עבודה זו מומנה על ידי גורדון ובטי מור קרן, ועל ידי היוזמה המדע UNCF / מרק.

1. צלחת הקרנת הכנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוסף LB 25 גרם צמיחה בינוני, אגר 15g ו 1.5g carboxymethyl תאית (CMC) המצע במים מזוקקים עד 1 ליטר ו החיטוי לעקר. </li><li style=";text-align:right;direction:rtl"> לאחר מעוקר, לאפשר בינוני להצטנן ולהוסיף אנטיביוטיקה המתאימה IPTG לריכוז סופי של 100μM. </li><li style=";text-align:right;direction:rtl"> יוצקים לתוך כל אחד בינוני 200 מ&quot;ל 5 צלחות פטרי גדול מרובע / מגשים המבדק (240 x 240 x 20 מ&quot;מ או יותר). אפשר צלחות להתייבש לחלוטין. </li></ol> 2. Endoglucanase הדור ספריה מסך <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> יצירת ספרייה של תחום קטליטי endoglucanase (ים). ניתן להשיג בדרכים רבות (למשל, אתר מכוונת או mutagenesis רקומבינציה אקראית, וכו &#39;) נקבעת על ידי החוקר. </li><li style=";text-align:right;direction:rtl"> Clone הספרייה לתוך וקטור כזה כי הם תחת שליטתה של האמרגן T7 עם המפעיל לאק, ומכילים רצף האות pelB עבור המיקוד periplasm. דוגמה וקטור מתאים pET22b (+) מ Novagen. </li><li style=";text-align:right;direction:rtl"> המרה הספרייה endoglucanase לתוך E. coli זן BL21 (DE3) ו צלחת מושבות אחת. </li><li style=";text-align:right;direction:rtl"> דגירה שינוי בן לילה ב 37 ° C. </li><li style=";text-align:right;direction:rtl"> בעזרת קיסמים סטרילי, לחסן שיבוטים לתוך 96 צלחות היטב היטב עמוק המכיל מדיום LB 400μL בתוספת אנטיביוטיקה מתאימה. לדגור על 37 מעלות צלזיוס לילה עם רועד בסל&quot;ד 250. </li><li style=";text-align:right;direction:rtl"> הוסף גליצרול סטרילי עד 96 תרבויות לילה טוב לריכוז סופי של 10% עבור אחסון לטווח ארוך ב -80 ° C. זו תהיה צלחת מאסטר. </li><li style=";text-align:right;direction:rtl"> באמצעות חותמת 96 פינים, לשכפל תרבויות צלחת לילה על גבי צלחות ההקרנה המכיל IPTG ו CMC. עד 5 סטים של תרבויות לילה יכול להיות מוטבע על כל צלחת ההקרנה. </li><li style=";text-align:right;direction:rtl"> לייבל צלחות ההקרנה כך זהות והכיוון של הצלחת 96 כל טוב ידוע. </li><li style=";text-align:right;direction:rtl"> דגירה הספריות חותמת בטמפרטורת החדר (17-25 מעלות) למשך הלילה או עד מושבות גלוי יצרו. </li><li style=";text-align:right;direction:rtl"> לשטוף צלחת עם מים מזוקקים עד כל עקבות המושבות יוסרו. </li><li style=";text-align:right;direction:rtl"> יוצקים קונגו האדום (CR; 0.5% במים) לצלחת שטף. הקפד להוסיף CR מספיק כדי לכסות את הצלחת. </li><li style=";text-align:right;direction:rtl"> דגירה 15 דקות בטמפרטורת החדר. לא יעלה על 15 דקות הדגירה CR. פעמים הדגירה ארוכים תגרום הילות פחות ברורים. </li><li style=";text-align:right;direction:rtl"> יוצקים את CR ומוסיפים כמות נדיבה (יותר ממספיק כדי לכסות את צלחת) של NaCl 1M. </li><li style=";text-align:right;direction:rtl"> דגירה 15 דקות בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> יוצקים את NaCl לחשוף הילות השפלה CMC. לקבלת רזולוציה טובה יותר של הילות, פעמים הדגירה כבר עם מכבסת NaCl NaCl או מרובים ייתכן שתידרש. </li><li style=";text-align:right;direction:rtl"> בעזרת קיסם סטרילי, להרים פעיל שיבוטים אנזימים מהצלחת האב אפיון נוסף. </li></ol> 3. נציג תוצאות: דוגמה readout עבור תפוקה גבוהה במסך זה מוצג באיור 3. המשובטים ניתן לזהות פעיל, פעיל חלושות, ופעיל מאוד בהתבסס על גודל של אזורי השפלה. תכונה זו שימושית במיוחד אם אנזים הפעילות ידוע נכלל בספריה כהפניה. כפי שמוצג כאן, זיהוי של אנזימים פעילים איתנה לשחזור. עם זאת, יש להיות מודעים לכך תיוג נאותה של לוחות ההקרנה הוא מאוד חשוב. החוקר חייב להיות מסוגל לזהות גרסאות פעיל לאחר כביסה המושבות משם, על מנת לאסוף אותם מהצלחת האב מאוחסנים -80 ° C לאפיון נוסף. לבסוף, קשה להעריך מראש את הקשר בין פעילות על מצעים מלאכותיים מול טבעיים. לכן, החוקר חייב לבדוק את כל שיבוטים חיובית על חומר תעשייתי רלוונטי לקביעת כושר חלבון. <img src="/files/ftp_upload/2942/2942fig1.jpg" alt="איור 1"/> באיור 1. מתווה סכמטי של תהליך המיון. תאים הופכים עם ספריה endoglucanase ו מצופה סלקטיבי עבור מושבות אחת. המשובטים הרים גדל בן לילה לאחסון (1) ב -80 ° C ו (2) ציפוי העתק על צלחות המכילות CMC ו IPTG לגרום ביטוי endoglucanase ב 96 צלחות היטב עמוק היטב. שיבוטים פעילים נקטפים מהצלחת האב מאוחסנים -80 ° C. <img src="/files/ftp_upload/2942/2942fig2.jpg" alt="איור 2"/> איור 2. שיבוט וביטוי של תחומים קטליטי פטרייתי ב E. coli. (א) וקטור Endoglucanase הביטוי. הגנים היו משובטים לתוך pET22b (+) (Novagen) תחת שליטה של ​​האמרגן T7. (ב) פאנל ימין: endoglucanase-לבטא BL21 (DE3) המושבות. פאנל משמאל: הילות השפלה CMC לאחר שטיפה ופיתוח עם קונגו האדום. <img src="/files/ftp_upload/2942/2942fig3.jpg" alt="איור 3"/> איור 3. תוצאות המדגם מן המסך האדום CMC / קונגו. תמונות שצולמו של צלחות ההקרנה לאחר שטיפה ופיתוח קונגו האדום. כל מושבות על זהצלחות creening היו בסדר גודל דומה.

<ol>
	<li>Atsumi, S., Hanai, T., Liao, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-fermentative+pathways+for+synthesis+of+branched-chain+higher+alcohols+as+biofuels.">Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels.</a> Nature. 451, 86-89 (2008).</li><li>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=Cellulases+and+biofuels.">Cellulases and biofuels.</a> Curr Opin Biotechnol. 20, 295-299 (2009).</li><li>Qin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+endoglucanase+II+from+Trichoderma+reesei+to+improve+the+catalytic+efficiency+at+a+higher+pH+optimum.">Engineering endoglucanase II from Trichoderma reesei to improve the catalytic efficiency at a higher pH optimum.</a> J Biotechnol. 135, 190-195 (2008).</li><li>Nakazawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+endoglucanase+III+(Cel12A)+from+Trichoderma+reesei.">Directed evolution of endoglucanase III (Cel12A) from Trichoderma reesei.</a> Appl Microbiol Biotechnol. 83, 649-657 (2009).</li><li>Heinzelman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+family+of+thermostable+fungal+cellulases+created+by+structure-guided+recombination.">A family of thermostable fungal cellulases created by structure-guided recombination.</a> Proc Natl Acad Sci. 106, 5610-5615 (2009).</li><li>Heinzelman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCHEMA+recombination+of+a+fungal+cellulase+uncovers+a+single+mutation+that+contributes+markedly+to+stability.">SCHEMA recombination of a fungal cellulase uncovers a single mutation that contributes markedly to stability.</a> J Biol Chem. 284, 26229-26233 (2009).</li><li>Lantz, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypocrea+jecorina+CEL6A+protein+engineering.">Hypocrea jecorina CEL6A protein engineering.</a> Biotechnol Biofuels. 8, 3-20 (2010).</li><li>Mahadevan, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-directed+mutagenesis+and+CBM+engineering+of+Cel5A+(Thermotoga+maritima).">Site-directed mutagenesis and CBM engineering of Cel5A (Thermotoga maritima).</a> FEMS Microbiol Lett. 287, 205-211 (2008).</li><li>Nakazawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+catalytic+domains+of+Trichoderma+reesei+endoglucanase+I,+II,+and+III,+expressed+in+Escherichia+coli.">Characterization of the catalytic domains of Trichoderma reesei endoglucanase I, II, and III, expressed in Escherichia coli.</a> Appl Microbiol Biotechnol. 81, 681-689 (2008).</li><li>Wang, H., Jones, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+Macrophomina+phaseolina+endoglucanase+(EGL+1)+gene+product+in+bacterial+and+yeast+expression+systems.">Properties of the Macrophomina phaseolina endoglucanase (EGL 1) gene product in bacterial and yeast expression systems.</a> Appl Biochem Biotechnol. 81, 153-160 (1999).</li><li>Howard, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme+activity+of+a+Phanerochaete+chrysosporium+cellobiohydrolase+(CBHI.1)+expressed+as+a+heterologous+protein+from+Escherichia+coli.">Enzyme activity of a Phanerochaete chrysosporium cellobiohydrolase (CBHI.1) expressed as a heterologous protein from Escherichia coli.</a> Afr J Biotechnol. 2, 296-300 (2003).</li><li>Howard, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+a+chimeric+Phanerochaete+chrysosporium+cellobiohydrolase+expressed+from+Escherichia+coli.">Characterisation of a chimeric Phanerochaete chrysosporium cellobiohydrolase expressed from Escherichia coli.</a> Afr J Biotechnol. 3, 349-352 (2004).</li><li>Teather, R. M., Wood, 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=Use+of+Congo+red-polysaccharide+interactions+in+enumeration+and+characterization+of+cellulolytic+bacteria+from+the+bovine+rumen.">Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen.</a> Appl Env Microbiol. 43, 777-780 (1982).</li><li>Gilkes, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mode+of+action+and+substrate+specificities+of+cellulases+from+cloned+bacterial+genes.">Mode of action and substrate specificities of cellulases from cloned bacterial genes.</a> J Bio Chem. 259, 10455-10459 (1984).</li></ol>

אין ניגודי אינטרסים הכריז.

פרוטוקול המתואר כאן מאפשר זיהוי מהיר תפוקה גבוהה של אנזימים פעילים, עם מניפולציה מינימלית. איתור פעילות רגיש למדי, איכותית משקף את היקף הפעילות בתוך התא. קלות השימוש שעושה שיטה זו מתאימה למגוון רחב של ספריות האנזים, מוגבל רק על ידי ביטוי פונקציונלית E. coli. יתר על כ...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> מגיב </th><th> חברה </th><th> מספר מוצר </th></tr><tr><td> IPTG </td><td> סיגמא </td><td> I1284 </td></tr><tr><td> קונגו אדום </td><td> סיגמא </td><td> C6277 </td></tr><tr><td> Carboxymethyl תאית </td><td> סיגמא </td><td> 360384 </td></tr><tr><td> NaCl </td><td> סיגמא </td><td> S1679 </td></tr><tr><td> Bacto-agar </td><td> BD Biosciences </td><td> 214030 </td></tr><tr><td> המבדק צלחות </td><td> Thermo Scientific </td><td> 240845 </td></tr></tbody></table>

high throughput screening of fungal endoglucanase activity in escherichia coli

Cellulase אנזימים (endoglucanases, cellobiohydrolases, ו β-glucosidases) hydrolyze תאית לסוכרים רכיב, אשר בתורו יכול להיות מומר כהלים דלק 1. הפוטנציאל הידרוליזה אנזימטית של ביומסה cellulosic לספק אנרגיה מתחדשת הגביר את המאמצים מהנדס cellulases עבור 2 דלק חסכוני הייצור. עניין מיוחד הם cellulases פטרייתי 3-8, אשר כבר נמצאים בשימוש תעשייתי עבור מזונות ועיבוד טקסטיל. זיהוי וריאנטים פעיל בין ספריה של cellulases מוטציה הוא קריטי לתהליך ההנדסה; מוטנטים פעילים ניתן לבדוק נוספת תכונות משופרות ו / או נתון mutagenesis נוספים. הנדסה יעיל של cellulases פטרייתי כבר הקשו על ידי היעדר כלים גנטית אורגניזמים מקומיים על ידי קשיים בהבעת האנזימים המארחים Heterologous. לאחרונה, פיתח Morikawa ועמיתיו שיטה לביטוי ב E. coli התחומים הקטליטית של endoglucanases מ ח jecorina 3,9, פטריות תעשייתי חשוב עם יכולת להפריש cellulases בכמויות גדולות. פונקציונלית E. הביטוי coli כבר דווח על cellulases מן פטריות אחרים, כולל Macrophomina phaseolina 10 ו Phanerochaete chrysosporium 11-12. אנו מציגים שיטה ההקרנה תפוקה גבוהה של פעילות endoglucanase פטרייתי ב E. coli. (איור 1) שיטה זו משתמשת חיידקים המשותף צבען האדום קונגו (CR) כדי להמחיש השפלה אנזימטית של תאית carboxymethyl (CMC) על ידי תאים גוברת על בינוני מוצק. Assay פעילות דורשת ריאגנטים זולה, מניפולציה מינימלית, נותן תוצאות חד משמעיות כמו אזורי השפלה (&quot;הילות&quot;) במקום מושבה. למרות מדד כמותי של הפעילות האנזימטית לא ניתן לקבוע לפי שיטה זו, מצאנו כי גודל הילה בקורלציה עם הפעילות האנזימטית הכולל בתא. אפיון נוסף של שיבוטים חיובי הפרט יקבע, כושר חלבון יחסית. מסורתית חיידקי כל CMC / CR תא פעילות מבחני 13 לערב לשפוך CMC אגר המכיל על מושבות, אשר כפופה זיהום צולב, או תרבויות דוגרים בבארות אגר CMC, המהווה פחות נגישה ניסויים בקנה מידה גדול. כאן אנו מדווחים על פרוטוקול משופר משנה לשטוף שיטות קיימות 14 לפעילות cellulase: התאים גדלו על צלחות אגר CMC מוסרים לפני מכתים CR. פרוטוקול שלנו מפחית באופן משמעותי את זיהום צולבות היא מדרגית, המאפשר הקרנת מהירה של אלפי שיבוטים. בנוסף ח אנזימים jecorina, יש לנו הביע הוקרן וריאנטים endoglucanase מן aurantiacus Thermoascus ו Penicillium decumbens (שמוצג באיור 2), טוען כי פרוטוקול זה הוא ישים אנזימים מתוך מגוון רחב של אורגניזמים.

Watch this Scientific Journal Video about High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli at JoVE.com

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

אנו מתארים עלות נמוכה, גבוה שיטת התפוקה למסך לפעילות endoglucanase פטרייתי ב<em&gt; א coli.</em&gt; השיטה מסתמכת על readout ויזואלית פשוטה של ​​השפלה המצע, אינו דורש טיהור האנזים, והיא ניתנת להרחבה. זה מאפשר הקרנה מהירה של ספריות גדולות של וריאנטים של האנזים.

הקרנת תפוקה גבוהה של פעילות Endoglucanase פטרייתיים ב Escherichia coli</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

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High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

20.5K Views.  California Institute of Technology. אנו מתארים עלות נמוכה, גבוה שיטת התפוקה למסך לפעילות endoglucanase פטרייתי ב א coli. השיטה מסתמכת על readout ויזואלית פשוטה של ​​השפלה המצע, אינו דורש טיהור האנזים, והיא ניתנת להרחבה. זה מאפשר הקרנה מהירה של ספריות גדולות של וריאנטים של האנזים.

Video: High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

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.

הקרנת תפוקה גבוהה של אנזימים פחמימות-משפיל שימוש רומן מסיסים chromogenic המצע Assay ערכות

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

Biochemistry

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as&#160;fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

ביטוי של רקומביננטי Cellulase Cel5A מ<em&gt; Reesei Trichoderma</em&gt; בצמחים טבק

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

Environment

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

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

GENPLAT: פלטפורמת Automated Discovery עבור ביומסה אנזימים ואופטימיזציה קוקטייל

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

Bioengineering

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.

מסך התפוקה גבוהה עבור Biomining פעילות cellulase מספריות Metagenomic

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

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

הקרנה ירוקה פלורסנט מבוסס חלבון ביטוי של החלבונים ממברנה ב<em&gt; Coli Escherichia</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

תועלת רבה פנים של Co-ביטוי חלבון ב<em&gt; Coli Escherichia</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

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.

פלטפורמה הגבוהה הקרנת מודולרי האוטומטי תפוקת Exopolysaccharide בשילוב עם ניתוח רגיש מאוד טביעות אצבע פחמימות

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).

הקרנה רב-אנזים באמצעות מערכת הקרנת אנזים הגנטי תפוקה גבוהה

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

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

הקרנה תפוקה גבוהה של חלבון מבוססת ירושה cerevisiae ס

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

Insoluble Chromogenic Biomass-Based Enzyme Screening: An Assay to Evaluate the Degradation Activity of Plant Biomass-Degrading Enzymes

Source: Sch&#252;ckel, J., et al. <a href="https://www.jove.com/v/54286">High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits.</a> J. Vis. Exp. (2016).
This video demonstrates an assay to screen enzymes effective in degrading complex polysaccharides in plant biomass using insoluble chromogenic biomass or ICB. The enzymes hydrolyze ICB to release soluble dye-linked oligosaccharides, and the enzyme activity is assessed by colorimetric analysis of the reaction product.

בדיקת אנזימים כרומוגנית בלתי מסיסה מבוססת ביומסה: בדיקה להערכת פעילות הפירוק של אנזימים מפרקים ביומסה צמחית

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

הקרנת תפוקה גבוהה של פעילות Endoglucanase פטרייתיים ב Escherichia coli

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