Ce travail a été financé en partie par le Département américain de l&#39;Energie des Grands Lacs Bioenergy Research Center (DOE Office of Science du BER DE-FC02-07ER64494) et accorder DE-FG02-91ER200021 du Département américain de l&#39;énergie, Bureau des sciences fondamentales de l&#39;énergie, Division des sciences chimiques, sciences de la terre et les biosciences. Nous remercions John Scott-Craig et Melissa Borrusch pour leurs contributions matérielles et conceptuelles.

1. La production d&#39;enzymes <ol><li> Produire des protéines dans Pichia pastoris par clonage des gènes correspondants dans des vecteurs ou des pPICZ pPICZα et sa transformation en P. pastoris. Cultiver des cellules dans 300 ml de lots dérouté flacons de 500 ml de méthanol à l&#39;induction de tous les h 24 (Banerjee et al., 2010a). </li><li> Concentré et dessaler les filtrats de culture par filtration à flux tangentiel. </li><li> Stocker les enzymes dans de petites aliquotes de 20% de glycérol à -80 ° C. La gamme de concentration est 1-10 mg / ml. </li></ol> 2. Conception de l&#39;expérience <ol><li> Entrez le nombre d&#39;enzymes pour être testés, leurs supérieurs et inférieurs des proportions (par exemple, une proportion plus faible de 5% pour les enzymes de base), et le type de conception expérimentale (par exemple, quadratique ou cubique) dans la conception-Expertsoftware. </li><li> Calculez le montant en ug de chaque enzyme à ajouter pour faire un chargement total de 15 mg / g de glucane, puis calculer le montant de chaque enzyme en ul à ajouter àchaque réaction. </li><li> Générer feuilles de calcul Excel en spécifiant matériel de laboratoire de source, puits de source, du matériel de laboratoire de destination, des puits de destination, et les volumes de transfert pour distribution d&#39;eau et d&#39;enzymes selon la conception expérimentale et l&#39;importer dans le logiciel Biomek FXP utilisant la fonction de transfert-de-fichier. </li></ol> 3. L&#39;hydrolyse enzymatique <ol><li> Suspendre la boue de la biomasse dans 50 mM citrate de sodium, pH 4,8, contenant 5 ug / ml de tetracycline et 5 ug / ml de cycloheximide, dans le réservoir à aubes. </li><li> Robotisé de pipette 200 pl de la suspension dans chaque puits d&#39;une plaque 96 puits de réaction en utilisant des puits profonds Span-8 embouts de pipettes 1000-ul avec la dernière 0.5 cm coupés. Mélangez le coulis une fois à 100 l / sec puis aspirer le même volume du réservoir de paddle et répartir dans les assiettes. </li><li> Distribuer les enzymes dans la même assiette en utilisant le programme est entré dans le FX Beckman. </li><li> Sceau des plaques avec capmats perçable. </li><li> Inversez et frappezles plaques à plusieurs reprises pour surmonter la tension de surface. Placez les plaques à 50 ° C pendant 48 h dans l&#39;hybridation incubateur tournant à 10 rpm. </li></ol> 4. Détermination de Glc et Xyl <ol><li> Centrifuger les plaques de réaction pendant 3 min à 1250 xg dans une centrifugeuse à godets oscillants. </li><li> Transférer 100 ul du surnageant dans réguliers plaques à 96 puits en utilisant la gousse AP96 du FX Biomek. </li><li> Incuber les plaques à 90-95 ° C pendant 10 min afin d&#39;inactiver les enzymes, puis centrifuger à 1250 xg pendant 30 sec. </li><li> Pour des mesures de Glc, transférer 12 ul des surnageants en emploi régulier plaques à 96 puits. Ajouter 192 ul de la glucose oxydase / peroxydase (goPod) réactif. Incuber les plaques à 50 ° C pendant 20 min et lire les absorbances à 510 nm dans un lecteur de microplaques. Blank est mélange réactionnel sans enzyme. </li><li> Pour des mesures Xyl, transfert 4 pl dans 384 puits. Ajouter les réactifs de dosage et de lire les plaques à 340 nm. Viergeest le mélange réactionnel sans enzyme. </li></ol> 5. Analyse des données <ol><li> Convertir les valeurs d&#39;absorbance à leur Glc% et des rendements sur la base des Xyl connue Glc et le contenu Xyl de la biomasse d&#39;origine. Importer ces pourcentages que les réponses dans le logiciel Design-Expert. Vérifiez la paramenters statistiques pour être sûr que les critères d&#39;un modèle robuste sont remplies. </li><li> Confirmer la meilleure prédiction du modèle expérimental. </li></ol> 6. Les résultats représentatifs avec GENPLAT: (1) Création de mélanges optimisés des individuels protéines pures. Les résultats indiquent que les enzymes sont importants, et dans quelles proportions, et aussi quelles enzymes ne sont pas importants. Certaines protéines qui sont abondantes dans le T. reesei sécrétome (Nagendran et al., 2009), comme Cip1 et CIP2, semblent jouer aucun rôle dans la libération Glc à partir de maïs prétraités par l&#39;expansion de fibres d&#39;ammoniaque (AFEX) ou alcalines hyd Stover<html> peroxyde d&#39;Rogen (AHP) (Banerjee et al., 2010b). D&#39;autre part, certaines protéines sont importantes inattendue. Par exemple, l&#39;endo-xylanases de familles Hydrolase glycosyle 10 et 11 sont toutes deux importantes pour Glc libération; une xylanase peut pas se substituer à l&#39;autre (Banerjee et al, 2010b, c).. Cel61A, une protéine mineure dans le T. reesei sécrétome, est très important pour Glc mais pas Xyl communiqué. Certaines enzymes sont importantes pour la libération des deux Glc et Xyl, alors que d&#39;autres ne sont nécessaires que pour l&#39;une ou l&#39;autre (Banerjee et al., 2010c). Un mélange synthétique contenant 16 éléments, chacun à une concentration de 0,94 mg / ml (c&#39;est à dire, un mélange non optimisé), a publié 38% de la disposition du Glc AFEX-prétraités DDG. Dans des conditions identiques hydrolyse, un mélange optimisé, dans laquelle les concentrations des 16 composantes variait de 0% à 32%, a publié 52% de la CGL (Fig. 2). Cette expérience illustre l&#39;utilité de construire des mélanges définis. _content &quot;&gt; (2) Création de cocktails optimisé pour un prétraitement différent / combinaisons substrat. actuelle des préparations commerciales sont cellulase&quot; one-size-fits-all &quot;. En réalité, le meilleur cocktail dépend à la fois le substrat et le prétraitement. Un seul produit tels que Accellerase 1000 donne des rendements différents de différents substrats prétraités de la même manière, et à partir du même substrat exposée à différents prétraitements (Fig. 1). Nous avons utilisé pour optimiser les mélanges GENPLAT des enzymes pures de prétraitement des matières premières et de multiples mutiple (Banerjee et al., 2010c). figure 2 montre comment les proportions optimales d&#39;enzymes de 16 enzymes pures diffère du maïs AFEX-prétraités canne, le maïs PLA-prétraités Stover et AFEX-prétraités DDG. Seulement 11 enzymes sont présentés dans la figure 2. optimale parce que la proportions des cinq autres (Cel61B, AbfB, Cip1, point d&#39;appel 2, et Cel12A) ont été trouvés à 0% (Banerjee et al., 2010c). (3) découverte de nouvelle enzyme </strong&gt;. La construction de mélanges optimisés des protéines pures est un processus continu. Comme de nouvelles protéines sont disponibles sous forme pure (à partir de sources commerciales, par l&#39;expression hétérologue, ou par purification), ils peuvent être testés systématiquement en les ajoutant à l&#39;ensemble actuel optimisé. Les nouvelles protéines qui contribuent à Glc ou Xyl communiqué deviennent alors partie intégrante d&#39;un nouveau jeu optimisé. De cette façon, GENPLAT devient une plate-forme pour l&#39;identification de nouvelles enzymes pour la déconstruction de la biomasse. Nous avons fait des extraits de champignons autres que T. reesei et A. Niger cultivés sur différents substrats. Un extrait particulièrement stimulé le rendement Glc lorsqu&#39;il est ajouté à notre noyau. La protéine responsable a été purifié et montré être un roman de glycosyl hydrolase pas présent dans toute préparation enzymatique commerciale (Banerjee et Walton, résultats non publiés). (4) Découverte de la meilleure des enzymes. Ayant montré avec GENPLAT laquelle les enzymes sont les plus importants de la dégradation d&#39;unparticulier la biomasse, nous avons une meilleure compréhension de laquelle les enzymes devrait être le centre d&#39;amélioration. Mieux exemples des enzymes essentielles pourraient être trouvés par la recherche dans la nature ou fabriqués par l&#39;ingénierie des protéines. (Une enzyme pourrait être «mieux» que son homologue de T. reesei de plusieurs façons, selon la propriété désirée, par exemple, l&#39;activité spécifique plus élevée, une plus grande synergie, une diminution de la sensibilité protéolytique, ou une meilleure stabilité thermique). GENPLAT fournit une plate-forme significative pour doser les enzymes potentiellement mieux, car il utilise un cocktail réaliste complexes (important car aucune enzyme biomasse fonctionne en vase clos) et un substrat réaliste (important car les résultats avec de la cellulose pure ou d&#39;autres substrats artificiels peuvent ne pas être pertinents pour lignocellulosiques naturelles matériaux). (5) l&#39;optimisation de l&#39;enzyme de commerce. Quantité suffisamment importante d&#39;enzymes pures n&#39;existent pas encore dans le monde réel. Jusqu&#39;à ce qu&#39;ils font, l&#39;éthanol Proproducteurs doivent compter sur des produits commerciaux tels que Accellerase 1000 et MultifectXylanase. Toutefois, les mélanges d&#39;enzymes commerciaux fonctionnent généralement mieux que tout autre individu, et GENPLAT peut être utilisé pour la conception optimisée de multiples cocktails enzymes commerciales. La figure 3 montre l&#39;utilisation de GENPLAT pour optimiser un mélange de quatre préparations enzymatiques commerciales. Les proportions optimales pour la libération de Glc d&#39;AFEX-prétraités DDG ont été trouvés à 47% Accellerase1000, 27% Multifect pectinase, 22% Novozyme 188, et 4% Multifect xylanase (Banerjee et al., 2010c). La figure 3B montre les différences dans le rendement du Glc AFEX-DDG avec quatre préparations enzymatiques; le mélange optimisé commerciale donne un rendement plus élevé que Glc Accellerase1000 seul, SpezymeCP seul, ou un mélange de 16 composants synthétiques. Cette expérience indique également que le mélange de 16 composant synthétique qui manque un ou plusieurs enzymes nécessaires à la libération optimale Glc, qui sont présents dans un ou plusieurs des enzymes commerciales. GENPLAT peut nous êtreED afin d&#39;identifier de tels composants. <img src="/files/ftp_upload/3314/3314fig1.jpg" alt="Figure 1"/> Figure 1. Pas de préparation enzymatique commerciale unique est optimale pour tous les supports et tous les prétraitements. Cinq substrats et trois prétraitements ont été comparés dans des conditions similaires de broyage (0,5 mm la taille des particules), le chargement d&#39;enzymes (15 mg / g de glucane), et les conditions de l&#39;hydrolyse (48 h, 50 ° C). A. La digestion des différents substrats prétraités AFEX- avec Accellerase 1000. Digestion B. canne de maïs prétraités par trois méthodes différentes avec Accellerase 1000 (voir Banerjee et al., 2010c). <img src="/files/ftp_upload/3314/3314fig2.jpg" alt="Figure 2"/> Figure 2. Proportions optimales de 11 enzymes pour la libération de Glc d&#39;AFEX-canne de maïs prétraités (AFEX-CS), alcalines d&#39;hydrogène peroxyde de maïs prétraités Stover (PLA-CS), ou AFEX-prétraitées séchées distillentvailleurs céréales (AFEX-DDG). Les données proviennent de Banerjee et al. (2010c). Numéros en haut des barres sont les rendements Glc que un pour cent du total Glc dans l&#39;échantillon de la biomasse. <img src="/files/ftp_upload/3314/3314fig3.jpg" alt="Figure 3"/> Figure 3. A. L&#39;utilisation de GENPLAT pour produire un mélange optimisé de quatre préparations enzymatiques commerciales pour la libération de Glc d&#39;AFEX-prétraités DDG. Multifect xylanase fait la plus petite contribution (4%) et a donc été omis dans ce diagramme ternaire. B. rendements Glc d&#39;AFEX-DDG avec des traitements enzymatiques différentes, dans des conditions identiques hydrolyse (15 enzymes glucane mg / g, 48 h, 50 ° C) . &quot;Quatre-composante commerciale&quot; a les proportions déterminées par l&#39;expérience le montre la figure. 3A.

<ol>
	<li>Anderson, M. J., Whitcomb, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> DOE Simplified: Practical Tools for Effective Experimentation. , (2007).</li><li>Banerjee, G., Car, S., Scott-Craig, J. S., Borrusch, M. S., Aslam, N., Walton, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+enzyme+mixtures+for+biomass+deconstruction:+production+and+optimization+of+a+core+set.">Synthetic enzyme mixtures for biomass deconstruction: production and optimization of a core set.</a> Biotechnol. Bioengineer. 106, 707-720 (2010).</li><li>Banerjee, G., Car, S., Scott-Craig, J. S., Borrusch, M. S., Bongers, M., Walton, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+multi-component+enzyme+mixtures+for+deconstruction+of+lignocellulosic+biomass.">Synthetic multi-component enzyme mixtures for deconstruction of lignocellulosic biomass.</a> Bioresour. Technol. 101, 9097-9105 (2010).</li><li>Banerjee, G., Car, S., Scott-Craig, J. S., Borrusch, M. S., Walton, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+optimization+of+enzyme+mixtures+for+deconstruction+of+diverse+pretreatment/biomass+feedstock+combinations.">Rapid optimization of enzyme mixtures for deconstruction of diverse pretreatment/biomass feedstock combinations.</a> Biotechnol. Biofuels. 3, 22-22 (2010).</li><li>Berlin, A., Maximenko, V., Gilkes, N., Saddler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+enzyme+complexes+for+lignocellulose+hydrolysis.">Optimization of enzyme complexes for lignocellulose hydrolysis.</a> Biotechnol. Bioeng. 97, 287-296 (2007).</li><li>Decker, S. R., Brunecky, R., Tucker, M. P., Himmel, M. E., Selig, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+screening+techniques+for+biomass+conversion.">High throughput screening techniques for biomass conversion.</a> Bioenerg. Res. 2, 179-192 (2009).</li><li>Gao, D., Chundawat, S. P., Krishnan, C., Balan, V., Dale, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixture+optimization+of+six+core+glycosyl+hydrolases+for+maximizing+saccharification+of+ammonia+fiber+expansion+(AFEX)+pretreated+corn+stover.">Mixture optimization of six core glycosyl hydrolases for maximizing saccharification of ammonia fiber expansion (AFEX) pretreated corn stover.</a> Bioresour. Technol. 101, 2770-2781 (2010).</li><li>Harris, P. V., Welner, D., McFarland, K. C., Re, E., Poulsen, J. C. N., Brown, K., Salbo, R., Ding, H., Vlasenko, E., Merino, S., Xu, F., Cherry, J., Larsen, S. Y., LoLeggio, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+lignocellulosic+biomass+hydrolysis+by+proteins+of+glycoside+hydrolase+family+61:+Structure+and+function+of+a+large,+enigmatic+family.">Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: Structure and function of a large, enigmatic family.</a> Biochemistry. 49, 3305-3316 (2010).</li><li>Kim, E., Irwin, D. C., Walker, L. P., Wilson, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factorial+optimization+of+a+six-cellulase+mixture.">Factorial optimization of a six-cellulase mixture.</a> Biotechnol. Bioeng. 58, 494-501 (1998).</li><li>King, B. C., Donnelly, M. K., Bergstrom, G. C., Walker, L. P., Gibson, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+microplate+assay+system+for+quantitative+evaluation+of+plant+cell+wall-degrading+enzyme+activity+of+fungal+culture+extracts.">An optimized microplate assay system for quantitative evaluation of plant cell wall-degrading enzyme activity of fungal culture extracts.</a> Biotechnol. Bioeng. 102, 1033-1044 (2009).</li><li>Nagendran, S., Hallen-Adams, H. E., Paper, J. M., Aslam, N., Walton, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+genomic+potential+for+secreted+plant+cell-wall-degrading+enzymes+in+the+ectomycorrhizal+fungus+Amanita+bisporigera,+based+on+the+secretome+of+Trichoderma+reesei.">Reduced genomic potential for secreted plant cell-wall-degrading enzymes in the ectomycorrhizal fungus Amanita bisporigera, based on the secretome of Trichoderma reesei.</a> Fung. Genet. Biol. 46, 427-435 (2009).</li><li>Rosgaard, L., Pedersen, S., Langston, J., Akerhielm, D., Cherry, J. R., Meyer, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+minimal+Trichoderma+reesei+cellulase+mixtures+on+differently+pretreated+barley+straw+substrates.">Evaluation of minimal Trichoderma reesei cellulase mixtures on differently pretreated barley straw substrates.</a> Biotechnol. Prog. 23, 1270-1276 (2007).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

Il est largement reconnu que la réduction du coût des enzymes est important pour le développement d&#39;une industrie d&#39;éthanol lignocellulosique économique. Actuellement disponibles cocktails enzymatiques commerciales sont des mélanges complexes et mal définies de nombreuses protéines (Nagendran et al., 2009), et ils sont adaptés pour une utilisation essentiellement sur l&#39;acide prétraité les tiges de maïs. Afin d&#39;accélérer le développement de cocktails enzymatiques mieux, plusieurs laboratoires ont développé des plates-formes à haut débit pour la découverte et la caractérisation d&#39;enzymes. Les efforts dans ce domaine ont intégré un ou plusieurs des propriétés suivantes ont également trouvé dans GENPLAT: robotique de distribution d&#39;enzymes et de boues de la biomasse, la conception statistique de l&#39;expérience et / ou la détermination automatisée de Glc et Xyl (Berlin et al, 2007; Decker et al. al, 2009;. Kim et al, 1998;. King et al, 2009).. GENPLAT étend ces efforts antérieurs, la plupart de manière significative dans la complexité des mélanges d&#39;enzymes qui peuvent être analysés à partir au plus 6 composantsles études antérieures à plus de 16 dans notre dernier travail (Banerjee et al., 2010c). D&#39;autres caractéristiques clés de GENPLAT sont l&#39;utilisation d&#39;une chambre de mélange billes (réservoir aube) qui peuvent garder boues tiges suspendues pendant la distribution; mélange en douceur lors de la digestion d&#39;ici la fin de plus en bout de rotation, et automatisé dosage colorimétrique du Glu et Xyl.

Equipements et logiciels utilisés: <ol><li> Design-Expert logiciel, la version 8.0 (Stat-Ease, Inc, Minneapolis, MN) ( <a href="http://www.statease.com">http://www.statease.com</a> ) </li><li> Biomek FXP automatisation des laboratoires poste de travail (Beckman-Coulter, Fullerton, CA) </li><li> Biomek FXP logiciel, la version 3.3 (Beckman-Coulter, Fullerton, CA) </li><li> Réservoir de Paddle (modèle VP-756C 1P100, V &amp; P Scientific Inc, San Diego) </li><li> Plaques, 96 de profondeur (1,5 ml) et (ABgene, Fisher Scientific, Epson, Royaume-Uni) </li><li> Capmats perçable (Etats-Unis scientifique, Ocala, Floride) </li><li> L&#39;hybridation rotatif incubateur (Modèle 5420, VWR) </li><li> Swinging-seau de centrifugeuse (Modèle 5810R, Eppendorf, Hambourg, Allemagne) </li><li> Assiettes, Costar de 96 puits (ABgene, Fisher Scientific, Epson, Royaume-Uni) </li><li> Assiettes, de 384 puits (BD Falcon, BD Biosciences, San Jose, CA) </li></ol> Réactifs utilisés: <ol><li> Pichia pastoris et les vecteurs: la souche X33, et pPICZ et pPICZα (Invitrogen, Carlsbad, CA) </li><li> T. reesei et les vecteurs: la souche QM9414 (Département américain de l&#39;Agriculture Centre national de recherche sur l&#39;utilisation agricole, Peoria, IL), et pCB1004-EV (Banerjee et al, 2010b). </li><li> Accellerase 1000 (1600844643 numéro de lot) (Genencor, Inc, Rochester, NY) </li><li> 50 mM de tampon citrate de sodium, pH 4,8 </li><li> Tétracycline stock: 10 mg / ml </li><li> Stock de cycloheximide: 10 mg / ml </li><li> GoPod test kit, catalogue K-gluc (Megazyme, Bray, Irlande) </li><li> Xylose test kit, le catalogue de K-xylose (Megazyme, Bray, Irlande) </li></ol>

genplat: an automated platform for biomass enzyme discovery and cocktail optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
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GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

GENPLAT (Plateforme Enzyme GLBRC) est une plate-forme automatisée pour la découverte et l&#39;optimisation des cocktails d&#39;enzymes de dégradation de la biomasse. Il peut être adapté à plusieurs matières premières et des mélanges d&#39;enzymes contenant de multiples composants.

GENPLAT: une plate-forme automatisée pour Discovery enzymatique de la biomasse et l&#39;optimisation Cocktail

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

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Bioengineering

15.3K vues.  Michigan State University. GENPLAT (Plateforme Enzyme GLBRC) est une plate-forme automatisée pour la découverte et l'optimisation des cocktails d'enzymes de dégradation de la biomasse. Il peut être adapté à plusieurs matières premières et des mélanges d'enzymes contenant de multiples composants.

Vidéo: GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

Microarrays polymères pour la découverte à haut débit des biomatériaux

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

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Bio-ingénierie

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells.1-3 In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes.4,5 In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion.6,7 Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells.8-12 This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.

This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.

Hydrophobe sel modifié Nafion pour l&#39;immobilisation d&#39;enzymes et de stabilisation

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

Une plate-forme automatisée polyvalent pour micro-échelle expériences de stimulation cellulaire

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

Development of an In Vitro Ocular Platform to Test Contact Lenses

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs.

Development of an In Vitro Ocular Platform to Test Contact Lenses

Current in vitro models for evaluating contact lenses (CLs) and other eye-related applications are severely limited. The presented ocular platform simulates physiological tear flow, tear volume, air exposure and mechanical wear. This system is highly versatile and can be applied to various in vitro analyses with CLs.

Développement d&#39;un<em&gt; In vitro</em&gt; Plateforme Ocular pour tester Lentilles de contact

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic ...

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

Conception et mise en œuvre d&#39;un Illuminating automatisé, la culture, et du système d&#39;échantillonnage pour Microbial optogénétiques Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

A Performance-testing Platform for a Conduction Micropump with an FR-4 Copper-clad Electrode Plate

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is fabricated. It is used to investigate the influence of chamber dimensions on the performance of a conduction micropump and to determine the reliability of the conduction pump when acetone is used as the working fluid. A testing platform is set up to evaluate conduction micropump performance under different conditions. When the chamber height is 0.2 mm, the pump pressure reaches its peak value.

This paper presents a protocol for the fabrication of a conduction micropump using symmetric planar electrodes on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) to test the influence of chamber dimensions on the performance of a conduction micropump.

Une plate-forme de test de performances pour une Conduction de Micropump avec une plaque cuivrée de FR-4 électrodes

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

Protocole générique pour l’optimisation de la Production de protéines hétérologues à l’aide de la technologie automatisée de MICROBIOREACTEUR

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Une plate-forme de Microfluidic ultra haute capacité pour le séquençage du génome de cellules individuelles

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Une nouvelle Technique chirurgicale comme fondement pour In Vivo partielle génie hépatique chez le Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Réprimer la Transcription du gène en redirigeant la machinerie cellulaire avec des modificateurs chimiques épigénétiques

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...