GH10 xylanase D from Penicillium funiculosum: biochemical studies and xylooligosaccharide production

<p>Abstract</p> <p>Background</p> <p>The filamentous fungus <it>Penicillium funiculosum </it>produces a range of glycoside hydrolases (GH). The <it>XynD </it>gene, encoding the sole <it>P. funiculosum </it>GH10 xylanase described so far, was cloned into the pPICZαA vector and expressed in methylotrophe yeast <it>Pichia pastoris</it>, in order to compare the results obtained with the <it>P. funiculosum </it>GH11 xylanases data.</p> <p>Results</p> <p>High level expression of recombinant XynD was obtained with a secretion of around 60 mg.L^-1. The protein was purified to homogeneity using one purification step. The apparent size on SDS-PAGE was around 64 kDa and was 46 kDa by mass spectrometry thus higher than the expected molecular mass of 41 kDa. The recombinant protein was N- and O-glycosylated, as demonstrated using glycoprotein staining and deglycosylation reactions, which explained the discrepancy in molecular mass. Enzyme-catalysed hydrolysis of low viscosity arabinoxylan (LVAX) was maximal at pH 5.0 with <it>K</it>m_(app)and <it>k_cat</it>/<it>K</it>m_(app)of 3.7 ± 0.2 (mg.mL^-1) and 132 (s^-1mg^-1.mL), respectively. The activity of XynD was optimal at 80°C and the recombinant enzyme has shown an interesting high thermal stability at 70°C for at least 180 min without loss of activity. The enzyme had an endo-mode of action on xylan forming mainly xylobiose and short-chain xylooligosaccharides (XOS). The initial rate data from the hydrolysis of short XOS indicated that the catalytic efficiency increased slightly with increasing their chain length with a small difference of the XynD catalytic efficiency against the different XOS.</p> <p>Conclusion</p> <p>Because of its attractive properties XynD might be considered for biotechnological applications. Moreover, XOS hydrolysis suggested that XynD possess four catalytic subsites with a high energy of interaction with the substrate and a fifth subsite with a small energy of interaction, according to the GH10 xylanase literature data.</p

Antioxidant and Antiproliferative Activities of Heated Sterilized Pepsin Hydrolysate Derived from Half-Fin Anchovy (Setipinna taty)

In this paper we studied the antioxidant and antiproliferative activities of the heated pepsin hydrolysate from a marine fish half-fin anchovy (HAHp-H). Furthermore, we compared the chemical profiles including the amino acid composition, the browning intensity, the IR and UV-visible spectra, and the molecular weight distribution between the half-fin anchovy pepsin hydrolysate (HAHp) and HAHp-H. Results showed that heat sterilization on HAHp improved the 1,1-diphenyl-2-picryl-hydrazil (DPPH) radical-scavenging activity and reducing power. In addition, the antiproliferative activities were all increased for HAHp-H on DU-145 human prostate cancer cell line, 1299 human lung cancer cell line and 109 human esophagus cancer cell line. The contents of free amino acid and reducing sugar of HAHp-H were decreased (P < 0.05). However, hydrophobic amino acid residues and the browning intensity of HAHp-H were increased. FT-IR spectroscopy indicated that amide I and amide III bands of HAHp-H were slightly modified, whereas band intensity of amide II was reduced dramatically. Thermal sterilization resulted in the increased fractions of HAHp-H with molecular weight of 3000–5000 Da and below 500 Da. The enhanced antioxidant and antiproliferative activities of HAHp-H might be attributed to the Maillard reaction

In Vitro Antioxidant Activities of Sulfated Derivatives of Polysaccharides Extracted from Auricularia auricular

In this research, two types of sulfated polysaccharide derivatives were successfully synthesized. Their antioxidant activities were investigated by employing various established in vitro systems. In addition, the degree of sulfation was evaluated using ion-chromatography and IR spectra. The results verify that, when employing scavenging superoxide radical tests, both the sulfation of acid Auricularia auricular polysaccharides (SAAAP) and the sulfation of neutral Auricularia auricular polysaccharides (SNAAP) derivatives possessed considerable antioxidant activity and had a more powerful antioxidant competence than that of the native non-sulfated polysaccharides (AAAP and NAAP). On the other hand, AAAP and NAAP exhibited stronger activity on scavenging both the hydroxyl radical and lipid peroxidation. Available data obtained with in vitro measurements indicates that the sulfated groups of AAAP and NAAP played an important role on antioxidant activity. In sum, the research demonstrates that the antioxidant activity of sulfated polysaccharide derivatives in vitro has a potential significance for seeking new natural antioxidant protective agents

Méthode d analyse des formes alcool et acétylée de l alpha-tocophérol. Etude de leur absorption intestinale par les cellules caco-2

La vitamine E est un micronutriment liposoluble synthétisé par les végétaux. Son activité antioxydante lui confère de nombreux effets bénéfiques, ce qui motive sa large utilisation en alimentation animale et humaine ainsi que dans les secteurs pharmaceutique et cosmétique. Des études internes à la société Adisseo ont montré que des poulets supplémentés avec de l'acétate d'a-tocophérol (Tac) excrètent les deux tiers de celui-ci. Il s'est établi, sur cette base, une collaboration entre Adisseo et l'IMRN afin d'améliorer l'absorption intestinale de Tac, forme produite et commercialisée par la firme multinationale. Cette thèse fait partie du programme de la collaboration. Suivant l'axe de recherche défini avec le partenaire industriel, nous avons mis en place une méthode d'analyse simultanée de l'a-tocophérol (Tol) et de Tac. Ces deux formes de vitamine E sont extraites d'une matrice (solutions aqueuses, micelles mixtes, plasma, tissus...) par de l'éthanol et de l'hexane, puis dosées par HPLC à l'aide d'une détection dans l'UV. La méthode est rapide, reproductible et sensible. Elle constitue, en outre, un moyen original et efficace pour mesurer l'activité estérasique sur Tac. Nos études in vitro ont montré que Tol et, à un degré moindre, Tac sont absorbés par la lignée cellulaire Caco-2. Par ailleurs, la présence d'une activité estérasique sur Tac a été mise en évidence dans les cellules Caco-2. L'optimisation des conditions de solubilisation a permis d'augmenter les quantités de vitamine E absorbées par les cellules Caco-2. Le criblage d'additifs a servi à améliorer davantage l'absorption des deux formes de vitamine E par les cellules Caco-2. Ces résultats ont été confirmés in vivo chez le porc et le poulet. Outre la contribution à la compréhension du processus d'absorption intestinale de la vitamine E, ce travail a abouti à une nouvelle formulation de vitamine E commercialisée depuis Janvier 2005 par la sodété Adisseo.AIX-MARSEILLE3-BU Sc.St Jérô (130552102) / SudocSudocFranceF

Etude de l'oxydation des lipides. Mise au point d'une méthode d'analyse par chimiluminescence et application aux produits à base d'amande

We developed a new analysis method to control lipoxidation in almond products based on the extraction of initial hydroperoxides by the mixture of Gum arabic-Dimethylsulfoxide and their detection by chemiluminescence (CL) This method and that of the TBARS (Thiobarbituric Acid Reactive Substances) were used to follow the lipoxydation during the manufacturing process of the calisson, and to study the effect of light during the storage, and that of temperature and water activity. The new method was transferred to the Roy Rene candy store to prevent rancidity of them products. The simultaneous use of CL and TBARS methods allowed having an overview on the progress of the lipoxidation in almond products. The used approach can be transposed for the other foodstuffs rich in unsaturated lipids or for in vivo analysis.AIX-MARSEILLE3-BU Sc.St Jérô (130552102) / SudocSudocFranceF

Etude du mécanisme d'action du sécrétome de Penicillium funiculosum sur la digestion des polysaccharides des blés Caphorn et Isengrain par la méthode in vitro TIM-I - Expression, caractérisation et étude de spécificité de l'endo-b(1,4)-xylanase D

Le Rovabio Excel est un cocktail enzymatique commercialisé par la société Adisseo et utilisé en nutrition animale pour augmenter la digestibilité des céréales comme le blé. Le but de notre étude a été dans un premier temps d'évaluer, de localiser dans l'espace et dans le temps et de comprendre l'effet du Rovabio sur la digestibilité des blés Caphorn et Isengrain à l'aide d'un modèle in vitro de digestion, le TIM-I. La digestion a été suivie à l'aide de la matière organique, des sucres réducteurs, des monosaccharides totaux et de certains produits finaux. La digestibilité de la matière organique pour les deux blés obtenue avec le T1M-1 est la même que celle obtenue chez le poulet. Le Rovabio améliore la digestibilité des deux blés principalement entre 180 et 360 min de digestion avec tous les marqueurs utilisés. L'effet positif du cocktail enzymatique sur le blé Caphom, est majoritairement jéjunal contrairement au blé Isengrain où l'effet est principalement iléal. Dans un deuxième temps et dans le but d'augmenter l'efficacité du Rovabio sur des substrats riches en glucides non digestibles de types arabinoxylanes, nous avons exprimé la xylanase D (GH10) de P. funiculosum dans le système eucaryote Pichia pastoris. Après l'avoir purifiée, caractérisée biochimiquement et comparée au niveau enzymologique aux autres xylanases du même champignon, nous avons évalué son efficacité hydrolytique sur les deux blés cités précédemment par la méthode TIM-I. Nous avons pu mettre en évidence un intérêt indéniable de cette enzyme qui représente à dose équivalente, environ 80 % de l'efficacité du cocktail enzymatique.AIX-MARSEILLE3-BU Sc.St Jérô (130552102) / SudocSudocFranceF

Insights into the fermentation biochemistry of Kombucha teas and potential impacts of Kombucha drinking on starch digestion

International audienceThe biochemistry of Kombucha fermentation was analyzed using green and black teas (GTK and BTK). Sucrose disappeared linearly in Kombucha mediums during the two week fermentation period for GTK (2.3 g.day(-1).L-1) but only during the first week for BTK (5.0 g.d(-1).L-1). The produced glucose and fructose formed disappeared faster in BTK than in GTK and in both fermentation mediums glucose was preferred to fructose as the carbon source. Ethanol, acetate equivalents and cellulose linearly increased during the fermentation (0.17, 0.35 and 0.50 g.d(-1).L-1 in GTK, and 0.15, 0.46 and 0.63 g.d(-1).L-1 in BTK, respectively). Likewise, the disappearance of glucose + fructose in Kombucha first linearly increased during the first stages of fermentation before it dropped at day 9 for GTK and at day 12 for BTK, suggesting a change of the carbon source. The protein fraction (\textless0.5 g/L) transiently increased during the fermentation of teas and so did a 60-kDa protein band in SDS-PAGE. In both GTK and BTK, only slight changes were observed in total phenolics and in the main tea flavanols. Nevertheless, the theaflavins moderately increased (more than 50% at day 15) and the thearubigins markedly decreased (more than 2-fold at day 15). The Kombucha beverage was able to strongly inhibit starch hydrolysis by porcine pancreatic alpha-amylase and the inhibition potency increased during fermentation progress. The active compounds were suspected to be monomeric and/or oligomeric phenolic compounds. After drinking a bowl of Kombucha beverage, the concentration of these phenolics could be sufficient for inhibiting, to a certain extent, pancreatic alpha-amylase in the small intestine, with a possible impact on starch digestion and on net absorbed glucose. (C) 2012 Elsevier Ltd. All rights reserved