title: Bioprocess and downstream optimization of recombinant bovine chymosin B in Pichia (Komagataella) pastoris under methanol-inducible AOXI promoter.
abstract: A clone of the methylotrophic yeast Pichia pastoris strain GS115 transformed with the bovine prochymosin B gene was used to optimize the production and downstream of recombinant bovine chymosin expressed under the methanol-inducible AOXI promoter. Cell growth and recombinant chymosin production were analyzed in flask cultures containing basal salts medium with biodiesel-byproduct glycerol as the carbon source, obtaining values of biomass level and milk-clotting activity similar to those achieved with analytical glycerol. The effect of biomass level at the beginning of methanol-induction phase on cell growth and chymosin expression was evaluated, determining that a high concentration of cells at the start of such period generated an increase in the production of chymosin. The impact of the specific growth rate on chymosin expression was studied throughout the induction stage by methanol exponential feeding fermentations in a lab-scale stirred bioreactor, achieving the highest production of heterologous chymosin with a constant specific growth rate of 0.01h(-1). By gel filtration chromatography performed at a semi-preparative scale, recombinant chymosin was purified from exponential fed-batch fermentation cultures, obtaining a specific milk-clotting activity of 6400IMCU/mg of chymosin and a purity level of 95%. The effect of temperature and pH on milk-clotting activity was analyzed, establishing that the optimal temperature and pH values for the purified recombinant chymosin are 37°C and 5.5, respectively. This study reported the features of a sustainable bioprocess for the production of recombinant bovine chymosin in P. pastoris by fermentation in stirred-tank bioreactors using biodiesel-derived glycerol as a low-cost carbon source.
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: 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: Matías
authors.initials: M
authors.lastname: Recúpero
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; 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; D061249:Batch Cell Culture Techniques; D056804:Biofuels; D018533:Biomass; D019149:Bioreactors; D002417:Cattle; D012085:Chymosin / Q000096:biosynthesis / Q000302:isolation & purification*; D005285:Fermentation; D015870:Gene Expression; D005990:Glycerol / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D000432:Methanol / Q000737:chemistry*; D010843:Pichia / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013696:Temperature
chemical_list: D056804:Biofuels; D011994:Recombinant Proteins; D012085:Chymosin; D005990:Glycerol; D000432:Methanol
keywords: Biodiesel-derived crude glycerol; Bovine chymosin B; Fed-batch fermentation; Methanol-inducible AOX1 promoter; Pichia pastoris; Protein purification