The effect of glycerol mixed substrate on the heterologous production of a Rhizopus oryzae lipase in Pichia pastoris system

• Published in: Biochemical engineering journal Vol. 57; pp. 30 - 37
• Main Authors: Arnau, Carolina, Casas, Carles, Valero, Francisco
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15.11.2011; Elsevier
• Subjects: Biological and medical sciences; Bioprocess monitoring; Biotechnology; enzyme activity; Fed-Batch culture; Fundamental and applied biological sciences. Psychology; Glycerol; methanol; Mixed substrate; Pichia pastoris; Rhizopus oryzae; Rhizopus oryzae lipase; sorbitol; Glycerol; Pichia pastoris; Fed-Batch culture; Rhizopus oryzae lipase; Mixed substrate; Bioprocess monitoring; Ascomycota; Zygomycota; Enzyme; Triacylglycerol lipase; Esterases; Carboxylic ester hydrolases; Fungi; Substrate; Rhizopus oryzae; Production; Hydrolases; Operating process; Culture; Semicontinuous; Monitoring
• ISSN: 1369-703X; 1873-295X
• DOI: 10.1016/j.bej.2011.08.004

► Mixed substrates are good strategy to increase protein production in Pichia pastoris. ► Methanol set-point and μ are the key parameters in the bioprocess production. ► Glycerol represses PAOX1 at μ highest than 0.04 h −1 in Mut s phenotype. ► Proteolytic activity was detected only when glycerol was used as co-substrate. ► Sorbitol was better co-substrate than glycerol for ROL production. A recombinant Rhizopus oryzae lipase producing Mut s Pichia pastoris strain was used as a model organism to study the effect of mixed substrates (glycerol and methanol) on the specific product productivity. Different fed-batch cultivations were performed under three constant specific growth rates (0.02, 0.05 and 0.1 h −1), maintaining a constant methanol concentration of 2 g l −1. At the lowest μ tested (0.02 h −1), the specific productivity was 1.23 and 1.61 fold higher and the specific methanol consumption rate ( q sMeOH) was 3 and 3.5 fold higher than values obtained when μ was 0.05 and 0.1 h −1, respectively. This implies a relation between the q sMeOH and the specific productivity, yielding higher specific productivities whenever the consumption of methanol is higher. Although glycerol was maintained under limiting conditions in all μ tested, when the relation between the μ Gly and μ MeOH was larger than 4, an important decrease on the maximal activity value was observed. Finally, a comparison under the same conditions using glycerol or sorbitol as co-substrates was also performed, obtaining better specific productivity when sorbitol was used. In addition, protease activity was detected when glycerol was used as co-substrate.