JoVE Journal > Biochemistry
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Abstract
Introduction
Protocol
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Biochemistry

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

Published: September 20, 2016
doi:
10.3791/54286
, ,
1Department for Plant and Environmental Sciences,University of Copenhagen, 2School of Agriculture, Food and Rural Development,Newcastle University

Summary

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.

Abstract

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.

Introduction

Techniques for mining genomes and metagenomes have developed rapidly in recent years, and so have medium- and high-throughput strategies for cloning and expressing recombinant enzymes. Furthermore, bioinformatic resources and associated depositories, such as (CAZy)1,2 have expanded greatly. However, there are considerable challenges inherent in the exploitation of microbial enzyme diversity for industrial purposes and the empirical determination of enzyme activities has now become a serious bottleneck. For example, it is estimated that, using current methods, we can safely predict the activities of no more than 4% of the proteins within the CAZy database. Although numerous methods are available for monitoring enzyme activities they all have some limitations. Well-established techniques based on chromatography combined with mass spectrometry are available for assessing the oligomeric fragments of glycosyl hydrolase (GH) activities3,4. However, these approaches are labor intensive and generally low-throughput. Methods based on the measurement of reducing sugars such as the dinitrosalicylic acid5 and Nelson-Somogyi6 assays are widely used for assessing GH activities. However, these assays have limited throughput and can be prone to side-reactions. Individual chromogenic polysaccharide substrates, such as azurine cross-linked (AZCL) are widely used for determination of enzyme activities, but purchasing all of the substrates separately and manually distributing the substrate powders within the assay plate can be cumbersome and costly7.

We have developed a new generation of chromogenic polymer hydrogel (CPH) substrates based on chlorotriazine dyes that, when used in conjunction with a 96-well filter plate, form a high-throughput assay system. Additional Insoluble Chromogenic Biomass (ICB) substrates were developed which provide information about substrate availability within complex polymer mixtures, such as those that exist in lignocellulosic biomass. Each substrate can be produced in one of four colors, and different colored substrates can be combined in a single well. In this protocol is shown that this methodology can be applied to a wide variety of polysaccharides and proteins and the potential for screening GHs, lytic polysaccharide monooxygenases (LPMOs) and proteases. Specific protocols are provided for the use of 96 well plates and representative results illustrate the high efficiency of the CPH and ICB substrate kits as tools for enzyme screening.

One significant advantage of the assay kits described, regardless of the substrate, is that the kits are ready to use within 15 minutes, after the activation step. This eliminates the need for time-consuming assembly of the assay from raw substrate materials as it is the case with some other methods7. The CPH and ICB substrates have excellent storage (at least one year at room temperature), pH and temperature stability 8 and require no specialized equipment or training. The CPH or ICB assays are based on 96-well filter plate within which the reaction with the enzyme is conducted. If the enzyme is active with a given substrate, soluble dyed oligomers are generated, producing a colored supernatant which can then be filtered into a regular clear-well 96-well plate using a vacuum manifold or a centrifuge 8.

The substrates are dyed with chlorotriazine dyes which absorb in the visible spectrum (VIS) range and individual colors (red, blue, yellow and green) can be resolved using linear regression if different CPH substrates of different colors are mixed in a single well, and the enzyme acts on more than one substrate. The resulting plate with the supernatants can be measured using a standard microtiter-plate reader capable of measuring absorbance in the VIS range. Mixing different substrates with different colors in one well increases the throughput of the assay system, to a total of 384 experiments in a 96-well plate (4 different substrates of different colors per well).

CPH substrates provide a valuable tool for assessing the specific activity of an enzyme while ICB substrates are used to evaluate the capacity of an enzyme to digest a component within the context of complex substrate mixtures that enzymes usually encounter within biomass. Although ICB substrates do not provide information about individual enzyme specificities, they are nonetheless useful tools for assessing the commercial performance of enzymes, cocktails or broths.

Protocol

96 çukurlu bir levha formatında CPH Substratlar 1. kromojenik tahlille Deney kiti plakasının etkinleştirme çalkalama olmaksızın oda sıcaklığında 10 dakika inkübasyon ile takip her kuyuya (kit ile elde edilmiş) 200 ul aktivasyon çözeltisi eklenerek deney kiti plakası (Malzeme listesi) (mavi CPH-ksilen içeren), 96-çukurlu bir filtre etkinleştirmek için kullanılır. günümüze kadar gelen aktivasyon çözeltisi kaldırmak için (iç boşluk bloğu ve herhangi bir standart bir toplama plakası gibi şeffaf 96 oyuklu tabanı ile), bir vakum manifoldu kullanılarak vakum uygulayın. Bunun yerine, vakum manifoldunun Bu aşama için 10 dakika boyunca 2700 x g'de santrifüje kullanmak da mümkündür. 100 ul steril su eklenerek CPH alt tabakaların yıkanması ve dengeleyici çıkması için vakum (ya da merkezkaç kuvveti) uygulanacaktır. Bu adımı iki kez daha tekrarlayın ve plakalar artık kullanıma hazırdır. Enzim reaksiyonu, NOT: Her zaman tampon içerirtek başına negatif kontrol olarak ve pozitif kontroller olarak mümkün olan, daha önce karakterize edilen enzimler durumunda. çoğaltır istatistiksel uygun sayıda kullanın. CPH substratlar 10.0'a pH 3.0 ve 180 uL geçmemelidir her çukuruna tampon ucu enzim çözeltisinin toplam hacmi arasındaki stabildir. Bitki ekstresi veya kültür brotu yerine de saflaştınlmış enzim çözeltisi kullanılabilir. 150 ul 100 mM sodyum asetat tamponu, pH 4.5 ve tahlil kiti plakasının her oyuğuna (1 U / ml 'lik bir nihai konsantrasyona kadar), 5 ul Endo -cellulase solüsyonu ekleyin. çalkalama sırasında reaksiyon plakasından herhangi bir olası kaçak toplamak için deney kiti plaka altında ürün plakası (mikrotitre plaka okuyucusu ile uyumlu berrak kuyucuklu plak) koyun. 150 rpm'de yatay çalkalayıcıda 30 dakika boyunca 25 ° C'de tahlil kiti plaka inkübe edin. Not: inkübasyon esnasında deney kiti plaka reaksiyonu Karıştırma tutarlı ve üreme elde edilmesi için çok önemlidirtekrarlanabilir sonucu. CPH substratlar 90 ° C 'ye kadar stabildir. CPH yüzeylerde bilinmeyen enzim konsantrasyonları içeren kültür broths test ederken inkübasyon süresi 24 saat kadar artırılmalıdır. Uygun enkübasyon süreleri enzim (ler) aktivitesine bağlı olduğuna dikkat edin, fakat 24 saat içinde tespit edilebilir bir etkinlik ise, genel olarak, enzim, test edilen alt-tabakanın bozmayan olasıdır. Bir aktif enzim renkli süpernatant olarak görünür çözünür kromojenik oligosakkaritler, içine CPH substrat çözünmeyen kromojenik polisakkaritler aşağılayıcı olduğunu. iç boşluk bloğu ile vakum manifoldunun içinde temiz ürün plaka koyun. üstte tahlil kiti plakası yerleştirin ve vakum (-60 kPa maksimum negatif basınç) uygulanır. 10 dakika boyunca 2700 x g'de santrifüje kullanmak da mümkündür. Not: Reaksiyon ürünü olarak renkli oligosakaritler ihtiva eden süzüntü, daha fazla analiz 8 ürün plaka artık </s> Yukarı. Algılama ve miktarının belirlenmesi Ürün bir levhanın her çukuruna sıvı hacmi yaklaşık görsel inceleme ile aynı olup olmadığını kontrol. Bir plaka okuyucusu kullanılarak mavi CPH-ksilan 595 nm'de toplama plakası absorbansı okuyun. veri analizi yaparken, bir enzim eklendi kuyulardan değerlerden tampon sadece negatif kontrol değerleri çıkarmak. Çoğaltmak kuyulardan 8'den ortalama değerini ve (SEM) araçlarının standart hata hesaplayın. 96 oyuklu bir formatta ICB Substratlar 2. kromojenik tahlille Enzim reaksiyonu, 150 ul 100 mM sodyum asetat tamponu, pH 4.5 ve 5 ekleme ul 31 U / ml endo -xylanase (de son enzim konsantrasyonu 1 U / ml), kırmızı ICB buğday samanı ihtiva eden deney kiti plakasının her oyuğuna Çözelti . Not: tahlil kiti levhalar (96-çukurlu bir filtre PLliteratürde tarif edildiği gibi ICB substratlar içeren ates) (Malzeme listesi bakınız) üretilmektedir. ICB substratlar 10.0'a pH 3.0 arasında bir pH aralığına sahip tampon stabildir. Her zaman, bir negatif kontrol olarak sadece tampon, bir pozitif kontrol olarak, ticari enzim ve yinelemeler istatistiksel uygun sayıda kullanımı bulunmaktadır. CPH alt-tabaka plaka gibi ICB alt tabakaların aktive ama vakumla süzülerek veya santrifüj, ardından 100 ul su ile üç kez yıkanarak dengeleyici kaldırın. sallayarak sırasında alt tabaka plaka herhangi bir potansiyel sızıntıyı toplamak için substrat plaka altında ürün tabak koydu. 2 saat süre ile, 150 rpm'de çalkalanarak 25 ° C'de bir reaksiyon inkübe edin. NOT: Bir aktif enzim renkli süpernatant olarak görünür çözünür oligosakkaritler, içine ICB substrat çözünmeyen kromojenik polisakkaritler aşağılayıcı olduğunu. ICB substratlar 90 ° C 'ye kadar stabildir. kuluçka tBöyle kültür sıvılarında olmayan saflaştırılmış enzimler kullanılması durumunda ime 24 saat kadar artırılmalıdır. iç boşluk bloğu ile vakum manifoldunun içinde ürün plaka koyun. üstte tahlil kiti plakası yerleştirin ve vakum (-60 kPa maksimum negatif basınç) uygulamak veya ürün plakasının kuyuya tahlil kiti plakadan ürünü süzmek için bir santrifüj kullanın. NOT: reaksiyon ürünü olarak renkli oligosakkaritler içeren süzüntü daha fazla analiz 8 toplama plakası artık. Algılama ve miktar Toplama plakanın her bir sıvı hacmi yaklaşık görsel inceleme ile aynı olup olmadığını kontrol. Bir plaka okuyucusu kullanılarak kırmızı ICB buğday samanı 517 nm'de toplama plakası absorbansı okuyun. veri analizi yaparken – tampon çıkarma – sadece negatif kontrol değerlerini kuyulardan değerlerden nerede bir enzimeklendi. Çoğaltmak kuyulardan 8'den ortalama değerini ve (SEM) araçlarının standart hata hesaplayın. NOT: bilinmiyor enzimler tarama durumda, enzimin aktivitesi dinamik aralık hakkında daha ayrıntılı veriler elde etmek amacıyla bir seyreltme serisi yapmanızı öneririz.

Representative Results

yüksek verimli ve bu tayinin çoklayıcı kapasitesi 96 oyuklu filtre plakası düzenlenmiş çözünmeyen kromojenik bir polimer (veya proteini), hidrojel (CPH) yüzeylere dayanır. Enzimler ayrıca negatif kontroller deney kiti plaka (Şekil 1 A) ilave edilir ve enzimler renkli bir süpernatant (Şekil 1B) üretilmesi karşılık gelen alt-tabakanın bozulmasına yol açar. Reaksiyon tamamlandıktan sonra, süpernatan berrak oyuklu ürün bir levhasına aktarılır ve absorbans, 96 oyuklu plakalar (Şekil 1C) için uygun bir spektrofotometre kullanılarak doğrudan ölçülebilir. Enzimin, farklı konsantrasyonlarda ksilanaz CPH-arabinoksilan bir doz tepkisi bir örneği (0,00-0,75 U / ml) azaltılması Enzim konsantrasyonu, görsel gözlenebilir burada Şekil 1D 'de gösterilmiştir. Daha detaylı bir spektrofotometrik kantitatifUS-PS enzim konsantrasyonu (Şekil 1E) karşı absorbansı çizmek için kullanılabilmektedir. Sinyal yoğunluğu, enzim aktivitesine karşılık gelir. Testin tekrarlanabilirliği hata çubukları ile gösterilir (ortalamanın standart hatasını, SEM, üç kopyaları). Bu testin tekrarlanabilirlik ilgili daha detaylı deneyler yerde 8 yayınlanmaktadır. CPH-arabinoksilan. A) CPH alt-tabaka ile (deney kiti plakasının bir şema Şekil 1. Xylanase tedavisi, örneğin, CPH-arabinoksilan gibi enzimler 1 ve 2 ve tamponun ilave edilmesinden önce 96 oyuklu filtre plakası yuvaları içine yüklenmiştir) -sadece kontrolü (enzim 1 endo -xylanase aktiviteye sahipti) C) vakum yardımlı Filtrat üzerine, enzim 1 ile CPH-arabinoksilan B) Parçalanma renkli süpernatant ürettiÜrün plaka için süpernatanların iyonu, absorbans spektrofotometrik olarak ölçülür, d) Ürün plakası 100 mM endo -β-1,4-ksilanaz farklı konsantrasyonları ile 4 farklı renkte CPH-arabinoksilan tedaviden sonra, reaksiyon ürünlerini ihtiva eden sodyum asetat tamponu, pH 4.5, oda sıcaklığında 60 dakika;. E) spectrofotometrisi kullanılarak D reaksiyon ürünlerinin Niceleme bu rakamın daha büyük bir versiyonunu görmek için lütfen buraya tıklayınız. Enzim taramasında bu testte kullanmak için farklı seçenekler vardır. Bir seçenek, örneğin tarama için farklı polisakaritler, bilinmeyen aktiviteli (saflaştırılmış) Endo -enzymes az sayıda içeren 96 çukurlu bir plaka kullanmaktır. Bu durumda sonuç, degrad olan polisakaritler gösterirHedef enzimi ile mümkün. Bu prensibi göstermek için, bir endo -cellulase 25 ° C 'de, farklı CPH substratlara karşı test edilmiştir. (1.0 U / ml ve 5 U / ml, 0.5 U / ml), üç farklı enzim konsantrasyonları 30 dakika inkübe edildi. Sonuç ürün plakası (Şekil 2A) açıkça görülebilir. Tedarikçi tarafından sağlanan -cellulase bu endo ürün levha ksiloglukan (demirhindi) için yan aktivite, arpa beta-glukan, glucomannan, huş ağacı ksilan ve galaktomannan düşük yan aktivitesini belirler. Buna uygun olarak, selülaz ilave aktivitesi CPH-β-glukan (arpa) karşı bulundu, CPH-ksiloglukan (demirhindi) CPH-galaktomannan (Şekil 2B) karşı CPH-ksilan (kayın) ve düşük faaliyet. Glucomannan test edilmemiştir. Önceki ile aynı koşullar altında pozitif kontroller olarak kullanılmıştır: Aynı CPH substratlar ticari olarak temin edilebilen enzim (0.5 U / ml ve 1.0 U / ml 0.1 U / ml, üç farklı enzim konsantrasyonları) ile sindirildideney. Bütün yüzeyler pozitif kontrol enzimi vasıtasıyla parçalanır ve sinyal yoğunluğu daha yüksek enzim konsantrasyonunun (Şekil 2C) ile orantılı olarak artış. Şekil 2'de ise, farklı CPH alt-tabakalar farklı konsantrasyonda, 30 dakika. A) bir endo -cellulase ile sindirilmiş, farklı CPH substratların ürünü plaka 25 ° C sıcaklıkta ajitasyon altında inkübe edildi. B) aktivitesi ve çeşitli yan etkinliğinin miktarının belirlenmesi Endo -cellulase. . E-LAMSE CPH-pachyman, CPH-kurdlan, CPH-, hata çubukları, üç yinelemeler C) karşılık gelen alt-tabaka CPH (endo-selülaz ve 2-HE-selüloz farklı ticari enzim aktivitesinin ortalamanın standart hatasını temsil eder β-glukan (arpa); E-XYAN4 ve CPH-ksilen, e-XEGP ve CP'H-ksiloglukan, e-BLAAM CPH-amiloz, E-BMACJ CPH-galaktomannan; Megazyme tüm enzimler). Hata çubukları iki kopyaları ortalamanın standart hatasını temsil etmektedir. Bu rakamın büyük halini görmek için lütfen buraya tıklayınız. Onlar kısmen biyokütle önemli bileşeni olan bitki hücre duvarlarında polisakkaritlerin doğal düzenleme korumak için çözünmez kromojenik biyokütle (ICB) yüzeylerde kromojenik alt tabaka repertuarına yararlı bir ek vardır. CPH ve usulü üzerinden alt tabakalar sıvı ortam içinde yetiştirildiğinde Phanerochaete chrysosporium salgılanmış enzimleri analiz için örnek kullanılmaktadır. Deney kiti plakasının plaka kurulumu 19 CPH substrat ve 5 ICB substratlar (her bir alt tabaka, Şekil 3B, 4 kuyu) ile birlikte, Şekil 3A'da gösterilmiştir. S. Chrysosporium cultivat olduÜç gün boyunca ed ve kültür süpernatanı analiz edilmiştir. Bu nedenle, 125 ul 200 mM tamponu her oyuğa 25 ul kültür yüzey eklendi aktarılmıştır. Üç farklı pH koşulları sodyum asetat tamponu pH 4.0 sodyum fosfat tamponu, pH 6.0 ya da pH 8.0 (Şekil 3C) kullanılarak test edilmiştir. Plaka 2 saat boyunca 25 ° C'de (150 rpm) çalkalanarak inkübe edildi. Reaksiyon ürünler ürün plaka (Şekil 3D) aktarılmış ve analiz edilmiştir. S. Chrysosporium çeşitli glukan, nişasta ve ksilanlar (Şekil 3E) bozulması için enzimleri üretilmektedir. Alt sinyal yarı selülozlar arabinan (şeker pancarı) ve peptik galaktan hem de RGI (soya fasulyesi) için tespit edilebilir. üretilen enzimler nötr ya da hafif bazik koşullar (pH 8.0) daha asidik koşullar (pH değeri 4.0) daha aktifti. ICB alt tabakasma karşı düşük faaliyeti (Şekil 3F)polisakaridler daha doğal bir bağlamda olduğunda, enzim etkinliği saf polisakarid ile aynı olmadığını gösterir ve ham ya da önceden tatbik edilmesi halinde ICB alt-tabakalar, enzim etkinlik ile ilgili daha gerçekçi bir görünüm ortaya yüzden işlenmiş bitki malzemesi. 19 CPH, 5 ICB substratlar. A), her bir alt-tabaka 4 kuyucuklu bir plaka ortamında bir düzeni ihtiva eden bir çok alt-tabaka plakası kullanılarak Phanerochaete chrysosporium bir 3 günlük sıvı kültürden bir kültür üstte yüzen Şekil 3. Tarama (gri arka = CPH yüzeyler, turuncu arka plan = ICB yüzeyler). B) substrat. C içeren tahlil plakasının Resim) Programı (o deneyde kullanılan tampon koşulları gösteren200 mM sodyum asetat pH 4.0, sodyum fosfat, pH 6.0 ve sodyum fosfat pH 8.0) d.) 25 ° C. D) absorbans 2 saat sonra ürün plakasının resmi 517 nm'de ve her bir münferit CPH alt-tabaka ve F için çizildi ilgili enzimler için) ICB substrat sonuçları. Bu rakamın büyük halini görmek için lütfen buraya tıklayınız. Kromojenik alt-tabakalar, aynı zamanda, tek enzimler kullanılarak ya da kokteyl enzim tek bir oyuk farklı renkli CPH alt tabakalar karışımı kullanılarak tedavi sonrası, reaksiyon süpernatant analiz ederek sinerjistik etkilerini incelemek için kullanılabilir. Şekil 4'te gösterilen örnekte, kırmızı CPH selüloz ve sarı CPH-ksilen substratlar yaklaşık ambalajlama birlikte karıştırılmıştır96 gözlü filtre plakası kuyuları al oranı. Şekil 4A, enzimsiz (kontrol), selüloz cel (2 U / ml), ksilanaz ksil (1 U / mL) ve bir oda sıcaklığında 1 saat işlemden sonra renkli reaksiyon ürünlerini gösteren her iki enzimin karışımı, 100 mM sodyum asetat tampon, pH 4.5 (3, her bir yaklaşım çoğaltır). Analizi için, reaksiyon ürünü, 700 nm'de (Şekil 4B) 350 nm absorbans spektrumunun tarama spektrofotometrik nicelleştirilmiştir. Genellikle yalnız gözle muayene farklı boyalar kaynaklanan absorbans spektrumları aynı zamanda her bozulması ölçüde daha doğru bir fikir vermesi için basit doğrusal regresyon 8 kullanılarak çözülebilir ancak kaydedilen enzim, bir veya birden fazla yüzeylerde hareket edip etmediğini bir göstergesidir verebilir karışımından alt-tabaka. esas karışımları gibi CPH substratlar kullanarak SCR sağlayan tahlilin verimi artırırBir deneyde (bir de) kadar 4 farklı yüzeylerde karşı Eening. Gösterilen örnekte kullanılan dört farklı yüzeyler vardır: mavi CPH-β-glukan (arpa), sarı CPH-ksilen (kayın), yeşil CPH-amiloz ve kırmızı CPH-pektik galaktan (acı bakla). Alt-tabaka plakanın düzeni Şekil 4C ve Şekil 4D'de deney plakasının bir resim gösterilir. Reaksiyon 25 ° C ve 150 rpm'de 30 dakika boyunca 100 mM sodyum asetat tampon, pH 4.5 gerçekleştirilmiştir. Ürün plakası beklendiği gibi artan enzim konsantrasyonu ile ilk single enzimler gelen CPH substrat (Şekil 4E) ve renkli süpernatant ile test edildi alındı ​​(Şekil 4F, satır 1A – 12D). Sıra E içeren CPH-ksilen ve mavi CPH-β-glukan endo -xylanase ve endo glükanaz ilgili enzimlerin farklı oranlarda bozulmuş iki farklı CPH substratlar san. Tepkimeden sonra, sonuç visib olduğule ürün plakasında: reaksiyon ürününün rengi koyu yeşil, mavi, daha endo glükanaz mevcut iken (Şekil 4F, 4E-6E) ve (endo -xylanase konsantrasyon arttıkça, daha hafif sarı-yeşil dönüştü Şekil 4F, 10-12E). Aynı iki substratlar kırmızı CPH-pektik galaktan ve sarı CPH-ksilan endo -galactanase ve endo -xylanase ile bozulmuş edildi satır F, görülür. Dört farklı renkli CPH alt-tabaka, bu (Şekil 4F, 1G-12F) ve tek enzimler, uygun CPH substrat bozulmuş ve ek ilave renkli reaksiyon ürünleri kombine alındı ​​enzimleri ile idi. Şekil 4. Farklı enzimler ile muamele, iki farklı CPH substrat kombinasyonu kırmızı CPH selüloz ve sarı CPH-ksilen. bir) Iki endo -cellulase (cel ile yüzeylerde) veya endo -xylanase (ksil) ya da her iki enzim. B) reaksiyon Süpernatantları absorbansı spektrumları tedavisinden sonra reaksiyon süpernatantlar. Farklı enzimler ile tedavi dört farklı CPH yüzeylerde kombinasyonu CPH substratlar içeren substrat plakasının C) Programı:. Mavi CPH-β-glukan (arpa), sarı CPH-ksilen (kayın), yeşil CPH-amiloz ve kırmızı CPH- pektik galaktan (bakla) farklı CPH substratlar içeren deney plakasının D) Resim eklendi enzimlerin oranını gösteren ürün plakası E) Programı (nominal konsantrasyonları (NC).:. glu = 1 U / ml endo glükanaz, = 1 U / ml endo -xylanase, amy = 5 U / ml endo-amilaz ve Gal = 0,5 U / ml endo -galactanase) xyl. ürün plakasının F) resmi 30 dakika inkübe edildikten sonra, 25 ° C'de. Bu rakamın büyük halini görmek için lütfen buraya tıklayınız. Yüzey Kaynak CPH-2-hidroksietilselüloz N / A (CPH-2-HE-selüloz) CPH-amilopektin patates CPH-amiloz patates CPH-arabinan şekerpancarı CPH-arabinoksilan buğday CPH-kazein sığır süt CPH-kitosan hayvansal kökenli CPH-kurdlan Alcaligenes faecalis CPH-dekstran Leuconostoc spp. </em> CPH-galaktomannan keçiboynuzu CPH-Laminarin Laminaria digitata CPH-lichenan İzlanda yosun CPH-metilselüloz N / A CPH-pachyman Poria cocos CPH-pektik galaktan patates CPH-pullulan Aureobasidium pullulans CPH-ramnogalakturonan I (RG I) 'in patates CPH-ramnogalakturonan I (Gal) * patates CPH-ramnogalakturonan soya fasulyesi CPH-ksilan kayın CPH-eksiloglukan demirhindi arpadan CPH-β-glukan arpa yulaftan CPH-β-glukan yulaf </td> mayadan CPH-β-glukan Maya ICB-Arabidopsis Rozet Arabidopsis yaprakları thaliana Col-0 (yetişkin bitki) ICB-Arabidopsis tohumları Arabidopsis thaliana ICB-küspe Saccharum officinarum (kurutulmuş yetişkin bitki, kök ve yaprak) ICB-kristalin selüloz (filtre kağıdı) Ticari Whatman 3MM Chr Kromatografi kağıdı ICB-çemen tohumu Trigonella spp. Tohumları ICB kendir Esrar spp. (Kurutulmuş yetişkin bitki, kök ve yaprak) ICB-acı bakla tohumu Lupinus tohum angustifolius ICB-polen P. pratense Phleum pratense polen ICB-ladin Picea spp. (Mimecburiyetine ağaç gövdesi) ICB tütün Nicotiana benthamiana yaprakları (fidan) ICB-buğday samanı Triticum spp. (Kurutulmuş yetişkin bitki, kök ve yaprak) ICB-söğüt Salix spp. (Kurutulmuş yetişkin bitki, öğütülmüş ağaç gövde) ICB-Sorgum Sorgum spp. (Yetişkin bitki yaprakları) * (çıkarıldı β-1,4-D-galaktan yan zincirler endo -β-1,4-D-Galaktanaz) Tablo 1. Mevcut Kromojenik Polimer Hidrojel (CPH) ve çözünmez Kromojenik Biyokütle (ICB) yüzeylerde listesi.

Discussion

Biz klorotriazin boyalar dayanmaktadır çok renkli CPH ve ICB yüzeylerde yeni nesil (Tablo 1 substratların tam listesi) kullanırken özel olarak tasarlanmış ticari tahlil kiti düzenlenmiş. Substratlann enzim sindirimi deney çözeltisi içinde tespit edilebilir ve bir plaka okuyucu 9 kullanılarak belirlenebilir küçük çözünür, boyanmış ürünleri elde edilir. Bu deney, endo -Oyunculuk enzimlerin değerlendirilmesi ve analiz duyarlılığı için tasarlanmış diğer yöntemler ekzo -Oyunculuk enzimler 11,12 için uygun iken Azurine çapraz bağlanmış (AZCL) kullanılarak bir 10 alt tabakalar benzerdir. CPH olarak ICB substratlar ekzo muhtemelen -enzymes tarafından da parçalanabilir olduğu gibi, bu deney kiti güçlüğü boyası ve çapraz bağlayıcı moleküller 8 kaynaklanan sterik engelleme ile endo -enzim aktivitesinin saptanması yer alır.

Tahlil bir 96-wel gerçekleştirilirl biçimi ve bireysel tepkiler kuyularda gerçekleşecek. Reaksiyon tekrarlanabilir verileri almak için plaka karıştırılmalıdır. Elde edilen süpernatanlar, her bir gözün absorbansı absorbans spektrometresi kullanılarak belirlenebilir ürün plakasına filtrelenmiştir. Temel ilkeler ve tahlilin düzeni deney alt-tabakalar ile bir deney plakasına (96 oyuklu filtre plakası) oluşur. Şekil 1 'de gösterilmektedir, ve enzimler ile inkübasyondan sonra, yüzer berrak oyuklu bir plakaya içinden süzülür ve absorbansları enzim özgüllük ve aktivite yarı kantitatif ölçümü sağlayan okunur. CPH substratlar kullanarak bu tarama deneyi, aynı zamanda çözünen reaksiyon ürünleri gece boyunca inkübasyondan sonra renkli bir hale oluşturarak bir agar plaka biçiminde kullanılabileceğini Bu gösterilmiştir. 8

gösterildiği gibi tahlil kitleri saflaştırılmış enzimler ve potansiyel yan aktivitelerini taranması için kullanılabilirŞekil 2,. Yan faaliyetlerinin tek bir enzim ve rasgele özgüllük değil, aynı zamanda analiz örneği, farklı enzimlerin bir karışımı ve bunların sinerjik etkisi incelenmesi gereken bir gerçeği ortaya çıkabilir. Bu önceki çalışmalarda gösterilmiştir Ek olarak, mantar 8 hem de endojen bitki enzimi ve bakteriler (yayınlanmamış veriler) gelen enzim kokteyl, bağırsak Mikrobiyota 13 ve kültür sıvılarında enzim kaynağı olarak kullanılabilir.

ICB substratların biyokütle arıza endüstriyel işlemlerde karşılaşılan hücre duvarı bileşenlerin kompleks karışımlarını arayabilir. Bu yüzeyler polisakkarit durumunu değerlendirmek ve etkin daha verimli bozulma çıkışı için bozulma kokteyller nasıl optimize hakkında bilgi sağlamak üzere tasarlanmıştır. Şekil l'de gösterilen gibi 3 CPH ve usulü üzerinden yüzeyler yan enzim tarama tarafında kullanılabilir – Enzim özgünlüğü ile ilgili bilgi zenginliği ortayaTercih edilen bir alt-tabaka (CPH) daha yakından doğa (ICB) bulunan bir makromoleküler düzeneği taklit tercih edilen alt-tabaka yanı sıra diğer bileşenleri içeren, daha doğal bir kompleksi içinde hem de D aktivitesi. Birden fazla renk kullanımı deneyinde yüksek verimlilik ve multiplexity arttırır çok substratlara karşı farklı enzim aktiviteleri eş zamanlı saptanması için izin verir. Farklı boyaların Spectra basit doğrusal regresyon ile ve çok substrat etkinliği tek başına görsel muayene ile görülebilir çoğu durumda çözülebilir. Bu tür bir deney ve sonuçlarının ardından bir örnek, Şekil 4'te gösterilmiştir.

Bu tahlil araç ve uygulama çok yönlülüğü çok iyi bilinmeyen faaliyetleri ile enzimler ve kültür sıvılarında ilk seviye taraması için uygundur. Bu testte en önemli yönleri, yüksek verimli yapısı, isteğe göre uyarlanabilirlik, kullanıcı ve esneklik kolaylığı vardır. akılda, w ileE araçları bu yeni seti büyük ölçüde iyileştirmek ve endüstriyel enzim tarama işlemleri yanı sıra akademik uygulamaları hızlandırmak inanıyoruz.

Disclosures

The authors have nothing to disclose.

Acknowledgements

Biz mükemmel teknik yardım için filme onun laboratuarlara erişim sağlanan Prof. J. Paul Knox (Leeds University, İngiltere), ve Susan E. Marcus teşekkür etmek istiyorum. JS WallTraC projesi (Avrupa Komisyonu Yedinci Çerçeve Programı (hibe anlaşması yok kabul. 263916) ve 21. yüzyıl için proje Biyokütle (Yenilik vakıf Danimarka, dava no .: 103408) SKK SET4Future projesi (Danimarka Stratejik Araştırma Kurumu) teşekkür ediyor, Stratejik Araştırmalar, Teknoloji ve Yenilik (Hibe Davası no .: 0603-00522B) Danimarka Konseyi Danimarka Konseyi tarafından kurulan Bio-Değer Stratejik ve Kompleks Polisakkarit Sistemleri (Hibe Case enzimatik Parçalanma Anlamak için Biyoloji odaklı yaklaşım yok .:. finansman için 107.279) Bu yazıda, sadece yazarların görüşlerini yansıtmaktadır Avrupa Birliği burada ihtiva bilgilerin herhangi bir şekilde kullanılmasından sorumlu değildir..

Materials

assay kit plates Glycospot customized assay kit plates
activation solution Glycospot for activating CPH substrates
350 ml receiver plate spacer block for vacuum manifold Pall Corporation 5015 spacer block
96-well MultiScreen HV filter plate, 0.45 µm, clear, non-sterile Millipore MSHVN4510 assay plate
96-Well Microplates, Polypropylene Greiner Bio-One 651201 collection plate after washing the substrates
Nunc™ MicroWell™ 96-Well Microplates Thermo Scientific 269620 product plate
Diaphragm pump MZ 2 NT Vacuubrand 732000 vacuum pump used with the vacuum manifold
Infors HT Ecotron Infors HT 4950132 (Buch & Holm) horizontal shaker
SpectraMax M5 Molecular Devices 10067-750 (VWR) 96-well plate absorbance reader
Vacuum manifold Pall Corporation 5017 vacuum manifold
endo-cellulase (EGII) (Trichoderma longibrachiatum) Megazyme E-CELTR cellulase [cel]
endo-β-1,4-mannanase (Cellvibrio japonicus)  Megazyme E-BMACJ mannanase [man]
endo-β-1,3-glucanase (Trichoderma spp.) Megazyme E-LAMSE β-glucanase [glu]
endo-b-1,4-D-galactanase (Aspergillus niger)  Megazyme E-EGALN galactanase [gal]
endo-β-1,4-xylanase M4 (Aspergillus niger)  Megazyme E-XYAN4 xylanase [xyl]
endo-xyloglucanase (GH5) (Paenibacillus sp.)  Megazyme E-XEGP xyloglucanase [xg]
α-amylase (Bacillus licheniformis)  Megazyme E-BLAAM amylase [amy]
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References

  1. Lombard, V., Ramulu, H. G., Drula, E., Coutinho, P. M., Henrissat, B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42 (Database issue), D490-D495 (2014).
  2. Cantarel, B. L., et al. The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res. 37, D233-D238 (2009).
  3. Agblevor, F. A., Murden, A., Hames, B. R. Improved method of analysis of biomass sugars using high-performance liquid chromatography. Biotechnol. Lett. 26 (15), 1207-1211 (2004).
  4. Black, G. E., Fox, A. Recent progress in the analysis of sugar monomers from complex matrices using chromatography in conjunction with mass spectrometry or stand-alone tandem mass spectrometry. J. Chromatogr. A. 720 (1-2), 51-60 (1996).
  5. Miller, G. L. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Anal. Chem. 31, 426-428 (1959).
  6. Somogyi, M. Notes on Sugar Determination. J. Biol. Chem. 195 (1), 19-23 (1952).
  7. Zantinge, J. L., Huang, H. C., Cheng, K. J. Microplate diffusion assay for screening of beta-glucanase-producing microorganisms. Biotechniques. 33 (4), 798 (2002).
  8. Kračun, S. K., et al. A new generation of versatile chromogenic substrates for high-throughput analysis of biomass-degrading enzymes. Biotechnol Biofuels. 8, (2015).
  9. Leemhuis, H., Kragh, K. M., Dijkstra, B. W., Dijkhuizen, L. Engineering cyclodextrin glycosyltransferase into a starch hydrolase with a high exo-specificity. J. Biotechnol. 103 (3), 203-212 (2003).
  10. Nyyssonen, M., et al. Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries. Front Microbiol. 4, 282 (2013).
  11. Sweeney, M. D., Xu, F. Biomass Converting Enzymes as Industrial Biocatalysts for Fuels and Chemicals: Recent Developments. Catalysts. 2 (2), 244-263 (2012).
  12. Biely, P., et al. Action of xylan deacetylating enzymes on monoacetyl derivatives of 4-nitrophenyl glycosides of beta-D-xylopyranose and alpha-L-arabinofuranose. J. Biotechnol. 151 (1), 137-142 (2011).
  13. Mackenzie, A. K., et al. A polysaccharide utilization locus from an uncultured bacteroidetes phylotype suggests ecological adaptation and substrate versatility. Appl Environ Microbiol. 81 (1), 187-195 (2015).

Tags

High-throughput ScreeningCarbohydrate-degrading EnzymesNovel Insoluble Chromogenic Substrate Assay KitsEnzyme DiscoveryBiomass DegradationChromogenic SubstratesEndo-cellulaseICB-wheat StrawAbsorbancePlate Reader

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Schückel, J., Kračun, S. K., Willats, W. G. T. High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits. J. Vis. Exp. (115), e54286, doi:10.3791/54286 (2016).
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