title: Production in stirred-tank bioreactor of recombinant bovine chymosin B by a high-level expression transformant clone of Pichia pastoris.
abstract: An intense screening of Pichia pastoris clones transformed with the gene of bovine chymosin under methanol-inducible AOX1 promoter was performed, obtaining a transformant clone with a higher milk-clotting activity value in comparison with our previous studies. The scaling of recombinant-chymosin production was carried out by a fed-batch strategy in a stirred-tank bioreactor using biodiesel-byproduct crude glycerol as the carbon source and pure methanol for the induction of chymosin expression, achieving a biomass concentration of 158 g DCW/L and a maximum coagulant activity of 192 IMCU/ml after 120 h of methanol induction. Recombinant bovine chymosin was purified from bioreactor-fermentation culture by a procedure including anion-exchange chromatography which allowed obtaining heterologous chymosin with high level of purity and activity; suggesting that this downstream step could be scaled up in a successful manner for chymosin purification. Thermoestability assay permitted to establish that unformulated recombinant chymosin could be stored at 5 °C without decrease of enzyme activity throughout at least 120 days. Finally, reiterative methanol-inductions of recombinant chymosin expression in bioreactor demonstrated that the reutilization of cell biomass overcame the low enzyme productivity usually reached by P. pastoris system.
authors.affiliation: Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECH), Universidad Nacional de San Martín (UNSAM)-Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), San Martín, 1650, Buenos Aires, Argentina. Electronic address: diegonoseda@yahoo.com.ar.
authors.forename: Diego Gabriel
authors.initials: DG
authors.lastname: Noseda
authors.affiliation: Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECH), Universidad Nacional de San Martín (UNSAM)-Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), San Martín, 1650, Buenos Aires, Argentina.
authors.forename: Matías
authors.initials: M
authors.lastname: Recúpero
authors.affiliation: Centro de Investigación y Desarrollo en Biotecnología Industrial, Instituto Nacional de Tecnología Industrial, Av. Gral Paz 5445, Edificio 51, San Martín, 1650, Buenos Aires, Argentina.
authors.forename: Martín
authors.initials: M
authors.lastname: Blasco
authors.affiliation: Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECH), Universidad Nacional de San Martín (UNSAM)-Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), San Martín, 1650, Buenos Aires, Argentina.
authors.forename: Joaquín
authors.initials: J
authors.lastname: Bozzo
authors.affiliation: Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECH), Universidad Nacional de San Martín (UNSAM)-Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), San Martín, 1650, Buenos Aires, Argentina; Laboratorio de Microbiología Industrial, Departamento de Ingeniería Química, Facultad de Ingeniería, Universidad de Buenos Aires, Pabellón de Industrias, Ciudad Universitaria, 1428, Buenos Aires, Argentina.
authors.forename: Miguel Ángel
authors.initials: MÁ
authors.lastname: Galvagno
mesh_terms: D000818:Animals; D056804:Biofuels / Q000032:analysis / Q000382:microbiology; D019149:Bioreactors; D002417:Cattle; D002852:Chromatography, Ion Exchange; D012085:Chymosin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D005285:Fermentation; D005990:Glycerol / Q000378:metabolism; D007218:Industrial Microbiology / Q000295:instrumentation; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D056804:Biofuels; D011994:Recombinant Proteins; D012085:Chymosin; D005990:Glycerol
keywords: Biodiesel-derived crude glycerol; Bovine chymosin; Fed-batch fermentation; Methanol-inducible AOX1 promoter; Pichia pastoris; Protein purification; Reiterative methanol-inductions