, Engineering and Technology 5 (2011) 4
World Academy of Science, Engineering and Technology, 5(4), 794-799
ISSN: 2070-3740
DOI: 10.5281/zenodo.1077739
Popis: 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achieved by the sol-gel method, using dimethyldimethoxysilane (DMDMOS) and tetramethoxysilane (TMOS) as precursors (unpublished results).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.
{"references":["D. J. Merkler, \"C-Terminal amidated peptides: Production by the in vitro\nenzymatic amidation of glycine-extended peptides and the importance of\nthe amide to bioactivity,\" Enzyme Microb. Technol., vo1. 16, pp. 450-\n456, 1994.","D. I. Chan, E. J. Prenner, H. J. Vogel, \"Tryptophan- and arginine-rich\nantimicrobial peptides: Structures and mechanisms of action,\"\nBiochimica et Biophysica Acta, vol. 1758, pp. 1184-1202, 2006.","J. James, B. K. Simpson, \"Application of enzymes in food processing,\"\nCrit. Rev. Food Sci. Nutr., vol. 36, no. 5, pp. 437-463, 1996.","T. Yoshimaru, K. Matsumoto, Y. Kuramoto, K. Yamada, M. Sugano,\n\"Preparation of Microcapsulated Enzymes for Lowering the Allergenic\nActivity of Foods,\" J. Agric. Food Chem., vol. 45, no. 10, pp. 4178-\n4182, 1997.","K. Sangeetha, V. B. Morris, T. E. Abraham, \"Stability and catalytic\nproperties of encapsulated subtilisin in xerogels of alkoxisilanes,\"\nApplied Catalysis A: General, vol. 341, no. 1-2, pp. 168-173, 2008.","K. S. Bisht, L. A. Henderson, R. A. Gross, D. L. Kaplan, G. Swift,\n\"Enzyme-catalyzed ring-opening polymerization of ¶Çêª-\npentadecalactone,\" Macromolecules, vol. 30, pp. 2705-2710, 1997.","M. T. Reetz, P. Tielmann, W. Wiesenhöfer, W. Könen, A. Zonta,\n\"Second generation sol-gel encapsulted lipases: robust heterogeneous\nbiocatalysts, \" Adv. Synth. Catal. Vol. 345, pp. 717-728, 2003.","S. Ota, S. Myyazaki, H. Matsuoka, K. Morisato, Y. Shintani, K.\nNakanishi, \"High-throughput protein digestion by trypsin-immobilized\nmonolithic silica with pipette-tip formula,\" J. Biochem. Biophys.\nMethods, vol. 70, pp. 57-62, 2007.","S. Xie, F. Svec, J. M. J. Frechet, \"Design of reactive porous polymer\nsupports for high throughput bioreactors: poly(2-vinyl-4,4-\ndimethylazlactone -co-acrylamide-co-ethylene dimethacrylate)\nmonoliths,\" Biotechnol. Bioeng., vol. 62, pp. 30-35, 1999.\n[10] L. Ferreira, M. A. Ramos, J. S. Dordick, M. H. Gil, \"Influence of\ndifferent silica derivatives in the immobilization and stabilization of a\nBacillus licheniformis protease (Subtilisin Carlsberg),\" Journal of\nMolecular Catalysis B: Enzymatic, vol. 21, pp. 189-199, 2003.\n[11] F. Peter, L. Poppe, C. Kiss, E. Sz¶Çäÿcs-B├¡ro, G. Preda, C. Zarcula, A.\nOlteanu, \"Influence of precursors and additives on microbial lipases\nstabilized by sol-gel entrapment,\" Biocatal.Biotransform., vol. 23, pp.\n251-260, 2005.\n[12] C. G. Boeriu, A. E. Frissen, E. Boer, K. van Kekem, D- J. van Zoelen, I.\nF. Eggen, \"Optimized enzymatic synthesis of C-terminal peptide amides\nusing subtilisin A from Bacillus licheniformis,\" Journal of Molecular\nCatalysis B: Enzymatic, vol. 66, pp. 33-42, 2010.\n[13] C. G. Boeriu, I. F. Eggen, D- J. van Zoelen, G. H. Bours, \"Selective\nenzymatic hydrolysis of C-terminal tert-butyl esters of peptides,\" in\nPeptides for Youth: The Proceedings of the 20th APS Symposium,\nMontreal, pp. 115-116, 2009,\n[14] C. G. Boeriu, I. F. Eggen, \"Selective enzymatic hydrolysis of C-terminal\ntert-butyl esters of peptides,\" WO 2007/082890 A1.\n[15] M. M. Bradford, \"Rapid and sensitive method for the quantitation of\nmicrogram quantities of protein utilizing the principle of protein-dye\nbinding,\" Anal. Biochem., vol. 72, pp. 248-254, 1976.\n[16] J. E. Celis, N. Carter, T. Hunter, K. Simons, J. V. Small, D. Shotton, Cell\nBiology: A Laboratory Handbook, 3th Edition, San Diego, Elsevier\nAcademic Press, 2006.\n[17] L. Betancor, H. R. Luckarift, \"Bioinspired enzyme encapsulation for\nbiocatalysis,\" Trends Biotechnol., vol. 26, no. 10, pp. 566-572, 2008.\n[18] I. F. Eggen, C. G. Boeriu, \"Process for the conversion of C-terminal\npeptide esters or acids to amides employing subtilisin in the present of\nammonium salts,\" WO 2009/000814."]}
Databáze: OpenAIRE
Externí odkaz: