Démarche d'optimisation d'un procédé de fabrication de comprimés via granulation humide

LYON1-BU Santé (693882101) / SudocSudocFranceF

Tablets Made from Paper—An Industrially Feasible Approach

Many orally administrated drugs exhibit poor bioavailability due to their limited solubility. The smartFilm technology is an innovative approach to improve the drug aqueous solubility, where the drug is embedded within the matrix of cellulose-based paper in an amorphous state, hence increasing its solubility. Despite its proven effectiveness, smartFilms, i.e., pieces of paper, exhibit limited flowability and are not easy to swallow, and thus oral administration is not convenient. In addition, there is a lack of knowledge of their mechanical behavior under compression. This study aimed to transform unloaded smartFilms, i.e., paper, into a flowable physical form and investigated its mechanical behavior when compressed. Granules made of paper were prepared via wet granulation and were compressed into tablets. The influence of using different amounts and forms of sucrose, as a binder, on the pharmaceutical properties of the produced granules and tablets was studied and the most suitable composition was identified by using instrumented die experiments. For this, the Poisson’s ratio and Young’s modulus were determined for different compaction force levels and the deformation behavior was estimated with the Heckel mathematical model. All granule batches showed good flowability with angle of repose values between 25–35°. Granule batches with ≤30% dry sucrose content produced tablets that fulfilled the European Pharmacopeia requirements, and the compaction behavior of the granules was found to be comparable to the behavior of classical binders and compression enhancers. Paper can be transferred into granules. These granules can be used as suitable intermediate products for the production of tablets made of paper in large, industrial scale

Design of a Protein with Improved Thermal Stability by an Evolution‐Based Generative Model

Efficient design of functional proteins with higher thermal stability remains challenging especially for highly diverse sequence variants. Considering the evolutionary pressure on protein folds, sequence design optimizing evolutionary fitness could help designing folds with higher stability. Using a generative evolution fitness model trained to capture variation patterns in natural sequences, we designed artificial sequences of a proteinaceous inhibitor of pectin methylesterase enzymes. These inhibitors have considerable industrial interest to avoid phase separation in fruit juice manufacturing or reduce methanol in distillates, averting chromatographic passages triggering unwanted aroma loss. Six out of seven designs with up to 30 % divergence to other inhibitor sequences are functional and two have improved thermal stability. This method can improve protein stability expanding functional protein sequence space, with traits valuable for industrial applications and scientific research

Caractérisation de trois enzymes de dégradation des pectines de type Rhamnogalactunonane I d’Aspergillus aculeatinus : spécificités biochimiques et potentiels applicatifs

International audienc

Putative pectate lyase PLL12 and callose deposition through polar CALS7 are necessary for long-distance phloem transport in Arabidopsis

In plants, the phloem distributes photosynthetic products for metabolism and storage over long distances. It relies on specialized cells, the sieve elements, which are enucleated and interconnected through large so-called sieve pores in their adjoining cell walls. Reverse genetics identified PECTATE LYASE LIKE 12 (PLL12) as critical for plant growth and development. Using genetic complementations, we established that PLL12 is required exclusively late during sieve element differentiation. Structural homology modeling, enzyme inactivation, and overexpression suggest a vital role for PLL12 in sieve element specific pectin remodeling. While short distance symplastic diffusion is unaffected, the pll12 mutant is unable to accommodate sustained plant development due to an incapacity to accommodate increasing hydraulic demands on phloem long distance transport as the plant grows – a defect that is aggravated when combined with another sieve element specific mutant callose synthase 7 (cals7). Establishing CALS7 as a specific sieve pore marker, we investigated the subcellular dynamics of callose deposition in the developing sieve plate. Using fluorescent CALS7 then allowed identifying structural defects in pll12 sieve pores that are moderate at the cellular level but become physiologically relevant due to the serial arrangement of sieve elements in the sieve tube. Overall, pectin degradation through PLL12 appears subtle in quantitative terms. We therefore speculate that PLL12 may act as a regulator to locally remove homogalacturonan thus potentially enabling further extracellular enzymes to access and modify the cell wall during sieve pore maturation.Peer reviewe

Three novel rhamnogalacturonan I- pectins degrading enzymes from Aspergillus aculeatinus: Biochemical characterization and application potential

International audienc

The specificity of pectate lyase VdPelB from Verticilium dahliae is highlighted by structural, dynamical and biochemical characterizations

International audiencePectins, complex polysaccharides and major components of the plant primary cell wall, can be degraded by pectate lyases (PLs). PLs cleave glycosidic bonds of homogalacturonans (HG), the main pectic domain, by β-elimination, releasing unsaturated oligogalacturonides (OGs). To understand the catalytic mechanism and structure/function of these enzymes, we characterized VdPelB from Verticillium dahliae. We first solved the crystal structure of VdPelB at 1.2 Å resolution showing that it is a right-handed parallel β-helix structure. Molecular dynamics (MD) simulations further highlighted the dynamics of the enzyme in complex with substrates that vary in their degree of methylesterification, identifying amino acids involved in substrate binding and cleavage of non-methylesterified pectins. We then biochemically characterized wild type and mutated forms of VdPelB. Pectate lyase VdPelB was most active on non-methylesterified pectins, at pH 8.0 in presence of Ca2+ ions. The VdPelB-G125R mutant was most active at pH 9.0 and showed higher relative activity compared to native enzyme. The OGs released by VdPelB differed to that of previously characterized PLs, showing its peculiar specificity in relation to its structure. OGs released from Verticillium-partially tolerant and sensitive flax cultivars differed which could facilitate the identification VdPelB-mediated elicitors of defence responses

The structural, dynamical and biochemical characterizations of Verticillium dahliae pectate lyase, VdPelB, highlight its specificities

Abstract Pectins, complex polysaccharides and major components of the plant primary cell wall, can be degraded by pectate lyases (PLs). PLs cleave glycosidic bonds of homogalacturonans (HG), the main pectic domain, by β-elimination, releasing unsaturated oligogalacturonides (OGs). To understand the catalytic mechanism and structure/function of these enzymes, we characterized VdPelB from Verticillium dahliae , a plant pathogen. We first solved the crystal structure of VdPelB at 1.2Å resolution showing that it is a right-handed parallel β-helix structure. Molecular dynamics (MD) simulations further highlighted the dynamics of the enzyme in complex with substrates that vary in their degree of methylesterification, identifying amino acids involved in substrate binding and cleavage of non-methylesterified pectins. We then biochemically characterized wild type and mutated forms of VdPelB. VdPelB was most active on non-methylesterified pectins, at pH 8 in presence of Ca 2+ ions. VdPelB-G125R mutant was most active at pH 9 and showed higher relative activity compared to native enzyme. The OGs released by VdPelB differed to that of previously characterized PLs, showing its peculiar specificity in relation to its structure. OGs released from Verticillium- partially tolerant and sensitive flax cultivars differed which could facilitate the identification VdPelB-mediated elicitors of defence responses

RHAMNOGALACTURONAN-I OLIGOGALACTURONIDE PROFILING BY HPSEC-MS/MS ELUCIDATES RGI STRUCTURE AND THE MODE OF ACTION OF ENZYMES

International audienceIn primary plant cell wall, cellulose microfibrils and hemicelluloses are embedded in a matrix of pectins. Depending on their chemical compositions and structures, four pectic domains can be described in including rhamnogalacturonan I (RGI) [1]. RGI consists of a backbone of repeating diglycosyl units (→2)-α-L-Rhap-(1→4)-α-D-GalpA-(1→), partially substituted at O-4 and/or O-3 positions of α-L-Rhap residues with notably side chains of →5)-α-L-arabinans-(1→ and →4)-β-D-galactans-(1→ [2]. Due to its complexity, the determination of RGI fine structure is challenging. We developed a sensitive analytical method based on the chromatographic separation of RGI-derived oligogalacturonides (OG-RGI), combined with accurate determination of their sizes and side-chains patterns using MS/MS [3,4]. This method can reveal the structure of RGI following its hydrolysis by specific enzymes such as rhamnogalacturonan hydrolases (RHG) and rhamnogalacturonan lyases (RGL). The three enzymes used in this study were expressed in Pichia pastoris heterologous system, purified by affinity chromatography, and their biochemical specificities determined. Using simple RGI-rich substrate, modified potato RG and more complex Camelina sativa mucilage, as well as two RHG and one RGL from Aspergillus aculeatinus we build an experimental OG-RGI library of 149 OGs. These OGs vary depending on their degree of polymerization (DP), degree of substitution as well as in their side-chains composition. Using a theoretical database, OG-RGI compounds were identified on the basis of their retention time, m/z and MS2 fragmentation patterns. We further showed that this method allows analyzing the structure of RGI from Arabidopsis cell wall and determining the specificities among multigenic families of fungal rhamnogalacturonan hydrolases. This new approach paves the way, through the analysis of mutants for RGI-degrading enzyme, for analyzing the contribution of RGI in modulating plant development