Diese Arbeit wurde teilweise durch das US Department of Energy Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494) und gewähren DE-FG02-91ER200021 aus dem US Department of Energy, Office of Basic Energy Sciences, Division finanziert of Chemical Sciences, Geosciences and Biosciences. Wir bedanken uns bei John Scott-Craig und Melissa Borrusch für ihre materielle und konzeptionelle Beiträge.

1. Enzymproduktion <ol><li> Produzieren Proteine ​​in Pichia pastoris durch Klonierung der entsprechenden Gene in Vektoren pPICZ oder pPICZα und die Umwandlung in P. pastoris. Wachsen Zellen in 300-ml Ansätzen in verblüffte 500-ml-Kolben mit Methanol Induktion alle 24 h (Banerjee et al., 2010a). </li><li> Konzentrieren Sie sich und entsalzen die Kultur Filtrate durch Tangentialflussfiltration. </li><li> Bewahren Sie die Enzyme in kleinen Portionen in 20% Glycerin bei -80 ° C. Der Konzentrationsbereich ist 1-10 mg / ml. </li></ol> 2. Design of Experiment <ol><li> Geben Sie die Anzahl der Enzyme getestet werden, deren obere und untere Proportionen (zB einen geringeren Anteil von 5% für Kern-Enzyme), und die Art des experimentellen Designs (zB quadratisch oder kubisch) in die Design-Expertsoftware. </li><li> Berechnen Sie die Menge in ug jedes Enzym hinzugefügt, um eine Gesamtbelastung von 15 mg / g Glucan zu machen, und dann berechnen die Höhe des jeweiligen Enzyms in ul zu ergänzenjede Reaktion. </li><li> Generate Excel-Arbeitsblätter Angabe Quelle Laborgeräte, Quelle Brunnen, Ziel-geräte, Ziel Brunnen und Transfervolumen für die Abgabe von Wasser und Enzymen nach dem experimentellen Design und importieren Sie es in der Biomek FXP Software mit dem Transfer-from-Datei-Funktion. </li></ol> 3. Enzymatische Hydrolyse <ol><li> Suspend der Biomasse Gülle in 50 mM Natriumcitrat, pH 4,8, enthält 5 pg / ml Tetracyclin und 5 pg / ml Cycloheximid, in dem Paddel Reservoir. </li><li> Robotically pipet 200 ul der Gülle in jede Vertiefung einer 96-Well-Deep-Well-Platten mit Reaktion Span-8-1000-ul Pipettenspitzen mit den letzten 0,5 cm abgeschnitten. Mix der Schlamm einmal bei 100 ul / sec und dann absaugen das gleiche Volumen aus dem Paddel Reservoir und verzichten in den Platten. </li><li> Dispense die Enzyme in die gleiche Platte mit dem Programm in den Beckman FX eingetragen. </li><li> Seal die Platten mit durchstechbarer CapMats. </li><li> Invert und tippen Sie aufdie Platten mehrfach zu überwinden Oberflächenspannung. Legen Sie die Platten bei 50 ° C für 48 Stunden in der Hybridisierung Inkubator rotierenden bei 10 Umdrehungen pro Minute. </li></ol> 4. Bestimmung von Glc und Xyl <ol><li> Centrifuge die Reaktion Platten für 3 min bei 1250 xg in einem Ausschwingrotor zentrifugiert. </li><li> Transfer 100 ul des Überstands in reguläre 96-Well-Platten mit dem AP96 pod des Biomek FX. </li><li> Die Inkubation bei 90-95 ° C für 10 min, um die Enzyme zu inaktivieren, und dann Zentrifuge bei 1250 xg für 30 sec. </li><li> Für Glc Messungen, Transfer 12 ul der Überstände in regelmäßigen 96-Well-Platten. Addieren Sie 192 hinzu ul der Glucose-Oxidase / Peroxidase (GOPOD) Reagenz. Die Inkubation bei 50 ° C für 20 min und die Absorbanz bei 510 nm in einem Mikroplatten-Reader. Blank ist Reaktionsgemisch ohne Enzym. </li><li> Für Xyl Messungen, Transfer 4 ul in 384-Well-Platten. Fügen Sie den Reagenzien des Assays und lesen Sie die Platten bei 340 nm. Leerwird das Reaktionsgemisch ohne Enzym. </li></ol> 5. Data Analysis <ol><li> Konvertieren Sie die Extinktionswerte auf ihre% Glc und Xyl Renditen auf den bekannten Glc und Xyl Inhalt der ursprünglichen Biomasse. Importieren Sie diese Prozentsätze wie die Reaktionen in den Design-Expert-Software. Überprüfen Sie die statistischen Parameter umfassen, um sicherzustellen, dass die Kriterien für eine robuste Modell erfüllt sind. </li><li> Bestätigen Sie das beste Modell Vorhersage experimentell. </li></ol> 6. Repräsentative Ergebnisse mit GENPLAT: (1) Erstellung von optimierten Mischungen der einzelnen reine Proteine. Die Ergebnisse zeigen, welche Enzyme wichtig sind, und in welchem ​​Verhältnis, und auch die Enzyme sind nicht wichtig. Einige Proteine, die reichlich vorhanden sind in der T. reesei Sekretom (Nagendran et al., 2009), wie Cip1 und Cip2, scheinen keine Rolle in Glc Lüftspiel aus Maisstroh vorbehandelt durch Ammoniak-Faser-Erweiterung (AFEX) oder alkalische hyd<html> rogen Peroxid (AHP) (Banerjee et al., 2010b). Auf der anderen Seite sind einige Proteine ​​unerwartet wichtig. Zum Beispiel sind Endo-Xylanasen von Glykosylhydrolase Familien 10 und 11 sowohl für Glc Freisetzung wichtig, eine Xylanase kann nicht für die anderen (. Banerjee et al, 2010b, c) zu ersetzen. Cel61A, eine kleine Protein in der T. reesei Sekretom, ist sehr wichtig für Glc aber nicht Xyl freizugeben. Einige Enzyme sind für die Freilassung der beiden Glc und Xyl wichtig, wohingegen andere nur für den einen oder anderen (Banerjee et al., 2010c) sind notwendig. Ein synthetisches Gemisch mit 16 Komponenten, die jeweils in einer Konzentration von 0,94 mg / ml (dh eine nicht-optimierte Mischung), veröffentlicht 38% der verfügbaren Glc von AFEX-vorbehandelte DDG. Unter identischen Bedingungen Hydrolyse, eine optimierte Mischung, in denen die Konzentrationen der 16 Komponenten im Bereich von 0% bis 32%, veröffentlicht 52% der Glc (Abb. 2). Dieses Experiment zeigt den Nutzen der Konstruktion definierte Mischungen. _content &quot;&gt; (2) Schaffung von optimierten Cocktails für unterschiedliche Vorbehandlung / Substrat-Kombinationen. Current kommerziellen Cellulase Präparate sind&quot; one-size-fits-all &quot;. In Wirklichkeit hängt die besten Cocktail-sowohl auf dem Substrat und der Vorbehandlung. Ein einzelnes Produkt wie Accellerase 1000 gibt unterschiedliche Erträge aus verschiedenen Substraten auf die gleiche Weise vorbehandelt, und aus dem gleichen Substrat ausgesetzt unterschiedlichen Vorbehandlungen (Abb. 1). Wir haben GENPLAT verwendet werden, um Mischungen von reinen Enzymen für mehrere Vorbehandlungen und Vielfaches Rohstoffe (Banerjee et optimieren al., 2010c). Abbildung 2 zeigt, wie die optimale Enzym Proportionen von 16 reine Enzyme für AFEX-vorbehandelte Maisstroh, AHP-vorbehandelten Maisstroh und AFEX-vorbehandelte DDG unterscheidet. Nur 11 Enzyme sind in Abb. gezeigt. 2, da die optimale Anteile der übrigen fünf (Cel61B, AbfB, Cip1, Cip2 und Cel12A) wurde festgestellt, dass 0% (Banerjee et al., 2010c). (3) Novel Enzym entdeckt </strong&gt;. Der Bau der optimierten Mischungen aus reinen Proteinen ist ein fortlaufender Prozess. Als neuer Proteine ​​in reiner Form (aus kommerziellen Quellen, durch heterologe Expression oder durch Reinigung) werden, können sie systematisch, indem sie in der aktuellen optimierten geprüft werden. Neuartige Proteine, die an Glc oder Xyl beitragen Release dann Teil einer neuen, optimierten gesetzt werden. Auf diese Weise wird GENPLAT eine Plattform für die Identifizierung neuer Enzyme für Biomasse Dekonstruktion. Wir haben Auszüge aus Pilzen außer T. gemacht reesei und A. niger auf unterschiedlichen Substraten gewachsen. Ein besonderes Extrakt steigerte Glc Ausbeute bei unseren Kern hinzugefügt. Die verantwortliche Protein wurde gereinigt und gezeigt, dass ein Roman Glykosylhydrolase nicht in jeder kommerziellen Enzympräparat (Banerjee und Walton, unveröffentlichte Ergebnisse) werden. (4) Entdeckung der besseren Enzyme. Nachdem mit GENPLAT die Enzyme sind die wichtigsten für den Abbau eines gezeigtinsbesondere Biomasse, haben wir ein besseres Verständnis für die Enzyme der Schwerpunkt für die Verbesserung sein sollte. Bessere Beispiele der kritischen Enzyme könnten durch die Suche in der Natur gefunden werden oder gemacht durch Protein-Engineering. (Ein Enzym könnte &quot;besser&quot; als ihre T. reesei homolog in mehrfacher Hinsicht abhängig von der gewünschten Eigenschaft, sank z. B. höhere spezifische Aktivität, größere Synergie, proteolytische Empfindlichkeit oder eine höhere thermische Stabilität). GENPLAT bietet eine sinnvolle Plattform für die Bestimmung von potentiell bessere Enzyme, weil es eine realistische komplexe Cocktail (wichtig, weil keine Biomasse Enzym arbeitet in Isolation) und eine realistische Substrat (wichtig nutzt, da die Ergebnisse mit reiner Cellulose oder anderen künstlichen Substraten möglicherweise nicht relevant für natürliche Lignozellulose Materialien). (5) Gewerbliche Enzym-Optimierung. Ausreichend große Mengen reiner Enzyme noch nicht in der realen Welt existieren. Bis sie es tun, Ethanol proProduzenten müssen auf kommerzielle Produkte wie Accellerase 1000 und MultifectXylanase verlassen. Allerdings Mischungen von handelsüblichen Enzymen in der Regel besser als jeder einzelne ein, und GENPLAT kann zur optimierten Cocktails von mehreren kommerziellen Enzyme Design. Abbildung 3 zeigt die Verwendung von GENPLAT um eine Mischung von vier kommerziellen Enzympräparate optimieren. Die optimale Proportionen für die Freigabe von Glc von AFEX-vorbehandelte DDG wurde festgestellt, dass 47% Accellerase1000, 27% Multifect Pektinase, 22% Novozyme 188 und 4% Multifect Xylanase (Banerjee et al., 2010c) werden. 3B zeigt die Unterschiede in Glc Ausbeute von AFEX-DDG mit vier Enzympräparate, die optimierte kommerzielle Mischung führt zu einer höheren Rendite als Glc Accellerase1000 allein SpezymeCP allein, oder ein 16-Komponente synthetischen Mischung. Dieses Experiment zeigt auch, dass die 16-Komponente synthetischen Mischung fehlt ein oder mehrere Enzyme für eine optimale Glc Release, das in einem oder mehreren der kommerziellen Enzyme benötigt werden. GENPLAT können unsed zu identifizieren solcher Komponenten. <img src="/files/ftp_upload/3314/3314fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Keine einzige kommerzielle Enzympräparation ist optimal für alle Untergründe und alle Vorbehandlungen. Fünf Substrate und drei Vorbehandlungen wurden unter ähnlichen Bedingungen von Schleifen (0,5 mm Korngröße) verglichen, Enzymbeladung (15 mg / g Glucan) und Hydrolysebedingungen (48 h, 50 ° C). A. Verdauung verschiedener AFEX-vorbehandelte Substrate mit Accellerase 1000. B. Verdauung von Maisstroh vorbehandelt durch drei verschiedene Methoden mit Accellerase 1000 (siehe Banerjee et al., 2010c). <img src="/files/ftp_upload/3314/3314fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Optimale Proportionen von 11 Enzymen für die Freisetzung von Glc von AFEX-vorbehandelte Maisstroh (AFEX-CS), alkalische Wasserstoffperoxid-vorbehandelte Maisstroh (AHP-CS) oder AFEX-vorbehandelte getrocknete destillierenERS &quot;Körner (AFEX-DDG). Die Daten stammen aus Banerjee et al. (2010c). Die Zahlen entlang der Oberseite der Balken sind die Glc Erträge als prozentualer Anteil der gesamten Glc in der Biomasse Probe. <img src="/files/ftp_upload/3314/3314fig3.jpg" alt="Abbildung 3"/> Abbildung 3. A. Die Verwendung von GENPLAT eine optimierte Mischung von vier kommerziellen Enzympräparate für die Freigabe von Glc von AFEX-vorbehandelte DDG zu produzieren. Multifect Xylanase aus den kleinsten Beitrag (4%) und wurde daher von dieser ternären Diagramm weggelassen. B. Glc Erträge aus AFEX-DDG mit verschiedenen Enzym-Behandlungen unter identischen Bedingungen Hydrolyse (15 mg / g Glucan Enzym, 48 h, 50 ° C) . &quot;Vier-Komponenten-commercial&quot; hat die Proportionen aus dem Experiment in Abb. bestimmt. 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>

Keine Interessenskonflikte erklärt.

Es ist allgemein anerkannt, dass die Senkung der Kosten von Enzymen ist wichtig, die Entwicklung eines wirtschaftlichen Lignocellulose Ethanol-Industrie. Die derzeit verfügbaren kommerziellen Enzym-Cocktails sind komplex und schlecht definierte Mischungen aus vielen Proteinen (Nagendran et al., 2009), und sie sind vorwiegend für den Einsatz auf den Säure-vorbehandelten Maisstroh angepasst. Um die Entwicklung besserer Enzym-Cocktails zu beschleunigen, haben mehrere Labors mit hohem Durchsatz-Plattformen für die Enzym-Entdeckung und Charakterisierung entwickelt. .; Decker et Roboter Abgabe von Enzymen und Biomasse Schlämmen, statistische Design of Experiments, und / oder automatisierten Bestimmung von Glc und Xyl (Berlin et al, 2007: Die Anstrengungen in diesem Bereich eine oder mehrere der folgenden Eigenschaften auch in GENPLAT gefunden eingebaut al, 2009;. Kim et al, 1998;. King et al, 2009).. GENPLAT erweitert diese früheren Bemühungen, die meisten deutlich in der Komplexität der Enzym-Mischungen, die aus maximal 6-Komponenten analysiert werden kann inden früheren Studien an mehr als 16 in unserem neuesten Werk (Banerjee et al., 2010c). Weitere wichtige Merkmale der GENPLAT sind die Verwendung von einem Wulst Mischkammer (Paddel-Reservoir), dass stover Schlämme während der Abgabe der Schwebe zu halten kann; schonende Durchmischung während der Verdauung von end-over-end Rotation und automatisierte kolorimetrischen Bestimmung von Glu und Xyl.

Ausrüstungen und Software zum Einsatz: <ol><li> Design-Expert-Software, Version 8.0 (Stat-Ease, Inc., Minneapolis, MN) ( <a href="http://www.statease.com">http://www.statease.com</a> ) </li><li> Biomek FXP Laborautomation Workstation (Beckman-Coulter, Fullerton, CA) </li><li> Biomek FXP-Software, Version 3.3 (Beckman-Coulter, Fullerton, CA) </li><li> Paddle-Reservoir (Modell VP 756C-1P100, V &amp; P Scientific Inc., San Diego) </li><li> Platten, 96-tiefe (1,5-ml) gut (Abgene, Fisher Scientific, Epson, UK) </li><li> Durchstechbare CapMats (USA Scientific, Ocala, FL) </li><li> Rotierende Hybridisierung Inkubator (Modell 5420, VWR) </li><li> Swinging-Becherzentrifuge (Model 5810R, Eppendorf, Hamburg, Deutschland) </li><li> Platten, Costar 96-well (Abgene, Fisher Scientific, Epson, UK) </li><li> Platten, 384-well (BD Falcon, BD Biosciences, San Jose, CA) </li></ol> Verwendete Reagenzien: <ol><li> Pichia pastoris und Vektoren: strain X33 und pPICZ und pPICZα (Invitrogen, Carlsbad, CA) </li><li> T. reesei und Vektoren: strain QM9414 (US Department of Agriculture National Center for Agricultural Utilization Research, Peoria, IL) und pCB1004-EV (Banerjee et al, 2010b). </li><li> Accellerase 1000 (Lot-Nummer 1600844643) (Genencor, Inc., Rochester, NY) </li><li> 50 mM Natriumcitrat-Puffer, pH 4,8, </li><li> Tetracycline Lager: 10 mg / ml </li><li> Cycloheximid Lager: 10 mg / ml </li><li> GOPOD Assay-Kit, Katalog K-GLUC (Megazyme, Bray, Irland) </li><li> Xylose-Assay-Kit, Katalog K-Xylose (Megazyme, Bray, Irland) </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 (GLBRC Enzyme Platform) ist eine automatisierte Plattform für die Entdeckung und Optimierung von Enzym-Cocktails für Biomasse Abbau. Es kann zu mehreren Rohstoffen und Mischungen von Enzymen, die mehrere Komponenten angepasst werden.

GENPLAT: eine automatisierte Plattform für Biomasse Enzym-Entdeckung und Cocktail-Optimierung

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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JoVE Journal

Bioengineering

15.3K Aufrufe.  Michigan State University. GENPLAT (GLBRC Enzyme Platform) ist eine automatisierte Plattform für die Entdeckung und Optimierung von Enzym-Cocktails für Biomasse Abbau. Es kann zu mehreren Rohstoffen und Mischungen von Enzymen, die mehrere Komponenten angepasst werden.

Serie: 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.

Polymer Microarrays für 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 ...

Forschung

Biotechnik

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 Salz-modifizierte Nafion zur Enzymimmobilisierung und Stabilisierung

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.

Eine vielseitige automatisierte Plattform für Mikro-Skala Handy Stimulation Experimente

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.

Entwicklung eines<em&gt; In Vitro</em&gt; Ocular Platform Kontaktlinsen zu Test

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.

Design und Implementierung eines automatisierten Illuminating, Anzucht und Probenahmesystem für Mikrobielle optogenetische Anwendungen

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.

Leistungstests Plattform für eine Wärmeleitung Mikropumpe mit einem FR-4 kupferummantelte Elektrodenplatte

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.

Generischen Protokoll zur Optimierung der heterologen Proteinproduktion mit automatisierten Microbioreactor Technologie

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.

Eine höchste Durchsatz mikrofluidischen Plattform für einzellige Genom-Sequenzierung

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.

Eine neuartige Operationstechnik als Grundlage für In Vivo teilweise Leber Engineering in der Ratte

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.

Gentranskription durch Umleiten zellulären Maschinerie mit chemischen epigenetische Modifikatoren zu unterdrücken

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