Selection of CalB immobilization method to be used in continuous oil transesterification: Analysis of the economical impact

Enzymatic transesterification of triglycerides in a continuous way is always a great challenge with a large field of applications for biodiesel, bio-lubricant, bio-surfactant, etc. productions. The lipase B from Candida antarctica (CalB) is the most appreciated enzyme because of its high activity and its non-regio-selectivity toward positions of fatty acid residues on glycerol backbone of triglycerides. Nevertheless, in the field of heterogeneous catalysis, we demonstrated that the medium hydrophilic nature of the support used for its commercial form (Lewatit VPOC1600) is a limitation. Glycerol is adsorbed onto support inducing drastic decrease in enzyme activity. Glycerol would form a hydrophilic layer around the enzyme resulting in diffusional limitations during triglyceride transfer to the enzyme. Accurel MP, a very hydrophobic macroporous polymer of propylene, was found not to adsorb glycerol. Immobilization conditions using this support were optimized. The best support was Accurel MP1001 (particle size<1000μm) and a pre-treatment of the support with acetone instead of ethanol enables the adsorption rate and the immobilized enzyme quantity to be maximized. An economical approach (maximization of the process net present value) was expanded in order to explore the impact of immobilization on development of an industrial packed bed reactor. The crucial ratio between the quantity of lipase and the quantity of support, taking into account enzyme, support and equipped packed bed reactor costs was optimized in this sense. The biocatalyst cost was found as largely the main cost centre (2–10 times higher than the investments for the reactor vessel). In consequence, optimal conditions for immobilization were a compromise between this immobilization yield (90% of lipase immobilized), biocatalyst activity, reactor volume and total investments

AutoBioTip: Towards automation of mechanobiological measurements by AFM

International audienceThe paradigm in bio-AFM is to measure a few dozen of cells and to draw fundamental biophysical conclusions. To apply these results in biology or medicine, it is essential to increase the statistics by measuring of significant number of cells and to automate the measurements. In this context, it is necessary to optimize the measurements, to reduce the results dispersity and to increase the experiment speed. Concerning the dispersity, we hypothesize that cells organized on patterns would have reduced Young’s modulus dispersion. We had the same hypothesis in regard to the indenter shape (cone vs. colloidal probe).Figure 1 shows that randomly shaped cells (A) present Young's moduli more dispersed than cells that have been forced into a square shape (B). The use of patterns therefore makes it possible to increase the homogeneity of the obtained results. We tested the effect of an indenter which probes a larger surface with a 5µm-diameter colloidal probe. The Young's modulus is smaller with the colloidal probe than with the pyramidal probe, but the dispersion is also smaller. There is therefore an advantage to use a colloidal probe in the automation process. To increase the measurement speed, we worked on two parameters, the tip velocity and the number of indentations per cell. We therefore evaluated the impact of the indentation velocity on the resulting mechanical properties. Young's modulus is higher with increasing velocity. But this trend is comparable for cells constrained by square patterns of fibronectin and those not constrained. A high indentation velocity can therefore be applied during automation. Finally, we compared the influence of the number of indentations (1,024; 256; 64 and 16) per cell . There is no significant difference in Young's modulus value between each condition. Automation can therefore be done with a reduced number of indentations per cell.To conclude, this optimization work opens the door to the implementation of automatic measurements on a large number of cells, which will allow us to probe a large number of cells in a controlled time. This will open the bio-AFM to statistical analyses on populations of cells of interest

AutoBioTip: Towards automation of mechanobiological measurements by AFM

International audienceThe paradigm in bio-AFM is to measure a few dozen of cells and to draw fundamental biophysical conclusions. To apply these results in biology or medicine, it is essential to increase the statistics by measuring of significant number of cells and to automate the measurements. In this context, it is necessary to optimize the measurements, to reduce the results dispersity and to increase the experiment speed. Concerning the dispersity, we hypothesize that cells organized on patterns would have reduced Young’s modulus dispersion. We had the same hypothesis in regard to the indenter shape (cone vs. colloidal probe).Figure 1 shows that randomly shaped cells (A) present Young's moduli more dispersed than cells that have been forced into a square shape (B). The use of patterns therefore makes it possible to increase the homogeneity of the obtained results. We tested the effect of an indenter which probes a larger surface with a 5µm-diameter colloidal probe. The Young's modulus is smaller with the colloidal probe than with the pyramidal probe, but the dispersion is also smaller. There is therefore an advantage to use a colloidal probe in the automation process. To increase the measurement speed, we worked on two parameters, the tip velocity and the number of indentations per cell. We therefore evaluated the impact of the indentation velocity on the resulting mechanical properties. Young's modulus is higher with increasing velocity. But this trend is comparable for cells constrained by square patterns of fibronectin and those not constrained. A high indentation velocity can therefore be applied during automation. Finally, we compared the influence of the number of indentations (1,024; 256; 64 and 16) per cell . There is no significant difference in Young's modulus value between each condition. Automation can therefore be done with a reduced number of indentations per cell.To conclude, this optimization work opens the door to the implementation of automatic measurements on a large number of cells, which will allow us to probe a large number of cells in a controlled time. This will open the bio-AFM to statistical analyses on populations of cells of interest

AutoBioTip: Towards automation of mechanobiological measurements by AFM

International audienceThe paradigm in bio-AFM is to measure a few dozen of cells and to draw fundamental biophysical conclusions. To apply these results in biology or medicine, it is essential to increase the statistics by measuring of significant number of cells and to automate the measurements. In this context, it is necessary to optimize the measurements, to reduce the results dispersity and to increase the experiment speed. Concerning the dispersity, we hypothesize that cells organized on patterns would have reduced Young’s modulus dispersion. We had the same hypothesis in regard to the indenter shape (cone vs. colloidal probe).Figure 1 shows that randomly shaped cells (A) present Young's moduli more dispersed than cells that have been forced into a square shape (B). The use of patterns therefore makes it possible to increase the homogeneity of the obtained results. We tested the effect of an indenter which probes a larger surface with a 5µm-diameter colloidal probe. The Young's modulus is smaller with the colloidal probe than with the pyramidal probe, but the dispersion is also smaller. There is therefore an advantage to use a colloidal probe in the automation process. To increase the measurement speed, we worked on two parameters, the tip velocity and the number of indentations per cell. We therefore evaluated the impact of the indentation velocity on the resulting mechanical properties. Young's modulus is higher with increasing velocity. But this trend is comparable for cells constrained by square patterns of fibronectin and those not constrained. A high indentation velocity can therefore be applied during automation. Finally, we compared the influence of the number of indentations (1,024; 256; 64 and 16) per cell . There is no significant difference in Young's modulus value between each condition. Automation can therefore be done with a reduced number of indentations per cell.To conclude, this optimization work opens the door to the implementation of automatic measurements on a large number of cells, which will allow us to probe a large number of cells in a controlled time. This will open the bio-AFM to statistical analyses on populations of cells of interest

Indentation of living cells by AFM tips may not be what we thought!

International audienceThe models used to calculate Young's moduli from atomic force microscopy (AFM) force curves consider the shape of the indentation. It is then assumed that the geometry of the indentation is identical to the geometry of the indenter, which has been verified for hard materials (E > 1 MPa). Based on this assumption, the force curves calculated by these models, for the same object with a given Young's modulus, are different if the indenter geometry is different. On the contrary, we observe experimentally that the force curves recorded on soft living cells, with pyramidal, spherical, or tipless indenters, are almost similar. This indicates that this basic assumption on the indentation geometry does not work for soft materials (E of the order of 5 kPa or less). This means that, in this case, the shape of the indentation is therefore different from the shape of the indenter. Indentation of living cells by AFM is not what we thought

Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyse thousands of cells

International audienceNanomechanical properties of cells could be considered as cellular biomarkers. The main method used to access the mechanical properties is based on nanoindentations measurements, performed with an operator manipulated Atomic Force Microscope (AFM) which is time-consuming, and expensive. This is one of the reasons preventing the transfer of AFM technology into clinical laboratories. In this presentation we report a methodology1 which includes an algorithm (transferred to a script, executed on a commercial AFM) able to automatically move the tip onto a single cell and through several cells to record force curves combined with a smart strategy of cell immobilization. Cells are placed into microwells of a microstructured polydimethylsiloxane (PDMS) stamp. Inside a classical 100x100 µm2 AFM field, 80 to 100 cells are immobilized. In an optimal configuration we were able to measure a population of 900 Candida albicans cells both unmodified and caspofungin treated in 4 h, which represents an unprecedented performance2. This strategy can be applied to cell arrays, proteins or glycan arrays. The big amount of data generated is compatible with the analysis by machine learning and will most probably generate unexpected understanding of the biological processes.References (max. 5): 1. Severac C.,, Proa-Coronado S., Formosa-Dague C., Martinez-Rivas A., Dague E. 2020 in Press Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans CellsJournal of Visualized Experiments e61315 URL: https://www.jove.com/video/61315 doi:10.3791/61315 2. Proa-Coronado S., Severac C., Martinez-Rivas A., Dague E. 2019Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyze thousands of cells.Nanoscale Horizons, DOI : 10.1039/c9nh00438fAcknowledgment: We want to acknowledge FONCYCYT of CONACYT (Mexico), the ministry of Foreign affairs of France and the Universit ́e Paris 13, though the financial support of the international collaborative ECOS-NORD project named Nano-palpation for diagnosis, No. 263337 (Mexico) and MI5P02 (France). AMR would like to thank the financial support of the SIP project No. 20195489, from IPN. SPC is supported by a PhD fellowship from CONACYT (No. 288029) and IPN through the cotutelle agreement to obtain double PhD certificate (IPN-UPS). ED is a researcher at Centre National de la Recherche Scientifique (CNRS)

Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyse thousands of cells

International audienceNanomechanical properties of cells could be considered as cellular biomarkers. The main method used to access the mechanical properties is based on nanoindentations measurements, performed with an operator manipulated Atomic Force Microscope (AFM) which is time-consuming, and expensive. This is one of the reasons preventing the transfer of AFM technology into clinical laboratories. In this presentation we report a methodology1 which includes an algorithm (transferred to a script, executed on a commercial AFM) able to automatically move the tip onto a single cell and through several cells to record force curves combined with a smart strategy of cell immobilization. Cells are placed into microwells of a microstructured polydimethylsiloxane (PDMS) stamp. Inside a classical 100x100 µm2 AFM field, 80 to 100 cells are immobilized. In an optimal configuration we were able to measure a population of 900 Candida albicans cells both unmodified and caspofungin treated in 4 h, which represents an unprecedented performance2. This strategy can be applied to cell arrays, proteins or glycan arrays. The big amount of data generated is compatible with the analysis by machine learning and will most probably generate unexpected understanding of the biological processes.References (max. 5): 1. Severac C.,, Proa-Coronado S., Formosa-Dague C., Martinez-Rivas A., Dague E. 2020 in Press Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans CellsJournal of Visualized Experiments e61315 URL: https://www.jove.com/video/61315 doi:10.3791/61315 2. Proa-Coronado S., Severac C., Martinez-Rivas A., Dague E. 2019Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyze thousands of cells.Nanoscale Horizons, DOI : 10.1039/c9nh00438fAcknowledgment: We want to acknowledge FONCYCYT of CONACYT (Mexico), the ministry of Foreign affairs of France and the Universit ́e Paris 13, though the financial support of the international collaborative ECOS-NORD project named Nano-palpation for diagnosis, No. 263337 (Mexico) and MI5P02 (France). AMR would like to thank the financial support of the SIP project No. 20195489, from IPN. SPC is supported by a PhD fellowship from CONACYT (No. 288029) and IPN through the cotutelle agreement to obtain double PhD certificate (IPN-UPS). ED is a researcher at Centre National de la Recherche Scientifique (CNRS)

High speed indentation measures by FV, QI and QNM introduce a new understanding of bionanomechanical experiments

International audienceStructural and mechanical mapping at the nanoscale by novel high-speed multiparametric Quantitative Imaging (QI) and PeakForce Quantitative Nanomechanical Mapping (PF-QNM) AFM modes was compared to the classical Force Volume (FV) mapping for the case of living Pseudomonas aeruginosa bacterial cells. QI and PF-QNM modes give results consistent with FV for the whole cells in terms of morphology and elastic modulus, while providing higher resolution and shorter acquisition time. As an important complement, the influence of scanning parameters on elastic modulus values was explored for small 0.2 2 m 2 central area on top of cells. The modulus decreases with the indentation depth due to the effect of the hard cell wall, while it increases vs. tip oscillation frequency, displaying viscoelastic behaviour of the living bacterial cells. The ability of different AFM modes to follow correctly the bacteria viscoelastic behaviour at high oscillation frequency was tested

Bacterial α-Glucan and Branching Sucrases from GH70 Family: Discovery, Structure–Function Relationship Studies and Engineering

International audienceGlucansucrases and branching sucrases are classified in the family 70 of glycoside hydrolases. They are produced by lactic acid bacteria occupying very diverse ecological niches (soil, buccal cavity, sourdough, intestine, dairy products, etc.). Usually secreted by their producer organisms, they are involved in the synthesis of α-glucans from sucrose substrate. They contribute to cell protection while promoting adhesion and colonization of different biotopes. Dextran, an α-1,6 linked linear α-glucan, was the first microbial polysaccharide commercialized for medical applications. Advances in the discovery and characterization of these enzymes have remarkably enriched the available diversity with new catalysts. Research into their molecular mechanisms has highlighted important features governing their peculiarities thus opening up many opportunities for engineering these catalysts to provide new routes for the transformation of sucrose into value-added molecules. This article reviews these different aspects with the ambition to show how they constitute the basis for promising future developments

Natural and engineered transglycosylases: Green tools for the enzyme-based synthesis of glycoproducts

International audienceAn increasing number of transglycosylase-based processes provide access to oligosaccharides or glycoconjugates, some of them reaching performance levels compatible with industrial developments. Nevertheless, the full potential of transglycosylases has not been explored because of the challenges in transforming a glycoside hydrolase into an efficient transglycosylase. Advances in studying enzyme structure/function relationships, screening enzyme activity, and generating synthetic libraries guided by computational protein design or machine learning methods should considerably accelerate the development of these catalysts. The time has now come for researchers to uncover their possibilities and learn how to design and precisely refine their activity to respond more rapidly to the growing demand for well-defined glycosidic structures