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.

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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).
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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

Universidad de Cartagena UDEC

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

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

Yeni çözünmez Kromojenik altyapı deneyiyle kitleri kullanılarak karbonhidrat parçalayıcı Enzimlerin yüksek verimli tarama

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

Biyokimya

High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the use of non-renewable energy sources. In order to identify which plants, out of a diverse pool, have the desired chemical traits for downstream applications, attributes, such as cellulose and lignin content, or monomeric sugar release following an enzymatic saccharification, must be compared. The experimental and data analysis protocols of the standard methods of analysis can be time-consuming, thereby limiting the number of samples that can be measured. High-throughput (HTP) methods alleviate the shortcomings of the standard methods, and permit the rapid screening of available samples to isolate those possessing the desired traits. This study illustrates the HTP sugar release and pyrolysis-molecular beam mass spectrometry pipelines employed at the National Renewable Energy Lab. These pipelines have enabled the efficient assessment of thousands of plants while decreasing experimental time and costs through reductions in labor and consumables.

Plant cell wall structure and chemistry traits are evaluated to identify ideal feedstocks for biofuels and bio-materials. Standard methods have limitations when applied to large data sets. These high-throughput pretreatment, enzyme saccharification, and pyrolysis-molecular beam mass spectrometry methods compare large numbers of biomass samples with decreased experimental time and cost.

Toplam Lignin, lignin Monomer ve enzimatik Şeker Yayın: Lignocellulosic Biyokütle rekalsitrantların Varyasyonları yüksek verimlilik Taraması

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the ...

Çevre

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

Diterpenoid Doğal Ürünlerin Enzimatik Üretimi ve Saflaştırılması için Özelleştirilebilir Bir Yaklaşım

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

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 ...

Biyomühendislik

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

Yüksek Verimli Ekran Metagenomic Kütüphaneler selülaz Aktivitesi Biomining

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

Biyoloji

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

Lignocellulosic Malzemeler için Yüksek verim Saccharification Testi

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

EMBL de MultiBac Protein Complex Üretim Platformu

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Otomatik Modüler Yüksek Verimli Ekzopolisakkarit Tarama Platformu Oldukça hassas Karbonhidrat Parmak İzi Analizi ile birleştiğinde

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

Çok enzim Yüksek verim Genetik Enzim Eleme Sisteminin Kullanılması Eleme

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...

Chromogenic Polymer Hydrogel-Based Enzyme Screening Assay: A High Throughput Method to Screen the Carbohydrate-Active Enzymes Using Synthetic Substrates

Source: Sch&#252;ckel, J., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/54286">High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits.</a> J. Vis. Exp. (2016).
In this video, we demonstrate a high throughput assay for carbohydrate-active enzyme screening using insoluble chromogenic polymer hydrogel substrates. The enzyme degrades these substrates into a soluble colored product that can be quantified by measuring the color intensity of the product.

Kromojenik Polimer Hidrojel Bazlı Enzim Tarama Testi: Sentetik Substratlar Kullanarak Karbonhidrat Aktif Enzimleri Taramak İçin Yüksek Verimli Bir Yöntem

Source: Sch&#252;ckel, J., et al. High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits. J. ...