title: A novel fed-batch based strategy for enhancing cell-density and recombinant cyprosin B production in bioreactors.
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 × 10(4) 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.
authors.affiliation: 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, pnsampaio@gmail.com.
authors.forename: P N
authors.initials: PN
authors.lastname: Sampaio
authors.forename: M S
authors.initials: MS
authors.lastname: Pais
authors.forename: L P
authors.initials: LP
authors.lastname: Fonseca
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry; D061249:Batch Cell Culture Techniques; D018533:Biomass; D019149:Bioreactors; D002244:Carbon / Q000737:chemistry; D002611:Cheese; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D005524:Food Technology / Q000379:methods*; D005690:Galactose / Q000737:chemistry; D005947:Glucose / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008892:Milk / Q000737:chemistry; D010944:Plants / Q000201:enzymology; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D002244:Carbon; D016282:Aspartic Acid Endopeptidases; C087364:cyprosin; D005947:Glucose; D005690:Galactose