Near infrared (NIR) spectroscopy was used to in situ monitoring the cultivation of two recombinant Saccharomyces cerevisiae strains producing heterologous cyprosin B. NIR spectroscopy is a fast and non-destructive technique, that by being based on overtones and combinations of molecular vibrations requires chemometrics tools, such as partial least squares (PLS) regression models, to extract quantitative information concerning the variables of interest from the spectral data. In the present work, good PLS calibration models based on specific regions of the NIR spectral data were built for estimating the critical variables of the cyprosin production process: biomass concentration, cyprosin activity, cyprosin specific activity, the carbon sources glucose and galactose concentration and the by-products acetic acid and ethanol concentration. The PLS models developed are valid for both recombinant S. cerevisiae strains, presenting distinct cyprosin production capacities, and therefore can be used, not only for the real-time control of both processes, but also in optimization protocols. The PLS model for biomass yielded a R(2)=0.98 and a RMSEP=0.46 g dcw l(-1), representing an error of 4% for a calibration range between 0.44 and 13.75 g dcw l(-1). A R(2)=0.94 and a RMSEP=167 Um l(-1) were obtained for the cyprosin activity, corresponding to an error of 6.7% of the experimental data range (0-2509 Um l(-1)), whereas a R(2)=0.93 and RMSEP=672 U mg(-1) were obtained for the cyprosin specific activity, corresponding to an error of 7% of the experimental data range (0-11,690 Um g(-1)). For the carbon sources glucose and galactose, a R(2)=0.96 and a RMSECV of 1.26 and 0.55 g l(-1), respectively, were obtained, showing high predictive capabilities within the range of 0-20 g l(-1). For the metabolites resulting from the cell growth, the PLS model for acetate was characterized by a R(2)=0.92 and a RMSEP=0.06 g l (-1), which corresponds to a 6.1% error within the range of 0.41-1.23 g l(-1); for the ethanol, a high accuracy PLS model with a R(2)=0.97 and a RMSEP=1.08 g l(-1) was obtained, representing an error of 9% within the range of 0.18-21.76 g l(-1). The present study shows that it is possible the in situ monitoring and prediction of the critical variables of the recombinant cyprosin B production process by NIR spectroscopy, which can be applied in process control in real-time and in optimization protocols. From the above, NIR spectroscopy appears as a valuable analytical tool for online monitoring of cultivation processes, in a fast, accurate and reproducible operation mode.
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http://dx.doi.org/10.1016/j.jbiotec.2014.07.454 | DOI Listing |
Sci Rep
July 2021
Plant Cell Biology Laboratory, Instituto de Tecnologia Química e Biológica António Xavier, ITQB NOVA, Universidade Nova de Lisboa, 2780-157, Oeiras, Portugal.
Cynara cardunculus L. or cardoon is a plant that is used as a source of milk clotting enzymes during traditional cheese manufacturing. This clotting activity is due to aspartic proteases (APs) found in the cardoon flower, named cyprosins and cardosins.
View Article and Find Full Text PDFAppl Microbiol Biotechnol
June 2018
Biocant, Biotechnology Innovation Center, Parque Tecnológico de Cantanhede, Núcleo 4, Lote 8, 3060-197, Cantanhede, Portugal.
The use of crude aqueous extracts of Cynara cardunculus flowers as coagulants in the production of high-quality sheep and goat cheeses-as are the cases of several Portuguese and Spanish cheese varieties with Protected Designation of Origin status-has been maintained since ancient times. The unique rheological attributes and sensory properties characteristic of these cheeses have always suggested that this plant coagulant (and, therefore, its isolated milk-clotting proteases) could be used as alternative rennet in the dairy industry, particularly suited for the production of sheep and goat cheeses. However, the lack of standardization of C.
View Article and Find Full Text PDFJ Ind Microbiol Biotechnol
January 2017
ISEL-Instituto Superior de Engenharia de Lisboa, Instituto Politécnico de Lisboa, Conselheiro Emídio Navarro Street, 1959-007, Lisbon, Portugal.
To increase the knowledge of the recombinant cyprosin production process in Saccharomyces cerevisiae cultures, it is relevant to implement efficient bioprocess monitoring techniques. The present work focuses on the implementation of a mid-infrared (MIR) spectroscopy-based tool for monitoring the recombinant culture in a rapid, economic, and high-throughput (using a microplate system) mode. Multivariate data analysis on the MIR spectra of culture samples was conducted.
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