A set-up procedure for the minimisation of signal noise of a capillary inlet mass spectrometer sy... more A set-up procedure for the minimisation of signal noise of a capillary inlet mass spectrometer system, enabling direct use of data without noise filtering or drift correction, is described. A reliable calibration method was developed, involving standard calibration mixtures determined by the extreme vertices design. This novel method was shown to be the most accurate in comparison with a number of commonly used methods. These procedures enabled reliable on-line fermenter headspace gas analysis. With a relatively inexpensive mass spectrometer, monitoring of a fermentation and accurate estimation of oxygen uptake rate, carbon dioxide evolution rate, respiratory quotient and biomass concentration was possible.
By expanded bed adsorption (EBA) it was possible to simultaneously recover and purify the heterol... more By expanded bed adsorption (EBA) it was possible to simultaneously recover and purify the heterologous cutinase directly from the crude feedstock. However, it was observed that in a highly condensed and consequently economically advantageous purification process as EBA, the cultivation step highly influences the following purification step. Thus, the yeast cultivation and cutinase purification by EBA cannot be considered as independent entities, and the understanding of the interactions between them are crucial for the development of a highly cost effective overall cutinase production process.From the cultivation strategies studied, one batch, one continuous and two fed-batch cultivations, the strategy that resulted in a more economical cutinase overall production process was a fed-batch mode with a feeding in galactose. This last cultivation strategy, exhibited the highest culture cutinase activity and bioreactor productivity, being obtained 3.8-fold higher cutinase activity and 3.0-fold higher productivity that could compensate the 40% higher cultivation medium costs when compared with a fed-batch culture with a feeding on glucose and galactose. Moreover, a 3.8-fold higher effective cutinase dynamic adsorption capacity and 3.8-fold higher effective purification productivity were obtained in relation to the fed-batch culture with the feeding on glucose and galactose. The cultivation strategy with a feeding on galactose, that presented 5.6-fold higher effective purification productivity, could also compensate the 32% effective adsorption capacity obtained with a continuous cultivation broth. Furthermore, a 205-fold higher cutinase activity, 24-fold higher bioreactor productivity and 6% of the cultivation medium costs were obtained in relation to the continuous culture.
The optimum conditions for the production of low methoxyl pectin using pectinmethylesterase (PME)... more The optimum conditions for the production of low methoxyl pectin using pectinmethylesterase (PME) from acerola (Malpighia glabra L.), immobilized in gelatin, have been established by factorial design and response surface methodology. In the case of the free enzyme, the optimum conditions for activity, within ranges adequate for food processing, are low NaCl concentrations (0.10 M), relatively high temperatures (55 °C) and slightly basic pH values (pH=9). The temperature and pH seem to have strong influence on the observed activity. In the immobilized enzyme, optimum NaCl concentration was 0.15 M, while the optimum pH remained at 9.0.
Although the metabolism and physiology of the growth of yeast strains has been extensively studie... more Although the metabolism and physiology of the growth of yeast strains has been extensively studied, many questions remain unanswered when the induced production of a recombinant protein is concerned. This work addresses the production of a Fusarium solani pisi cutinase by a recombinant Saccharomyces cerevisiae strain induced through the use of a galactose promoter. It was observed that whenever the strain needed to activate biosynthetic pathways, either for cutinase synthesis, or for the synthesis of the enzymes required for galactose intake, acetate production occurred. The on-line detection of acetate in the medium might prove useful for the control and the supervision of recombinant protein production processes using yeast. The volumes of acid and base added to control the pH throughout the time course of the cultivations were used to calculate an on-line estimator for acetate concentration.
Ultra high fructose syrup is of increasing importance, because of its sweetening properties. Fruc... more Ultra high fructose syrup is of increasing importance, because of its sweetening properties. Fructose is often obtained by glucose isomerization. However, this process has a relatively low conversion (around 45%), and it uses a complex purification scheme. An alternative process involves the hydrolysis of natural products, such as inulin, a polymer which contains between 5 and 65 molecules of fructose. The use of enzymes in such hydrolysis reactions would add the benefit of mild reaction conditions and a better product profile. In this work, the enzymatic hydrolysis of inulin has been studied in a membrane reactor (EMR). A first set of experiments aimed to find the optimum operating conditions, in terms of pH (a value of 4.5 was obtained as optimum), substrate concentration (a maximum of 100 g/L is recommended, because of the ease of inulin precipitation), and substrate/enzyme ratio. All experiments were conducted at 50 °C. Then, enzymes were immobilized in order to be able to reuse them, which would result in a more economically feasible process. As immobilization supports both hollow fibre membranes and particulates have been used and compared, and methods of improving the amount and/or the stability of the immobilized enzymes have also been proposed and tested. The results show that the EMR is capable to retaining more than 50% of the initial activity that the enzyme has in solution over at least five repeated reaction cycles. This work can provide a preliminary basis for the production of fructose syrups with a commercial inulinase preparation immobilized onto a membrane.
A set-up procedure for the minimisation of signal noise of a capillary inlet mass spectrometer sy... more A set-up procedure for the minimisation of signal noise of a capillary inlet mass spectrometer system, enabling direct use of data without noise filtering or drift correction, is described. A reliable calibration method was developed, involving standard calibration mixtures determined by the extreme vertices design. This novel method was shown to be the most accurate in comparison with a number of commonly used methods. These procedures enabled reliable on-line fermenter headspace gas analysis. With a relatively inexpensive mass spectrometer, monitoring of a fermentation and accurate estimation of oxygen uptake rate, carbon dioxide evolution rate, respiratory quotient and biomass concentration was possible.
By expanded bed adsorption (EBA) it was possible to simultaneously recover and purify the heterol... more By expanded bed adsorption (EBA) it was possible to simultaneously recover and purify the heterologous cutinase directly from the crude feedstock. However, it was observed that in a highly condensed and consequently economically advantageous purification process as EBA, the cultivation step highly influences the following purification step. Thus, the yeast cultivation and cutinase purification by EBA cannot be considered as independent entities, and the understanding of the interactions between them are crucial for the development of a highly cost effective overall cutinase production process.From the cultivation strategies studied, one batch, one continuous and two fed-batch cultivations, the strategy that resulted in a more economical cutinase overall production process was a fed-batch mode with a feeding in galactose. This last cultivation strategy, exhibited the highest culture cutinase activity and bioreactor productivity, being obtained 3.8-fold higher cutinase activity and 3.0-fold higher productivity that could compensate the 40% higher cultivation medium costs when compared with a fed-batch culture with a feeding on glucose and galactose. Moreover, a 3.8-fold higher effective cutinase dynamic adsorption capacity and 3.8-fold higher effective purification productivity were obtained in relation to the fed-batch culture with the feeding on glucose and galactose. The cultivation strategy with a feeding on galactose, that presented 5.6-fold higher effective purification productivity, could also compensate the 32% effective adsorption capacity obtained with a continuous cultivation broth. Furthermore, a 205-fold higher cutinase activity, 24-fold higher bioreactor productivity and 6% of the cultivation medium costs were obtained in relation to the continuous culture.
The optimum conditions for the production of low methoxyl pectin using pectinmethylesterase (PME)... more The optimum conditions for the production of low methoxyl pectin using pectinmethylesterase (PME) from acerola (Malpighia glabra L.), immobilized in gelatin, have been established by factorial design and response surface methodology. In the case of the free enzyme, the optimum conditions for activity, within ranges adequate for food processing, are low NaCl concentrations (0.10 M), relatively high temperatures (55 °C) and slightly basic pH values (pH=9). The temperature and pH seem to have strong influence on the observed activity. In the immobilized enzyme, optimum NaCl concentration was 0.15 M, while the optimum pH remained at 9.0.
Although the metabolism and physiology of the growth of yeast strains has been extensively studie... more Although the metabolism and physiology of the growth of yeast strains has been extensively studied, many questions remain unanswered when the induced production of a recombinant protein is concerned. This work addresses the production of a Fusarium solani pisi cutinase by a recombinant Saccharomyces cerevisiae strain induced through the use of a galactose promoter. It was observed that whenever the strain needed to activate biosynthetic pathways, either for cutinase synthesis, or for the synthesis of the enzymes required for galactose intake, acetate production occurred. The on-line detection of acetate in the medium might prove useful for the control and the supervision of recombinant protein production processes using yeast. The volumes of acid and base added to control the pH throughout the time course of the cultivations were used to calculate an on-line estimator for acetate concentration.
Ultra high fructose syrup is of increasing importance, because of its sweetening properties. Fruc... more Ultra high fructose syrup is of increasing importance, because of its sweetening properties. Fructose is often obtained by glucose isomerization. However, this process has a relatively low conversion (around 45%), and it uses a complex purification scheme. An alternative process involves the hydrolysis of natural products, such as inulin, a polymer which contains between 5 and 65 molecules of fructose. The use of enzymes in such hydrolysis reactions would add the benefit of mild reaction conditions and a better product profile. In this work, the enzymatic hydrolysis of inulin has been studied in a membrane reactor (EMR). A first set of experiments aimed to find the optimum operating conditions, in terms of pH (a value of 4.5 was obtained as optimum), substrate concentration (a maximum of 100 g/L is recommended, because of the ease of inulin precipitation), and substrate/enzyme ratio. All experiments were conducted at 50 °C. Then, enzymes were immobilized in order to be able to reuse them, which would result in a more economically feasible process. As immobilization supports both hollow fibre membranes and particulates have been used and compared, and methods of improving the amount and/or the stability of the immobilized enzymes have also been proposed and tested. The results show that the EMR is capable to retaining more than 50% of the initial activity that the enzyme has in solution over at least five repeated reaction cycles. This work can provide a preliminary basis for the production of fructose syrups with a commercial inulinase preparation immobilized onto a membrane.
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Papers by Bruno Ferreira