A novel fed-batch based strategy for enhancing cell-density and recombinant cyprosin B production in bioreactors |
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Authors: | P N Sampaio M S Pais L P Fonseca |
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Institution: | 1. BioFIG, Unit of Molecular Biology and Plant Biotechnology, Plant Systems Biology Laboratory, Institute of Applied Science and Technology, Faculty of Sciences of University of Lisbon, Campo Grande, 1749-016, Lisbon, Portugal 2. Department of Bioengineering, Instituto Superior Técnico (IST), University of Lisbon, Av Rovisco Pais, 1049-001, Lisbon, Portugal 3. Institute for Biotechnology and Bioengineering (IBB), Centre for Biological and Chemical Engineering, IST, UL, Lisbon, Portugal
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Abstract: | Nowadays, the dairy industry is continuously looking for new and more efficient clotting enzymes to create innovative products. Cyprosin B is a plant aspartic protease characterized by clotting activity that was previously cloned in Saccharomyces cerevisiae BJ1991 strain. The production of recombinant cyprosin B by a batch and fed-batch culture was compared using glucose and galactose as carbon sources. The strategy for fed-batch cultivation involved two steps: in the first batch phase, the culture medium presented glucose 1 % (w/v) and galactose 0.5 % (w/v), while in the feed step the culture medium was constituted by 5 % (w/v) galactose with the aim to minimize the GAL7 promoter repression. Based on fed-batch, in comparison to batch growth, an increase in biomass (6.6-fold), protein concentration (59 %) and cyprosin B activity (91 %) was achieved. The recombinant cyprosin B was purified by a single hydrophobic chromatography, presenting a specific activity of 6 × 104 U·mg?1, corresponding to a purification degree of 12.5-fold and a recovery yield of 16.4 %. The SDS-PAGE analysis showed that recovery procedure is suitable for achieving the purified recombinant cyprosin B. The results show that the recombinant cyprosin B production can be improved based on two distinct steps during the fed-batch, presenting that this strategy, associated with a simplified purification procedure, could be applied to large-scale production, constituting a new and efficient alternative for animal and fungal enzymes widely used in cheese making. |
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