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- Effect of Saccharomyces cerevisiae fermentation conditions on expanded bed adsorption of heterologous cutinasePublication . Calado, Cecília; Cabral, Joaquim M.S.; Fonseca, Luís P. P.The effect of culture media composition, and fermentation conditions and strategy on the growth and cutinase production of recombinant Saccharomyces cerevisiae and subsequent cutinase purification by expanded bed adsorption (EBA) was studied. The reduction in the amount of yeast extract used as nitrogen source from 20 g dm−3 to 10 g dm−3 in batch cultures led to a 29% decrease in the heterologous cutinase production, while the 5% cutinase dynamic adsorption capacity (q5%) on the cation Streamline SP XL was increased 6.7‐fold. By dilution of the whole fermentation broth, performed with the lowest yeast extract content, which reduces conductivity, the q5% was additionally increased by 1.9‐fold. After implementation of a fed‐batch strategy the cutinase concentration, cutinase yield on carbon source, cutinase yield on nitrogen source and productivity were increased by 10.8‐, 2.9‐, 5.3‐ and 6.4‐fold, respectively, in relation to the previously‐mentioned batch fermentation. However, the increased cutinase production was compromised by heterologous protein loss during the EBA recovery operation and the cutinase dynamic adsorption capacity and purification productivity decreased by 90% and 75%, respectively. Thus, target protein production by S cerevisiae fermentation and a downstream process with EBA cannot be considered as separate entities, where the understanding of the factors that affect the interactions among them are crucial towards optimization of the overall production process of heterologous proteins.
- Optimisation of culture conditions and characterisation of cutinase produced by recombinant Saccharomyces cerevisiaePublication . Calado, Cecília; Taipa, M. Ângela; Cabral, Joaquim M.S.; Fonseca, Luís P. P.The optimum culture dissolved oxygen concentration and culture pH for the production of cutinase from Fusarium solani pisi by the recombinant Saccharomyces cerevisiae SU50 strain was investigated. Dissolved oxygen concentrations in the fermentation culture of 5, 30 and 60% of air saturation were evaluated. A high cutinase production, specific cutinase activity and the highest cutinase yield on biomass and the highest specific cutinase production rate were obtained with a 5% of air saturation, which could have impact on process economics. Furthermore, at a low dissolved oxygen concentration, the specific growth rate, specific cutinase production rate, cutinase yield on biomass, cutinase activity and specific cutinase activity were increased when the pH control was changed from 5.25 to 6.25. The cutinase, accumulated in the yeast culture presents two glycosilated isoforms with molecular weights of 22.8 and 24.9 kDa measured by SDS-PAGE. Furthermore, cutinase in clarified culture samples presents a linear relationship between estereolytic and lypolitic activity and a high stability at room temperature.
- Effect of pre-fermentation on the production of cutinase by a recombinant Saccharomyces cerevisiaePublication . Calado, Cecília; Monteiro, Sandra M. S.; Cabral, Joaquim M.S.; Fonseca, Luís P. P.The importance of controlling the expression of heterologous cutinase in a recombinant Saccharomyces cerevisiae SU50 strain was investigated. Maximum specific growth rate and the biomass yield increased 1.91 and 1.16 fold, respectively, when cutinase production was induced by galactose in a pre-fermentation step. However, only 19% of specific cell activity was obtained in comparison to other fermentations following a pre-fermentation step without induction of cutinase expression. Thus, the pre-fermentation step was performed using a selective medium not containing galactose, and the fermentation was performed with a cheaper and complex non-selective medium containing galactose. Under these conditions, and with the aim of maximising the specific cutinase activity, a pre-fermentation with low volume and high density of viable cells must be used. However, due to the low pre-fermentation volume, low yeast cell concentrations and low specific cell activities were obtained after 96 h of fermentation. Otherwise, when the aim was to maximise cutinase yield and productivity, a pre-fermentation volume of 10% (v/v) in relation to fermentation and in the exponential growth phase with a cell concentration between 1.1 and 1.8 g dcw/l should be used. A higher pre-fermentation volume, such as 20% (v/v), would still be economical in the case of a pre-fermentation with low cell density or low cell viability.
- Development of a fed-batch cultivation strategy for the enhanced production and secretion of cutinase by a recombinant Saccharomyces cerevisiae SU50 strainPublication . Calado, Cecília; Almeida, Claúdio; Cabral, Joaquim M.S.; Fonseca, Luís P. P.Saccharomyces cerevisiae SU50 strain was cultivated with different concentrations of glucose and galactose with the aim of increasing cutinase activity, cutinase yield on the carbon source, and bioreactor productivity. Cultivations in shake flasks with galactose as the sole carbon source, with sugar concentrations between 10 and 40 g/l, exhibited growth-associated cutinase production and a constant specificity of cutinase secretion. Furthermore, as the galactose concentration increased to values higher than 15 g/l, a progressively higher maximum specific galactose consumption rate and a consequent higher alcoholic fermentation occurred, resulting in progressively lower biomass yields on the carbon source and cutinase yields on biomass. Using high glucose and galactose concentrations in a well-aerated bioreactor resulted in a high biomass productivity (0.5 g dcw/l/h), a high cutinase yield on biomass (21.5 U/mg dew), a final high cutinase secretion efficiency (97%), and plasmid stability (99%). Based on these studies, a two phase fed-batch cultivation strategy was developed. A batch phase with high glucose and galactose concentrations, followed by a fedbatch with a constant feed rate with galactose as the sole carbon source in order to minimize the repression of the GAL 7 promoter, were established. The feed rate was estimated to maintain a pre-determined concentration of galactose (20 g/l) on the culture medium in order to maximize the efficiency of cutinase secretion and minimize the galactose alcoholic fermentation. By this cultivation strategy, enhancements of 3.6-fold in cutinase activity, 1.2-fold in cutinase yield on the carbon source, and 8.7-fold culture productivity were obtained in relation to a batch cultivation performed in shake flasks with 20 g/l of galactose.
- A flow injection analysis system for on‐line monitoring of cutinase activity at outlet of an expanded bed adsorption column almost in real timePublication . Almeida, Cláudio F.; Calado, Cecília; Bernardino, Susana A.; Cabral, Joaquim M.S.; Fonseca, Luís P. P.Expanded bed adsorption (EBA) was used to recover, concentrate and purify Fusarium solani pisi cutinase, secreted by a recombinant Saccharomyces cerevisiae strain, directly from a whole fermentation culture. A flow injection analysis (FIA) system for monitoring Fusarium solani pisi cutinase based on microencapsulation of p‐nitrophenylbutyrate (p‐NPB) in a micellar system (sodium cholate, tetrahydrofuran, phosphate buffer) was developed for monitoring this target enzyme at the outlet tube of the EBA column. Slight differences in yeast cultivation conditions during cutinase production may influence the fermentation performance, which affects directly the adsorption of cutinase during the loading step and consequently the efficiency of the EBA process. This effect can be especially relevant when it is necessary to stop the application of feedstock to the EBA column when the outlet concentration (A) of the desired product is lower than 5% of the feed concentration (Ao). Excellent correlations between the FIA system and the off‐line analytical method for monitoring cutinase activity during the different EBA steps were obtained. Additionally, the blocking/fouling of the sample injector and tubes of the FIA system initially observed were eliminated due to the excellent surfactant properties of the sodium cholate contained in the phosphate buffer and used to dilute the enzyme samples. This FIA system was shown to be a powerful analytical tool for monitoring cutinase activity almost in real time (45–60 s), maximizing enzyme adsorption while minimizing product loss and consequently maximizing the recovery yield of the product. Copyright © 2006 Society of Chemical Industry
- Production of wild‐type and peptide fusion cutinases by recombinant Saccharomyces cerevisiae MM01 strainsPublication . Calado, Cecília; Mannesse, Maurice; Egmond, Maarten; Cabral, Joaquim M.S.; Fonseca, Luís P. P.This study focused on the growth of Saccha‐romyces cerevisiae MM01 recombinant strains and the respective production of three extracellular heterologous cutinases: a wild‐type cutinase and two cutinases in which the primary structure was fused with the peptides (WP)2 and (WP)4, respectively. Different cultivation and strategies were tested in a 2‐L shake flask and a 5‐L bioreactor, and the respective cell growth and cutinase production were analyzed and compared for the three yeast strains. The highest cutinase productions and productivities were obtained in the fed‐batch culture, where wild‐type cutinase was secreted up to a level of cutinase activity per dry cell weight (specific cell activity) of 4.1 Umg−1 with activity per protein broth (specific activity) of 266 Umg−1, whereas cutinase‐(WP)2 was secreted with a specific cell activity of 2.1 Umg−1 with a specific activity of 200 Umg−1, and cutinase‐(WP)4 with a specific cell activity of 0.7 Umg−1 with a specific activity of 15 Umg−1. The results indicate that the fusion of hydrophobic peptides to cutinase that changes the physical properties of the fused protein limits cutinase secretion and subsequently leads to a lower plasmid stability and lower yeast cell growth. These effects were observed under different cultivation conditions (shake flask and bioreactor) and cultivation strategies (batch culture versus fed‐batch culture).