Fed-Batch Production of Saccharomyces cerevisiae L-Asparaginase II by Recombinant Pichia pastoris MUTs Strain
| Autor: | Tajindar Basi, Juan Carlos Flores-Santos, Omar Pillaca-Pullo, Marcela V. Pimenta, David Rodrigues, Luís P. Fonseca, Gisele Monteiro, Karin Torres-Obreque, Ignacio Sánchez-Moguel, André Moreni Lopes, Adalberto Pessoa, Attilio Converti |
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| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Histology lcsh:Biotechnology Saccharomyces cerevisiae Biomedical Engineering Biomass Bioengineering 02 engineering and technology law.invention Pichia pastoris 03 medical and health sciences chemistry.chemical_compound law lcsh:TP248.13-248.65 Glycerol Food science L-Asparaginase Original Research chemistry.chemical_classification FERMENTAÇÃO biology Chemistry Bioengineering and Biotechnology heterologous protein production high cell density culture 021001 nanoscience & nanotechnology biology.organism_classification Enzyme assay defined medium 030104 developmental biology Enzyme biology.protein Recombinant DNA Fermentation fed-batch fermentation 0210 nano-technology Biotechnology |
| Zdroj: | Frontiers in Bioengineering and Biotechnology, Vol 7 (2019) Frontiers in Bioengineering and Biotechnology Repositório Institucional da USP (Biblioteca Digital da Produção Intelectual) Universidade de São Paulo (USP) instacron:USP |
| ISSN: | 2296-4185 |
| Popis: | L-Asparaginase (ASNase) is used in the treatment of acute lymphoblastic leukemia, being produced and commercialized only from bacterial sources. Alternative Saccharomyces cerevisiae ASNase II coded by the ASP3 gene was biosynthesized by recombinant Pichia pastoris MUTs under the control of the AOX1 promoter, using different cultivation strategies. In particular, we applied multistage fed-batch cultivation divided in four distinct phases to produce ASNase II and determine the fermentation parameters, namely specific growth rate, biomass yield, and enzyme activity. Cultivation of recombinant P. pastoris under favorable conditions in a modified defined medium ensured a dry biomass concentration of 31 gdcw.L−1 during glycerol batch phase, corresponding to a biomass yield of 0.77 gdcw.gglycerol-1 and a specific growth rate of 0.21 h−1. After 12 h of glycerol feeding under limiting conditions, cell concentration achieved 65 gdcw.L−1 while ethanol concentration was very low. During the phase of methanol induction, biomass concentration achieved 91 gdcw.L−1, periplasmic specific enzyme activity 37.1 U.gdcw-1, volumetric enzyme activity 3,315 U.L−1, overall enzyme volumetric productivity 31 U.L−1.h−1, while the specific growth rate fell to 0.039 h−1. Our results showed that the best strategy employed for the ASNase II production was using glycerol fed-batch phase with pseudo exponential feeding plus induction with continuous methanol feeding. |
| Databáze: | OpenAIRE |
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