Francielle Herrmann Mobayed1, Juliane Carraro Nunes2, Adriano Gennari1,3, Bruna Coelho de Andrade1,3, Matheus Loch Velvites Ferreira2, Paolla Pauli2, Gaby Renard4, Jocelei Maria Chies5, Giandra Volpato2, Claucia Fernanda Volken de Souza6,7. 1. Laboratório de Biotecnologia de Alimentos, Universidade do Vale do Taquari - Univates, Av. Avelino Tallini, 171, ZC, Lajeado, RS, 95914-014, Brazil. 2. Curso de Biotecnologia, Instituto Federal de Educação, Ciência e Tecnologia do Rio Grande do Sul - IFRS, Campus Porto Alegre, Porto Alegre, RS, Brazil. 3. Programa de Pós-Graduação em Biotecnologia, Universidade do Vale do Taquari - Univates, Lajeado, RS, Brazil. 4. Centro de Pesquisa em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brazil. 5. Quatro G Pesquisa & Desenvolvimento Ltda, Porto Alegre, RS, Brazil. 6. Laboratório de Biotecnologia de Alimentos, Universidade do Vale do Taquari - Univates, Av. Avelino Tallini, 171, ZC, Lajeado, RS, 95914-014, Brazil. claucia@univates.br. 7. Programa de Pós-Graduação em Biotecnologia, Universidade do Vale do Taquari - Univates, Lajeado, RS, Brazil. claucia@univates.br.
Abstract
OBJECTIVE: The aim of the present study was to evaluate the efficiency of lactose derived from cheese whey and cheese whey permeate as inducer of recombinant Kluyveromyces sp. β-galactosidase enzyme produced in Escherichia coli. Two E. coli strains, BL21(DE3) and Rosetta (DE3), were used in order to produce the recombinant enzyme. Samples were evaluated for enzyme activity, total protein content, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis after induction with isopropyl-β-D-1-thiogalactoside (IPTG) (0.05 and 1 mM) and lactose, cheese whey, and cheese whey permeate solutions (1, 10, and 20 g/L lactose) at shake-flask cultivation, and whey permeate solution (10 g/L lactose) at bioreactor scale. RESULTS: The highest specific activities obtained with IPTG as inducer (0.05 mM) after 9 h of induction, were 23 and 33 U/mgprotein with BL21(DE3) and Rosetta(DE3) strains, respectively. Inductions performed with lactose and cheese whey permeate (10 and 20 g/L lactose) showed the highest specific activities at the evaluated hours, exhibiting better results than those obtained with IPTG. Specific activity of recombinant β-galactosidase using whey permeate (10 g/L lactose) showed values of approximately 46 U/mgprotein after 24-h induction at shake-flask study, and approximately 26 U/mgprotein after 16-h induction at bench bioreactor. CONCLUSIONS: The induction with cheese whey permeate was more efficient for recombinant β-galactosidase expression than the other inducers tested, and thus, represents an alternative form to reduce costs in recombinant protein production.
OBJECTIVE: The aim of the present study was to evaluate the efficiency of lactose derived from cheese whey and cheese whey permeate as inducer of recombinant Kluyveromyces sp. β-galactosidase enzyme produced in Escherichia coli. Two E. coli strains, BL21(DE3) and Rosetta (DE3), were used in order to produce the recombinant enzyme. Samples were evaluated for enzyme activity, total protein content, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis after induction with isopropyl-β-D-1-thiogalactoside (IPTG) (0.05 and 1 mM) and lactose, cheese whey, and cheese whey permeate solutions (1, 10, and 20 g/L lactose) at shake-flask cultivation, and whey permeate solution (10 g/L lactose) at bioreactor scale. RESULTS: The highest specific activities obtained with IPTG as inducer (0.05 mM) after 9 h of induction, were 23 and 33 U/mgprotein with BL21(DE3) and Rosetta(DE3) strains, respectively. Inductions performed with lactose and cheese whey permeate (10 and 20 g/L lactose) showed the highest specific activities at the evaluated hours, exhibiting better results than those obtained with IPTG. Specific activity of recombinant β-galactosidase using whey permeate (10 g/L lactose) showed values of approximately 46 U/mgprotein after 24-h induction at shake-flask study, and approximately 26 U/mgprotein after 16-h induction at bench bioreactor. CONCLUSIONS: The induction with cheese whey permeate was more efficient for recombinant β-galactosidase expression than the other inducers tested, and thus, represents an alternative form to reduce costs in recombinant protein production.
Authors: Antonio De León-Rodríguez; Dulce Rivera-Pastrana; Emilio Medina-Rivero; José Luis Flores-Flores; Alejandro Estrada-Baltazar; Leandro G Ordóñez-Acevedo; Ana P Barba de la Rosa Journal: Biomol Eng Date: 2006-11-09