Bu çalışma, ABD Enerji Departmanı, Temel Enerji Bilimler, Bölüm Dairesi, ABD Enerji Great Lakes Biyoenerji Araştırma Merkezi Departmanı (DOE Bilim BER Office DE-FC02-07ER64494) ve hibe DE-FG02-91ER200021 tarafından finanse edildi Kimyasal Bilimler, Yer Bilimleri ve Biosciences. John Scott-Craig ve Melissa Borrusch Biz onların maddi ve kavramsal katkıları için teşekkür ederim.

1. Enzim Üretimi <ol><li> PPICZ veya pPICZα vektörler ilgili genlerin klonlanması ve P. dönüşüm Pichia pastoris proteinlerin üretin pastoris. Metanol indüksiyonu ile şaşkına 500 ml&#39;lik şişeler içinde 300 ml gruplar halinde hücrelerin büyümesine, her 24 saat (Banerjee ve ark, 2010a). </li><li> Konsantre ve teğetsel akış filtrasyon ile kültür filtratlar desalt. </li><li> -80% 20 gliserol küçük alikotları enzimler Mağaza ° C Mg / ml konsantrasyon aralığında 1-10. </li></ol> 2. Deney Tasarımı <ol><li> Giriş test enzimlerin sayısı, üst ve daha düşük oranlarda (çekirdek enzimler için% 5 daha düşük bir oranda) ve deneysel tasarım Tasarım Expertsoftware içine türü (örneğin, kuadratik veya kübik). </li><li> 15 mg / g glukan toplam yükleme yapmak eklenecek her enzim mikrogram miktarını hesaplayın ve sonra eklemek için ul her bir enzimin miktarını hesaplamakHer reaksiyon. </li><li> Excel çalışma sayfaları, deneysel tasarım ve Biomek FXP yazılım aktarmak dosya transferi işlevini kullanarak göre su ve enzimler dağıtımı için kaynak Laboratuvar, kaynak kuyuları, hedef Laboratuvar, hedef kuyuları, ve transfer birimleri belirterek oluşturun. </li></ol> 3. Enzimatik Hidroliz <ol><li> Raketi rezervuar, 5 mg / ml tetrasiklin ve 5 mg / ml Siklohekzimidsiz içeren 50 mM sodyum sitrat, pH 4.8 biyokütle bulamaç askıya alınması. </li><li> 96-kuyu derin kuyu reaksiyon plakaların her kuyuya bulamaç robot pipetlemeyin 200 ul Açıklığı-8 kesilmiş son 0.5 cm ile 1000-ul pipetlemeyin ipuçları. 100 ul / sn bulamaç kez karıştırın ve daha sonra aynı kürek rezervuar hacmi aspirat ve tabak içine dağıtmak. </li><li> Beckman FX girilen programını kullanarak aynı plaka enzimler koyun. </li><li> Delinebilen capmats plakaları. </li><li> Ters çevirin ve dokununplakaların yüzey gerilimi aşmak için birkaç kez. 50 ° C&#39;de 48 saat süreyle 10 rpm&#39;de melezleşme inkübatör dönen plakalar yerleştirin. </li></ol> 4. GLC ve Xyl Belirlenmesi <ol><li> Sallanan bir kova santrifüj 1250 xg az 3 dakika için reaksiyon plakaları santrifüjleyin. </li><li> 100 ul süpernatantı Biomek FX AP96 bölmesini kullanarak düzenli 96-kuyucuğu içine aktarın. </li><li> 90-95 az plakalar ° C&#39;de enzimleri inaktive ve daha sonra 30 sn için 1250 xg&#39;de santrifüj için 10 dk. </li><li> GLC ölçümleri için, düzenli olarak 96-kuyucuğu süpernatantlar 12 ul aktarın. Glukoz oksidaz / peroksidaz (GOPOD) reaktif 192 ul ekleyin. Plakalar için 50 ° C&#39;de 20 dakika inkübe edin ve bir mikroplak okuyucu 510 nm de absorbans okunur. Boş hiçbir enzim ile reaksiyona karışımıdır. </li><li> Xyl ölçümleri için, 384-kuyucuğu 4 ul transfer. Tahlil reaktifler ve 340 nm&#39;de plakaları okuyun. Boşhiçbir enzim reaksiyon karışımı. </li></ol> 5. Veri Analizi <ol><li> Absorbans değerleri bilinen GLC ve orijinal biyokütle Xyl içeriği temel alarak% GLC ve Xyl verim dönüştürün. Tasarım Uzman yazılımı tepkiler olarak bu yüzdeler içe aktarın. Istatistiksel paramenters sağlam bir model için kriterleri yerine getirmiş olduğundan emin olmak için kontrol edin. </li><li> Deneysel olarak en iyi modeli tahmin onaylayın. </li></ol> 6. GENPLAT Temsilcisi Sonuçlar: (1) bireysel saf proteinlerin optimize edilmiş bir karışım oluşturulması. Sonuçları enzimler önemli olduğu ve hangi oranlarda, hangi enzimlerin önemli değildir . T. bol Bazı proteinler Cip1 ve Cip2 reesei secretome (Nagendran ve ark, 2009), amonyak fiber genişleme (AFEX) ya da alkalin HYD tarafından tedavi öncesi mısır koçanı GLC sürümde hiçbir rol oynadığı görülmektedir<html> oksidatif stresin (AHP) (Banerjee ve ark, 2010b). Öte yandan, bazı proteinler beklenmedik önemlidir. Örneğin, 10 ve 11 glikozil hidrolaz aileleri endo-xylanases GLC serbest bırakılması için iki önemli olan bir xylanase diğer (. Banerjee ve ark, 2010b, c) yerini alamaz. T., küçük bir protein Cel61A reesei secretome GLC için çok önemli ama serbest Xyl değil. Bazı enzimler ise diğerleri sadece birini ya da diğerini (Banerjee ve ark. 2010C) için gerekli, hem de GLC ve Xyl serbest bırakılması için önemlidir. 16 bileşenler, her bir konsantrasyon 0.94 mg / ml (yani, olmayan bir optimize karışımı) içeren bir sentetik karışım, AFEX önceden tedavi DDG kullanılabilir GLC% 38&#39;i piyasaya sürdü. , Aynı hidroliz koşullar altında, optimum bir karışım, 16 bileşenlerin konsantrasyonları,% 0 ile%% 32 arasında değişmektedir (Şekil 2)% 52 GLC yayınladı. Bu deney tanımlanan karışımları inşa yararını göstermektedir. _content &quot;farklı tedavi öncesi / substrat kombinasyonları için en uygun kokteyller&gt; (2) Yaratılış Mevcut ticari selülaz hazırlıkları&quot; tek tip uyan tüm &quot;Gerçekte, en iyi kokteyl substrat ve tedavi öncesi hem de bağlıdır. tek bir ürün Accellerase 1000, farklı yüzeyler, aynı şekilde ön işlem ve farklı ön işlemleri (Şekil 1) maruz aynı substrat farklı verim verir gibi birden fazla ön işlemleri ve mutiple hammadde (Banerjee ve saf enzimlerin karışımlar optimize etmek için GENPLAT kullanmış ve arkadaşları, 2010C). Şekil 2 16 saf enzimlerin optimal enzim oranlarda ön tedavi-AFEX mısır koçanı, AHP-tedavi öncesi mısır koçanı ve AFEX ön tedavi DDG için nasıl farklı olduğunu gösterir. Sadece 11 enzimler Şekil 2 çünkü optimal diğer beş (Cel61B AbfB, Cip1, Cip2 ve Cel12A) oranları% 0 olarak bulundu (Banerjee ve ark. 2010C). (3) Roman enzim keşif </strong&gt;. Saf proteinlerin optimize edilmiş karışımlar inşaatı devam eden bir süreçtir. Yeni proteinler saf formu (ticari kaynaklardan, heterolog ifadeye göre, ya da arıtma) kullanılabilir hale geldikçe, onlar sistematik olarak optimize set ekleyerek test edilebilir. GLC veya Xyl katkıda Roman proteinlerin optimize edilmiş yeni bir set parçası haline bırakın. Bu şekilde, GENPLAT biyokütle Yapısızlaştırmanın için yeni enzimlerin belirlenmesi için bir platform haline geldi. Biz diğer mantarlar T. daha özleri yaptık reesei ve A. Nijer farklı yüzeyler üzerinde büyüdü. Temel eklendiğinde belirli bir özü GLC verimi artırdı. Sorumlu proteinin saflaştırılmış ve bir roman glikozil hidrolaz (Banerjee ve Walton, yayınlanmamış sonuçlar), herhangi bir ticari enzim hazırlık mevcut değil olduğu gösterilmiştir. Daha iyi enzimler (4) Discovery. Bir bozulma enzimlerin en önemli GENPLAT ile gösterilir.Özellikle biyokütle, enzimler iyileştirilmesi için odak noktası olması gereken daha iyi bir anlayış var. Kritik enzimler daha iyi örnekler doğada arayarak ya da protein mühendisliği tarafından yapılmış olabilir. (Bir enzim istenilen özelliğine bağlı olarak çeşitli şekillerde T. reesei homolog, daha &quot;iyi&quot; olabilir, örneğin, yüksek spesifik aktivite, daha fazla sinerji, proteolitik hassasiyet, veya gelişmiş termal stabilite azalma). Saf selüloz veya diğer yapay substratlar sonuçlar doğal lignocellulosic ile ilgili olmayabilir, çünkü gerçekçi karmaşık bir kokteyl (biyokütle enzim izolasyon çalıştığı için önemli) ve önemli gerçekçi bir substrat (kullanır, çünkü GENPLAT, potansiyel olarak daha iyi enzimler assaying için anlamlı bir platform sağlar malzemeler). (5) Ticari enzim optimizasyonu. Yeterince büyük miktarlarda saf enzimlerin henüz gerçek dünyada yok. Yapıncaya kadar, etanol producers gibi Accellerase 1000 ve MultifectXylanase gibi ticari ürünler güvenmek gerekir. Ancak, ticari enzimler karışımları genellikle herhangi bir birey daha iyi performans ve GENPLAT birden fazla ticari enzimlerin optimize kokteyller tasarım için kullanılabilir. Şekil 3, dört ticari enzim preparatları bir karışımı optimize etmek için GENPLAT kullanımı gösterir. AFEX ön tedavi DDG GLC serbest bırakılması için en uygun oranlarda,% 47 Accellerase1000,% 27,% 22 Multifect Pektinaz Novozyme 188 ve% 4 Multifect xylanase (Banerjee ve ark. 2010C) olarak bulunmuştur. Şekil 3B dört enzim preparatları ile AFEX-DDG GLC verim arasındaki farklılıklar gösterir; optimize edilmiş ticari karışımı yüksek GLC verim verir, tek başına Accellerase1000 daha SpezymeCP tek başına veya bir 16-bileşenli sentetik karışım. Bu deney aynı zamanda 16-bileşenli sentetik karışımı için gerekli ticari enzimlerin bir veya daha fazla mevcut en iyi GLC açıklaması, bir veya daha fazla enzimler eksik olduğunu gösterir. GENPLAT bize.ed gibi bileşenler tespit etmek. <img src="/files/ftp_upload/3314/3314fig1.jpg" alt="Şekil 1"/> Şekil 1: Tek başına hiç bir ticari enzim hazırlık tüm yüzeyleri ve tüm ön işlemleri için en uygunudur. Beş yüzeyleri ve taşlama (0.5 mm parçacık boyutu) benzer koşullar altında üç ön işlemleri karşılaştırıldığında, enzim yükleme (15 mg / g glukan) ve hidroliz koşulları (48 saat, 50 ° C) farklı AFEX ön işlem substratlar A. Sindirim Accellerase 1000 ile mısır koçanı B. Sindirim Accellerase 1000 ile üç farklı yöntem (Banerjee ve ark. 2010C) ile ön işlem. <img src="/files/ftp_upload/3314/3314fig2.jpg" alt="Şekil 2"/> Şekil 2 AFEX-tedavi öncesi mısır koçanı (AFEX CS), alkalin hidrojen peroksit tedavi öncesi mısır koçanı (AHS-CS), ya da kurutulmuş AFEX ön tedavi GLC serbest bırakılması için 11 enzimlerin optimal oranlarda damıtmakers &#39;tahıl (AFEX DDG). Veriler Banerjee ve ark.. (2010C). Çubukların üstündeki sayıları, toplam GLC biyokütle örnek bir yüzdesi olarak GLC verim. <img src="/files/ftp_upload/3314/3314fig3.jpg" alt="Şekil 3"/> Şekil 3. A. AFEX-ön tedavi DDG GLC serbest bırakılması için dört ticari enzim preparatları optimize edilmiş bir karışımı üretmek için GENPLAT kullanın. Multifect xylanase en küçük katkısı (% 4) ve bu nedenle bu üçlü diyagram atlandı. Özdeş hidroliz (15 mg / g glukan enzim, 48 saat, 50 ° C) koşulları altında farklı enzim tedaviler ile B. AFEX-DDG GLC verimleri . &quot;Dört bileşenli ticari&quot; Şekil deney belirlenen oranlara sahiptir. 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>

Çıkar çatışması ilan etti.

Bu yaygın enzimlerin maliyetini azaltarak ekonomik bir lignocellulosic etanol endüstrisinin gelişimi için önemli olduğu kabul edilmektedir. Şu anda mevcut ticari enzim kokteyller birçok proteinleri (Nagendran ve ark, 2009) karmaşık ve kötü tanımlanmış karışımları ve özellikle asit-tedavi öncesi mısır koçanı kullanmak için uyarlanmıştır. Daha iyi enzim kokteyller gelişimini hızlandırmak için, bazı laboratuvarlar enzim keşif ve karakterizasyonu için yüksek verimli platformlar geliştirdik. Decker et robot, enzimler ve biyokütle çamurlar dağıtım, istatistiksel deney tasarımı ve / veya otomatik olarak belirlenmesi GLC ve Xyl (Berlin ve ark, 2007: Bu alandaki çabalar da GENPLAT bulunan aşağıdaki özelliklerden bir veya daha fazla dahil diğerleri, 2009; Kim ve ark, 1998; King ve ark, 2009). GENPLAT en az 6 bileşenleri analiz edilebilir enzim karışımlarının karmaşıklığı en önemlisi, bu daha önceki çabaları uzanıren son çalışma fazla 16 daha önceki çalışmalarda (Banerjee ve ark. 2010C). Üzerinde uç uç dönme sindirim sırasında nazik karıştırma; ve Glu ve Xyl otomatik kolorimetrik tespiti GENPLAT Ek temel özellikleri, dağıtım sırasında askıya koçanı çamurlar tutabilir boncuk karıştırma haznesinin kullanımı (kürek rezervuar).

Kullanılan araç ve gereçler ve yazılım: <ol><li> Tasarım Uzman yazılım, Sürüm 8.0 (Stat-kolaylığı, Inc, Minneapolis, MN) ( <a href="http://www.statease.com">http://www.statease.com</a> ) </li><li> Biomek FXP laboratuvar otomasyon iş istasyonu (Beckman Coulter, Fullerton, CA) </li><li> Biomek FXP yazılımı, Sürüm 3.3 (Beckman Coulter, Fullerton, CA) </li><li> Paddle rezervuar (Model VP 756C-1P100, V &amp; P Scientific Inc, San Diego) </li><li> Tabaklar, 96-derin (1,5 ml) (Abgene, Fisher Scientific, Epson, Birleşik Krallık) </li><li> Delinebilen capmats (ABD Bilimsel, Ocala, FL) </li><li> Döner melezleşme inkübatör (Model 5420, VWR) </li><li> Sallanır kovası santrifüj (Model 5810R, Eppendorf, Hamburg, Almanya) </li><li> Tabaklar, Costar 96 (Abgene, Fisher Scientific, Epson, İngiltere) </li><li> Tabaklar, 384-(BD Falcon, BD Biosciences, San Jose, CA) </li></ol> Reaktifler kullanılır: <ol><li> Pichia pastoris ve vektörler: suşu X33 ve pPICZ ve pPICZα (Invitrogen, Carlsbad, CA) </li><li> T. reesei ve vektörleri: suşu QM9414 (ABD Dışişleri Bakanlığı Tarımsal Kullanımı Ulusal Tarım Araştırma Merkezi, Peoria, IL) ve pCB1004-EV (Banerjee ve ark, 2010b) </li><li> Accellerase 1000 (lot numarası 1600844643) (Genencor, Inc, Rochester, NY) </li><li> 50 mM sodyum sitrat tamponu, pH 4.8 </li><li> Tetrasiklin stok: 10 mg / ml </li><li> Cycloheximide stok: 10 mg / ml </li><li> GOPOD testi kiti, katalog K-GLUC (Megazyme, Bray, İrlanda) </li><li> Ksiloz testi kiti, katalog K-ksiloz (Megazyme, Bray, İrlanda) </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).
<div id="myEventWatcherDiv">
	 </div>

Watch this Scientific Journal Video about GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization at JoVE.com

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

GENPLAT (GLBRC Enzim Platformu) biyokütle bozulması için enzim kokteyller keşif ve optimizasyonu için otomatik bir platform. Bu birden fazla hammadde ve birden fazla bileşen içeren enzimlerin karışımları adapte edilebilir.

GENPLAT: Biyokütle Enzim Discovery ve Kokteyl Optimizasyonu Otomatik Platform

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

genplat-an-automated-platform-for-biomass-enzyme-discovery-cocktail

Research

JoVE Journal

Bioengineering

15.4K Görüntüleme.  Michigan State University. GENPLAT (GLBRC Enzim Platformu) biyokütle bozulması için enzim kokteyller keşif ve optimizasyonu için otomatik bir platform. Bu birden fazla hammadde ve birden fazla bileşen içeren enzimlerin karışımları adapte edilebilir.

Video: 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.

Biyomalzemelerin Yüksek Verimli Discovery için Polimer Mikroarray&#39;ler

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

Biyomühendislik

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.

Enzim immobilizasyonu ve stabilizasyonu için hidrofobik Tuz-modifiye Nafion

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.

Mikro ölçekli Hücre Stimülasyon deneyler için Çok Yönlü Otomatik Platformu

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

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.

Mikrobiyal optogenetic Uygulamaları Tasarım ve Otomatik Aydınlatıcı, kültürleme ve Örnekleme Sistemi Uygulaması

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.

Performans Test platformu ve tel Micropump FR-4 bakır kaplı elektrot plaka ile için

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.

Otomatik Microbioreactor teknolojisini kullanarak Contegra Protein üretim duruma getirilmesi için genel iletişim kuralı

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.

Tek hücreli genom sıralama için bir üretilen iş Ultrahigh mikrosıvısal Platform

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.

Sıçan Vivo içinde kısmi karaciğer Dairesi'nde için bir temel olarak yeni bir cerrahi teknik

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.

Kimyasal epigenetik değiştiriciler ile hücresel makine yeniden yönlendirerek Gen transkripsiyonu bastırmak

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

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Otomatik bir Mikrobioreactor sistemi kullanılarak Çin hamster yumurtlama hücrelerinden bir monoklonal antikor arıtma ve analitik

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...