Renato Froidevaux

Continuous production of a peptidic fraction containing the intermediate opioid peptide LVV-haemorphin-7 (LVVh-7) by peptic hydrolysis of bovine haemoglobin in a continuous membrane reactor

Peptic hydrolysis of native bovine haemoglobin at pH 3 yields the LVV‐haemorphin‐7 (Leu‐Val‐Val‐Tyr‐Pro‐Trp‐Thr‐Gln‐Arg‐Phe; LVVh‐7) opioid peptide corresponding to the residues‐31–40 fragment of the β‐chain of haemoglobin. This peptide is intermediate in the course of batch hydrolysis and is rapidly degraded. Indeed, it shows an optimum at 3% degree of hydrolysis (i.e. 2 min of reaction time). The hydrolysis was carried out in a continuous membrane reactor with a space time (ratio of the flux to the reactor volume) set to 2 min (corresponding to optimum LVVh‐7 production). This process allows the continuous production of a constant fraction of intermediate peptides containing LVVh‐7 for 48 min.

Highly sensitive terahertz spectroscopy in microsystem

We herein present a microfluidic system dedicated to THz spectroscopy of aqueous solutions. This device is able to reach a state of the art sensitivity of 5 mg mL−1, while requiring only small sample quantities and a low power source. Hydration of BSA, lysozyme and chymotrypsine proteins is studied inside microchannels and hydration numbers are computed, showing that the developed system is well suited for quantitative analysis. Moreover, coupled with advanced chemometrics algorithms, this system can become a tool for fundamental research and for the understanding of biochemical processes. Here, the hydration shell structure is discussed by chemometric analysis of the measured absorption spectra. Two spectral behaviours are observed and can be explained by Molecular Dynamics simulations. This methodology could be considered as a key element of a lab-on-a-chip for biological liquid metrology.

Advancement in intermediate opioid peptide production in an enzymatic membrane reactor assisted by solvent extraction

Abstract The objective of this work is the future concept of a continuous multiphase enzyme membrane reactor to produce and extract an intermediate opioid peptide (LVVH-7) from a complex hydrolysate of bovine hemoglobin. We report in the first part of this work the production of adecolorized (heme free) fraction of intermediate peptide containing LVVH-7 in a continuous enzymatic membrane reactor. The space time of our reactor was fixed up to 2 minutes corresponding to the necessary time to generate in batch hydrolysis our intermediates peptides. The hydrolysis has been carried out continuously for 46 minutes. In the second part of this work, a solvent mixture of water butan-2-ol octan-1-ol displaying a very good extraction selectivity of LVVH-7 extraction in course of peptic hemoglobin hydrolysis has been designed and optimized. The mixture design used to optimization gave the best results for the mixture composed of 45% water, 45% butan-2-ol and 10% octan-1-ol.

Ion-pairing separation of bioactive peptides using an aqueous/octan-1-ol micro-extraction system from bovine haemoglobin complex hydrolysates

The ion-pair concept was applied on complex haemoglobin hydrolysates to extract two opioid peptides, LVV-haemorphin-7 and VV-haemorphin-7, in an aqueous/octan-1-ol micro-extraction system in the presence of alkyl-sulfonic acid as a surfactant agent and in relation to the haemorphin physico-chemical properties (charge, hydrophobicity). The effect of combined alkyl chain length/aqueous phase pH and the haem behaviour during the extraction, on the haemorphin recovery yield and enrichment has been determined. It has proved that transport over the organic phase is mediated by the alkyl-sulfonic acids, whatever be the aqueous phase pH. However, increasing both the alkyl chain length and the pH in the aqueous phase shows an haemorphin enrichment ratio increase but a recovery decrease of the extracted opioid peptides in the organic phase. Therefore, the best conditions to extract LVVh-7 and VVh-7 are the use of the octane-sulfonic acid at aqueous phase pH of 5 or 7 and the octane or the heptane-sulfonic acid with an aqueous phase pH of 5 or 7 respectively. In these conditions, a partition coefficient of 1.64 and 1.60 respectively for LVVh-7 and VVh-7 are obtained and represent about 40 times that acquired without agent.

Hybrid Catalysis: A Suitable Concept for the Valorization of Biosourced Saccharides to Value-Added Chemicals

Through biomass valorization, sugars can become a major carbon resource for the production of fuels and chemicals by using catalysis. Hybrid catalysis, a direct combination of biocatalysis and chemocatalysis, may yield innovative solutions. 5‐Hydroxymethylfurfural (5‐HMF) is a platform molecule derived from glucose, for which fructose is a key intermediate. To overcome the thermodynamic enzymatic equilibrium between glucose and fructose, two methodologies based on hybrid catalysis are described herein. In the first method, the glucose isomerase and fructose‐to‐5‐HMF dehydration chemocatalysts are simultaneously implemented in a multiphasic way to imply the complexation and transport of fructose. The second method proposes a route through sorbitol, obtained by the hydrogenation of glucose. Sorbitol is then enzymatically converted into fructose, whereas regeneration of the cofactor is performed in situ with an organometallic complex as a chemocatalyst.

Diffusion based kinetic selectivity modulation of enzymatic proteolysis in a microfluidic reactor: experimental analysis and stochastic modeling

Microreactors enable new experimental ways for enzyme engineering. In this context, we show that the liquid–liquid parallel laminar flows in microchannels cause a kinetic selectivity modification of proteolytic enzymatic reaction involving hemoglobin and pepsin, reaction that generates numerous bioactive peptides at different advancement state. Here we show that this diffusion based kinetic modulation induces an altered peptides appearance kinetics for a part of the initial substrate population. Indeed, microfluidic and conventional batch experiments performed in the same reaction conditions lead to strong differences in the resulting peptidic profiles obtained by reversed-phase high-performance liquid chromatography. Such differences are explained by the laminar flow diffusive conditions inside microchannels and are supported by a stochastic algorithm based on the Michaelis–Menten equation. Several bioactive peptides are identified by mass spectrometry and show the potential of such methodology in peptides screening from a complex proteolysis but also for selective peptides preparation by microfluidics.

Optimization of Peptide Separation from Complex Peptide Mixture in a Foaming-Draining System

The physico-chemical environment of the separation, applied on a complex peptide hydrolysate obtained from the hydrolysis of bovine hemoglobin by pepsin, to recover an antimicrobial peptide fraction was shown to influence its recovery efficiency and selectivity. The objective of the present work was to evaluate experimental conditions that maximize the adsorption capacity of the antimicrobial peptide fraction on liquid/air interface through the use of an experimental design methodology. A first step comprised a study on several factors thanks to a screening matrix to acquire basic knowledges on these variables and their influence on the extraction selectivity. Hydrolysate and salt concentrations, pH of both the hydrolysate and draining solutions were established as the four main factors. Then a composite matrix was studied to identify with response surface modelling the optimum conditions for fractionation.

From sequential chemoenzymatic synthesis to integrated hybrid catalysis: Taking the best of both worlds to open up the scope of possibilities for a sustainable future

Enzymatically catalyzed reactions are highly efficient but higher scale extrapolation is limited to a certain class of stable and easily recyclable enzymes. Homogeneously and/or heterogeneously catalyzed reactions are often limited by the multiphasic nature of catalytic systems and/or variations in the distribution of active sites. For these reasons, the development of highly selective and efficient hybrid catalysts is especially needed, and it is now well admitted that disruptive innovation is highly desirable in process engineering, (bio)catalyst design and materials science. Optimal integration of all types of catalysis is the key for efficient global implementation of sustainable processes. This review gives an overview of all the pathways that integrate chemical and biological catalysis, beginning with combined chemoenzymatic processes in cascade to progressively open the field of hybrid catalysis in a one-pot concomitant execution. We have emphasized the factors that have to be considered to understand the catalytic synergy between these systems. A better understanding of hybrid catalytic systems is an essential tool not only in the choice of the best route of their implementation but also in the synthesis of a wide range of fine chemicals in many fields, with a particular emphasis on their production from renewables.

Sustainable efficient way for opioid peptide LVV-h7 preparation from enzymatic proteolysis in a microfluidic-based reaction-extraction process with solvent recycling

LVV-h7 (LVVYPWTQFR) is a bioactive peptide that can be obtained from blood as waste of food industry, more precisely from hemoglobin hydrolysis by pepsin. This opioid peptide belongs to the hemorphins family and have strong physiological effects that bring its use in pharmaceutics and various therapeutic treatments attractive, in particular for substituting its costly chemically synthetized analogous. Hemoglobin hydrolysis by pepsin generates a huge variety of peptides among whose LVV-h7 can be purified by liquid-liquid extraction (LLE). Herein, selective preparation of this peptide is proposed by a microfluidic-based continuous reaction-separation process. Hemoglobin hydrolysis in microreactor was firstly coupled to LVV-h7 LLE in octan-1-ol and then coupled to LVV-h7 back LLE in acidic water. This continuous process allowed to prepare pure LVV-h7, as confirmed by liquid chromatography and mass spectrometry. The microfluidic circuit also allowed octan-1-ol recycling in a closed loop, making this method more sustainable than similar biphasic batch process.