Name: defining substrate specificities for lipase and phospholipase candidates
Uploaded: 2016-11-23T12:30:00
Description: Watch this Scientific Journal Video about Defining Substrate Specificities for Lipase and Phospholipase Candidates at JoVE.com

Questo lavoro è stato sostenuto da sovvenzioni dal Consejo Nacional de Ciencias y Tecnología-Messico (CONACYT-Messico) (82614, 153998, 253549, e 178.359 in Investigación Científica Básica e 118 in Investigación en Fronteras de la Ciencia) e dalla Dirección General de Asuntos de Personal Académico-Universidad Nacional Autónoma de México (UNAM DGAPA-; PAPIIT IN202616, IN203612).

1. Clone e iperespressione del gene strutturale per lipasi prevista <ol><li> Utilizzando la reazione a catena della polimerasi (PCR) 14 e oligonucleotidi specifici (Tabella 1) 15, amplificare il gene di interesse (smc01003, smc00930 o smc00171), previsto per codificare per una lipasi o fosfolipasi, dal DNA genomico dell&#39;organismo ospite (ie , S. meliloti). <ol><li> Introdurre siti specifici di restrizione (con la sequenza creazione...

<ol>
	<li>Nelson, D. L., Cox, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lehninger, Principles of Biochemistry. , (2013).</li><li>Jaeger, K. E., Eggert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipases+for+biotechnology.">Lipases for biotechnology.</a> Curr. Opin. Biotechnol. 13 (4), 390-397 (2002).</li><li>Dowhan, W., Bogdanov, M., Mileykovskaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+roles+of+lipids+in+membranes.">Functional roles of lipids in membranes.</a> Biochemistry of Lipids, Lipoproteins and Membranes. , 1-37 (2008).</li><li>Dowhan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+for+membrane+phospholipid+diversity:+why+are+there+so+many+lipids?.">Molecular basis for membrane phospholipid diversity: why are there so many lipids?.</a> Annu. Rev. Biochem. 66, 199-232 (1997).</li><li>De Rudder, K. E. E., Thomas-Oates, J. E., Geiger, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhizobium+meliloti+mutants+deficient+in+phospholipid+N-methyltransferase+still+contain+phosphatidylcholine.">Rhizobium meliloti mutants deficient in phospholipid N-methyltransferase still contain phosphatidylcholine.</a> J. Bacteriol. 179, 6921-6928 (1997).</li><li>Geiger, O., R&#246;hrs, V., Weissenmayer, B., Finan, T. M., Thomas-Oates, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulator+gene+phoB+mediates+phosphate+stress-controlled+synthesis+of+the+membrane+lipid+diacylglyceryl-N,N,N-trimethylhomoserine+in+Rhizobium+(Sinorhizobium)+meliloti.">The regulator gene phoB mediates phosphate stress-controlled synthesis of the membrane lipid diacylglyceryl-N,N,N-trimethylhomoserine in Rhizobium (Sinorhizobium) meliloti.</a> Mol. Microbiol. 32 (1), 63-73 (1999).</li><li>Klug, R. M., Benning, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+enzymes+of+diacylglyceryl-O-4'-(N,N,N,-trimethyl)homoserine+biosynthesis+are+encoded+by+btaA+and+btaB+in+the+purple+bacterium+Rhodobacter+sphaeroides.">Two enzymes of diacylglyceryl-O-4'-(N,N,N,-trimethyl)homoserine biosynthesis are encoded by btaA and btaB in the purple bacterium Rhodobacter sphaeroides.</a> Proc. Natl. Acad. Sci. USA. 98 (10), 5910-5915 (2001).</li><li>Zavaleta-Pastor, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sinorhizobium+meliloti+phospholipase+C+required+for+lipid+remodeling+duringphosphorus+limitation.">Sinorhizobium meliloti phospholipase C required for lipid remodeling duringphosphorus limitation.</a> Proc. Natl. Acad. Sci. USA. 107 (1), 302-307 (2010).</li><li>Pech-Canul, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FadD+is+required+for+utilization+of+endogenous+fatty+acids+released+from+membrane+lipids.">FadD is required for utilization of endogenous fatty acids released from membrane lipids.</a> J. Bacteriol. 193 (22), 6295-6304 (2011).</li><li>Banerji, S., Flieger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patatin-like+proteins:+a+new+family+of+lipolytic+enzymes+present+in+bacteria?.">Patatin-like proteins: a new family of lipolytic enzymes present in bacteria?.</a> Microbiology. 150 (Pt 3), 522-525 (2004).</li><li>Sahonero-Canavesi, D. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid-releasing+activities+in+Sinorhizobium+meliloti+include+unusual+diacylglycerol+lipase.">Fatty acid-releasing activities in Sinorhizobium meliloti include unusual diacylglycerol lipase.</a> Environ. Microbiol. 17 (9), 3391-3406 (2015).</li><li>Fischer, M., Pleiss, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Lipase+Engineering+Database:+a+navigation+and+analysis+tool+for+protein+families.">The Lipase Engineering Database: a navigation and analysis tool for protein families.</a> Nucl. Acid. Res. 31 (1), 319-321 (2003).</li><li>Sigrist, C. J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+and+continuing+developments+at+PROSITE.">New and continuing developments at PROSITE.</a> Nucleic Acids Res. 41 (Database issue), D344-D347 (2013).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+Chain+Reaction:+Basic+Protocol+Plus+Troubleshooting+and+Optimization+Strategies.">Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies.</a> J. Vis. Exp. (63), e3998 (2012).</li><li>Untergasser, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer3+-+new+capabilities+and+interfaces.">Primer3 - new capabilities and interfaces.</a> Nucl. Acids Res. 40 (15), e115 (2012).</li><li>Studier, F. W., Rosenberg, A. H., Dunn, J. J., Dubendorff, J. W. <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+T7+RNA+polymerase+to+direct+expression+of+cloned+genes.">Use of T7 RNA polymerase to direct expression of cloned genes.</a> Methods Enzymol. 185, 60-89 (1990).</li><li>Miller, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Experiments in Molecular Genetics. , (1972).</li><li>Desjardins, P., Hansen, J. B., Allen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvolume+Protein+Concentration+Determination+using+the+NanoDrop+2000c+Spectrophotometer.">Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer.</a> J. Vis. Exp. (33), e1610 (2009).</li><li>Bligh, E. G., Dyer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+for+total+lipid+extraction+and+purification.">A rapid method for total lipid extraction and purification.</a> Can. J. Biochem. Physiol. 37 (8), 911-917 (1959).</li><li>Molecular Dynamics. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Phosphorimager SI User&#180;s Guide. , (1994).</li><li>Dixon, M., Webb, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Enzymes: Third Edition. , (1979).</li><li>Rudolph, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression,+characterization,+and+mutagenesis+of+the+Yersinia+pestis+murine+toxin,+a+phospholipase+D+superfamily+member.">Expression, characterization, and mutagenesis of the Yersinia pestis murine toxin, a phospholipase D superfamily member.</a> J. Biol. Chem. 274 (17), 11824-11831 (1999).</li><li>Kato, S., Yoshimura, T., Hemmi, H., Moriyama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+analysis+of+a+novel+lipolytic+enzyme+YvdO+from+Bacillus+subtilis.">Biochemical analysis of a novel lipolytic enzyme YvdO from Bacillus subtilis.</a> Biosci. Biotechnol. Biochem. 74 (4), 701-706 (2010).</li><li>Peppelenbosch, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinome+profiling.">Kinome profiling.</a> Scientifica (Cairo). , (2012).</li><li>Manafi, M., Kneifel, W., Bascomb, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorogenic+and+chromogenic+substrates+used+in+bacterial+diagnostics.">Fluorogenic and chromogenic substrates used in bacterial diagnostics.</a> Microbiol. Rev. 55 (3), 335-348 (1991).</li><li>Kuznetsova, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme+genomics:+Application+of+general+enzymatic+screens+to+discover+new+enzymes.">Enzyme genomics: Application of general enzymatic screens to discover new enzymes.</a> FEMS Microbiol. Rev. 29 (2), 263-279 (2005).</li><li>Scopes, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protein Purification, Principles and Practice. , (2010).</li><li>Geiger, O., Lopez-Lara, I. M., Sohlenkamp, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylcholine+biosynthesis+and+function+in+bacteria.">Phosphatidylcholine biosynthesis and function in bacteria.</a> Biochim. Biophys. Acta. 1831 (3), 503-513 (2013).</li></ol>

Nel corso degli ultimi 20 anni, genomi di molti organismi sono stati sequenziati e anche se una ricchezza di dati sequenza del genoma è stato generato, interpretazione funzionale è in ritardo e ostacola quindi la nostra comprensione della funzione del genoma. funzioni geniche in genomi sono spesso assegnati in base alla somiglianza con geni di funzione nota o di insorgenza di motivi conservati. Tuttavia, la precisa funzione di un dato gene spesso non è noto. Soprattutto, previsto geni strutturali per enzimi non posso...

Lipidi glicerolo-based come trigliceridi e fosfolipidi (glycero) costituiscono importanti e probabilmente il più noti classi di lipidi 1. Trigliceridi (tag) sono grassi o oli, che di solito fungono da lipidi di stoccaggio, e quindi come potenziali fonti di energia e di carbonio. TAG possono essere degradati dalla lipasi, che sono spesso secrete dall&#39;organismo produzione di digerire variabili esterne e renderli disponibili come fonti di carbonio. Inoltre, lipasi sono stati ampiamente studiati negli anni, grazie alle loro importanti applicazioni biotecnologiche 2. A causa della loro natura anfifilica e la loro forma quasi cilindrica, proprietà di membrana formante (glycero) fosfolipidi esporre e generalmente costituiscono i principali componenti lipidici di membrana doppo strato 3. In microrganismi semplici, come il batterio Escherichia coli, solo tre varianti principali del gruppo testa, fosfatidilglicerolo (PG), cardiolipina (CL), e phosphatidylethanolamine (PE) è riscontrabile, anche se si deve essere consapevoli che ciascuno di essi può essere sostituito con un considerevole numero di differenti catene grassi acil al sn -1 o sn posizione -2 dando luogo ad un grande numero di differenti specie molecolari 4 . Altri batteri possono avere altri fosfolipidi in aggiunta o al posto. Ad esempio, Sinorhizobium meliloti, un batterio del suolo, che è in grado di formare una radice nodulo simbiosi azotofissatrice con l&#39;erba medica legume (Medicago sativa), contiene in aggiunta al PE un secondo fosfolipide zwitterionico, fosfatidilcolina (PC) 5. Inoltre, i lipidi non contenenti fosforo o glicerolo potrebbe essere parte anfifilico e la forma della membrana cellulare. Ad esempio, dalle condizioni di crescita fosforo limitativo, in S. meliloti, (glycero) fosfolipidi sono in gran parte sostituiti da lipidi di membrana che non contengono fosforo, cioè, sulfolipids, lipidi ornitina, e diacylglyceryl trimethylhomoserine (DGTS) 6. Nei batteri, DGTS è formata da diacilglicerolo (DAG) in un percorso in due fasi 7, ma la fonte per la generazione di DAG non era chiaro. Esperimenti di pulse-chase suggerito che il PC potrebbe essere un precursore per DGTS 8 e utilizzando la metodologia descritta in questo manoscritto abbiamo potuto identificare un fosfolipasi C (PLCP, SMc00171) che si forma in condizioni di fosforo-limitante e che può convertire il PC in DAG e phosphocholine 8. In uno studio separato, abbiamo scoperto che un sintetasi acil-CoA (FADD) mutante -carente di S. meliloti o di Escherichia coli accumulato acidi grassi liberi entrando fase stazionaria della crescita 9. Sebbene questi acidi grassi sembravano essere derivato da lipidi di membrana, la fonte precisa per gli acidi grassi liberi o l&#39;enzima (s) liberandoli non erano noti. Anche in questo caso, utilizzando la strategia delineata in questo manoscritto, due 10 (fosfo) lipasi Patatin-like (SMc00930 e SMc01003), che ha contribuito alla formazione di acidi grassi liberi a S. meliloti 11 sono stati previsti. Sorprendentemente, SMc01003 utilizzato DAG come substrato convertirlo monoacilglicerolo e infine glicerolo e acidi grassi liberi 11. Pertanto, SMc01003 è una lipasi DAG (DGLA). Sebbene un certo numero di algoritmi esistenti per prevedere il potenziale (fosfo) lipasi 12,13, la loro funzione precisa e ruolo fisiologico non è generalmente noto. Qui descriviamo un protocollo, per clonare e iperespressione di lipasi (fosfo) previsti o potenziali. Questo manoscritto spiega come saggi enzimatici possono essere sviluppati e ottimizzati per la (fosfo) lipasi sovraespresso utilizzando substrati cromogeni artificiali. Forniamo esempi di come con un metodo enzimatico ottimizzato il vero (fosfo) substrato lipasi può essere incontrato e di come queste scoperte potrebbero arricchire la nostra comprensione della fisiologia microbica.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chloroform</td>
			<td>JT Baker</td>
			<td>9180-03</td>
			<td>TLC analysis &#38; Lipid extraction</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>JT Baker</td>
			<td>9070-03</td>
			<td>TLC analysis &#38; Lipid extraction</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>JT Baker</td>
			<td>9507-05</td>
			<td>TLC analysis &#38; Lipid extraction</td>
		</tr>
		<tr>
			<td>Hexanes</td>
			<td>JT Baker</td>
			<td>9309-02</td>
			<td>TLC analysis &#38; Lipid extraction</td>
		</tr>
		<tr>
			<td>Diethylether</td>
			<td>Sigma</td>
			<td>32203</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>bidistilled water</td>
			<td>&#160;ANY</td>
			<td>&#160;NA</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Sigma</td>
			<td>T-1503</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Baker</td>
			<td>9535-02</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Baker</td>
			<td>3624-01</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X-100</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>ANY</td>
			<td>NA</td>
			<td>Bacterial growth, 10g tryptone + 5g yeast extract + 10g NaCl per liter of bidistilled water</td>
		</tr>
		<tr>
			<td>tryptone</td>
			<td>Becton Dickinson and Company</td>
			<td>211705</td>
			<td>Bacterial growth</td>
		</tr>
		<tr>
			<td>yeast extract</td>
			<td>Becton Dickinson and Company</td>
			<td>212750</td>
			<td>Bacterial growth</td>
		</tr>
		<tr>
			<td>TY broth</td>
			<td>ANY</td>
			<td>NA</td>
			<td>Bacterial growth, 8g tryptone + 3g yeast extract + 66mg CaCl2 2 H20 per liter of bidistilled water</td>
		</tr>
		<tr>
			<td>CaCl2 2 H2O</td>
			<td>Baker</td>
			<td>1332-01</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>isopropyl-&#946;-D-thiogalactoside (IPTG)</td>
			<td>Invitrogen</td>
			<td>15529-019</td>
			<td>Bacterial growth</td>
		</tr>
		<tr>
			<td>Diethanolamine</td>
			<td>Sigma</td>
			<td>D-8885</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>MnCl2</td>
			<td>Sigma</td>
			<td>221279</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>Phospholipase A2 snake venom</td>
			<td>Sigma</td>
			<td>P0790</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>Phospholipase C Clostridium perfingrens</td>
			<td>Sigma</td>
			<td>P7633</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>Bis-p-nitrophenyl phosphate</td>
			<td>Sigma</td>
			<td>07422AH</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>p-nitrophenyl stearate</td>
			<td>Sigma</td>
			<td>N3627</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>p-nitrophenyl dodecanoate</td>
			<td>Sigma</td>
			<td>61716</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>p-nitrophenyl decanoate</td>
			<td>Sigma</td>
			<td>N0252</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>p-nitrophenyl palmitate</td>
			<td>Sigma</td>
			<td>N2752</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>p-nitrophenyl butyrate</td>
			<td>Sigma</td>
			<td>N9876</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>p-nitrophenyl octanoate</td>
			<td>Sigma</td>
			<td>21742</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>Acetic Acid, sodium salt [1-14C]</td>
			<td>Perkin Elmer</td>
			<td>NEC084</td>
			<td>Bacterial growth</td>
		</tr>
		<tr>
			<td>dimethylsulfoxide (DMSO)</td>
			<td>JT Baker</td>
			<td>9224-01</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>Aluminium HPTLC silica gel 60 plates. Silica gel HPTLC plates size 20 x 20 cm, 25 sheets.</td>
			<td>Merck</td>
			<td>105547</td>
			<td>TLC analysis &#38; Lipid extraction</td>
		</tr>
		<tr>
			<td>Spectrometer UV/VIS Lambda 35</td>
			<td>Perkin Elmer</td>
			<td>NA</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>Storm 820 Phosphorimager</td>
			<td>Molecular Dynamics</td>
			<td>NA</td>
			<td>Photostimulable Luminescence scanner&#160;</td>
		</tr>
		<tr>
			<td>Multipurpose Scintillation Counter</td>
			<td>Beckman Coulter</td>
			<td>NA</td>
			<td>Radioactivity Quantification</td>
		</tr>
		<tr>
			<td>French Pressure Cell</td>
			<td>ThermoSpectronic</td>
			<td>NA&#160;</td>
			<td>Breakage of cells</td>
		</tr>
		<tr>
			<td>chromatography paper 3MM Chr</td>
			<td>Whatman</td>
			<td>3030917</td>
			<td>TLC analysis</td>
		</tr>
		<tr>
			<td>Sinorhizobium meliloti 1021our</td>
			<td>reference 34</td>
			<td></td>
			<td>studied strain</td>
		</tr>
		<tr>
			<td>Escherichia coli BL21 (DE3) pLysS Competent cells</td>
			<td>Novagen</td>
			<td>69451</td>
			<td>protein expression strain</td>
		</tr>
		<tr>
			<td>pET9a vector</td>
			<td>Novagen</td>
			<td>69431</td>
			<td>protein expression vector</td>
		</tr>
		<tr>
			<td>pET17b vector</td>
			<td>Novagen</td>
			<td>69663</td>
			<td>protein expression vector</td>
		</tr>
		<tr>
			<td>sterile polystyrene round-bottom tube (14 ml) Falcon</td>
			<td>Becton Dickinson</td>
			<td>352057</td>
			<td>radiolabeling of bacterial cultures</td>
		</tr>
		<tr>
			<td>polypropylene microcentrifuge tubes (1.5 ml)</td>
			<td>Eppendorf</td>
			<td>30125.15</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>1,2-dipalmitoyl-sn-glycerol</td>
			<td>Sigma</td>
			<td>D9135</td>
			<td>lipid standard</td>
		</tr>
		<tr>
			<td>L-&#945;-phosphatidylcholine, dipalmitoyl</td>
			<td>Sigma</td>
			<td>P6267</td>
			<td>lipid standard</td>
		</tr>
		<tr>
			<td>DL-&#945;-monopalmitin</td>
			<td>Sigma</td>
			<td>M1640</td>
			<td>lipid standard</td>
		</tr>
		<tr>
			<td>palmitic acid</td>
			<td>Sigma</td>
			<td>P0500</td>
			<td>lipid standard</td>
		</tr>
	</tbody>
</table>

defining substrate specificities for lipase and phospholipase candidates

I microrganismi producono un ampio spettro di lipasi (fosfo) che vengono secreti per rendere substrati esterni disponibili per l&#39;organismo. In alternativa, altri (fosfo) lipasi possono essere fisicamente associate con l&#39;organismo producendo causando un turnover di lipidi intrinseche e spesso dando origine ad un rimodellamento delle membrane cellulari. Sebbene potenziali (fosfo) lipasi possono essere previsti con un certo numero di algoritmi quando la sequenza del gene / proteina è disponibile, prova sperimentale delle attività enzimatiche, specificità di substrato e potenziali funzioni fisiologiche è spesso non è stata ottenuta. Questo manoscritto descrive l&#39;ottimizzazione delle condizioni di analisi per i potenziali (fosfo) lipasi con specificità di substrato sconosciute e come impiegare queste condizioni ottimali nella ricerca per il substrato naturale di un rispettivo (fosfo) lipasi. Usando substrati cromogeni artificiali, come i derivati p -nitrophenyl, può aiutare a rilevare un minoreattività enzimatica per un (fosfo) lipasi previsto in condizioni standard. Avendo riscontrato un attività enzimatica tale minore, i parametri distinti di un saggio enzimatico possono essere variate per ottenere un&#39;idrolisi più efficiente del substrato artificiale. Dopo aver determinato le condizioni in cui un enzima funziona bene, una varietà di potenziali substrati naturali devono essere analizzati per la loro degradazione, un processo che può essere seguita con metodi cromatografici distinti. La definizione di specificità di substrato per nuovi enzimi, fornisce spesso ipotesi per un potenziale ruolo fisiologico di questi enzimi, che poi possono essere testati sperimentalmente. Seguendo queste linee guida, siamo stati in grado di identificare una fosfolipasi C (SMc00171) che degrada fosfatidilcolina di phosphocholine e diacylglycerol, in una fase cruciale per il rimodellamento delle membrane nel batterio Sinorhizobium meliloti dalle condizioni di fosforo-limitante della crescita. Per due predetto patatin-come fosfolipasi (SMc00930 e SMc01003) dello stesso organismo, potremmo ridefinire le loro specificità di substrato e chiarire che SMc01003 è una lipasi diacilglicerolo.

Attività di specifici PC fosfolipasi C SMc00171 con Bis- p -nitrophenyl fosfato Estratti privi di cellule ottenute da E. coli BL21 (DE3) pLysS x, che avevano smc00171 espressi, sono stati studiati per la loro capacità di idrolizzare bis- p esteri -nitrophenyl fosfato, utilizzando un saggio enzimatico spettrofotometrica, misurando la -NP <e...

Watch this Scientific Journal Video about Defining Substrate Specificities for Lipase and Phospholipase Candidates at JoVE.com

Defining Substrate Specificities for Lipase and Phospholipase Candidates

Many predicted (phospho)lipases are poorly characterized with regard to their substrate specificities and physiological functions. Here we provide a protocol to optimize enzyme activities, search for natural substrates, and propose physiological functions for these enzymes.

La definizione del substrato Specificità per lipasi e fosfolipasi candidati

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. ...

The overall goal of this procedure is to accelerate the detection of physiological substrates for potential lipases and phosopholipases. This method can help answer key questions in the Entomology field, such as how phospholipases contribute to lipid remodeling in biological membranes. The main advantage of this technique is that the optimization of enzymatic activity and the search for the natural substrate for the enzyme can be performed independently.Though this method can provide insight into substrate specificity of bacterial lipases and phospholipases, it can also be modified to study hydrolases of any other model organisms. We first had the idea for this method when we searched for an enzyme activity that could degrade the intrinsic membrane lipid phosphatidylcholine inorhizobium meliloti. To prepare cell-free protein extracts, defrost bacterial cell suspensions and store them on ice.Then, pass the cell suspensions three times through a cold pressure cell at 20, 000 pounds per square inch. Remove the in-tact cells and cell debris by centrifugation at 5, 000 times g for 30 minutes, at four degrees Celsius. After centrifugation, prepare aliquots of 100 and 500 microliters from the supernatant for subsequent analysis.Set up the previously-optimized enzyme assay using the pipetting schemes tabulated in the text protocol. For an initial coverage of distinct enzyme activities, use artificial substrates that yield a colored product upon hydrolysis, such as para-nitrophenol or PNP derivatives. Follow the time course for an increase of absorbance at 405 nanometers, due to the formation of PNP in a spectrophotometer at 30 degrees Celsius over a five-minute period.Finally, perform calculations as described in the text protocol. For radio labeling of lipids, prepare an overnight pre-culture of an organism of interest in five milliliters of the desired culture medium and grow at 30 degrees Celsius. From the pre-culture, inoculate into 20 millimeters of fresh medium in a 100-millimeter culture flask, to obtain an initial optical density at 620 nanometers of 0.3 for the culture.Next, transfer a one-milliliter aliquot of the culture to a 14-milliliter sterile polystyrene round bottom tube under sterile conditions. Add one microcurie of 114 c acetate to the one-milliliter culture. Then, incubate the liquid culture under agitation at 30 degrees Celsius for a period of 24 hours.At the end of the incubation period, transfer the culture to a 1.5 milliliter microcentrifuge tube and centrifuge at 12, 000 times g at room temperature for five minutes. Resuspend the pellet in 100 microliters of water. At this point, store the cell suspension at minus 20 degrees Celsius or immediately continue with the extraction of polar lipids.To perform the extraction of polar lipids, add 375 microliters of a two to one methanol to chloroform solution to the 100 microliters of aqueous cell suspension. Vortex the sample for 30 seconds before incubating for five minutes at room temperature. Following incubation, centrifuge for five minutes at 12, 000 times g at room temperature.Then, transfer the supernatant to a new 1.5 milliliter microcentrifuge tube. Next, add 125 microliters of chloroform and 125 microliters of water to the supernatant. After vortexing for 30 seconds, centrifuge the sample for one minute at 12, 000 times g at room temperature.Transfer the lower chloroform phase to a fresh tube. Then, dry the solution with a stream of nitrogen gas. Dissolve the dried lipids in 100 microliters of chloroform to methanol solution.For separation of neutral polar lipids by thin layer chromatography, apply three microliter aliquots of the lipid samples, on a high performance thin-layer chromatography silica gel aluminum sheet, starting two centimeters from the edges of the plate. If multiple samples are analyzed in one-dimensional chromatography, keep a distance of at least 1.5 centimeters between the different sample application spots. Transfer the mobile phase to a TLC developing chamber, internally coated with chromatography paper.Cover with a glass plate to let the chamber saturate for 30 minutes. Transfer the HPTLC Silica Gel aluminum sheet with the dried lipid samples to the chamber. Allow the plate to develop for 30 minutes in a closed chamber before removing the plate.Then, let the solvents dry off in a flow hood for two hours. Once the developed TLC sheet is dry, incubate it with a photostimulable luminescence screen in a closed cassette for three days. Then, expose the incubated screen to a PSL scanner and acquire a virtual image of the separated radio-labeled lipids.To determine diacylglycerol, or DAG, lipase activity, first add 5, 000 CPM of 14 C-labeled DAG to a 1.5 milliliter microcentrifuge tube. Also, add a solution of Triton X-100 to the tube and mix by vortex. Dry the solution under a stream of nitrogen.For a final 100 microliter assay, add Tris-HCl buffer, a sodium chloride solution, and bi-distilled water. Vortex for five seconds. Initiate the reaction by adding five microliters of enzyme to the solution.Incubate the solution at 30 degrees Celsius for four hours. Stop the reaction by the addition of 250 microliters of methanol and 125 microliters of chloroform, before extracting lipids as described earlier in this video. Proceed to analyze neutral polar lipids by 1DTLC and subsequent PSL imaging as before.Although the predicted phosopholipase SMc01003 shows activity with artificial para-nitrophenol acyl esters, the activity is only double or triple of the background activity present in cell-free extracts of Escherichia coli, in which no foreign gene had been expressed. This indicates that para-nitrophenyl acyl esters don't seem to be good substrates for SMc01003. Whereas cell-free extracts of E.Coli cannot degrade diacylglycerol, extracts of strains in which the predicted phospholipase, SMc01003, had been expressed, caused a degradation of diacylglycerol.Though SMc01003 was predicted to be a phospholipase, it is actually a diacylglycerol lipase that degrades diacylglycerol to monoacylglycerol and one fatty acid. It then degrades monoacylglycerol further to glycerol and another fatty acid. Once mastered, this technique can be done in a few weeks if it's performed properly.After watching this video, you should have a good understanding of how to optimize enzyme activities of lipases and phospholipases, and how to continue with the search for their physiological substrates. A crucial advantage of this procedure is that the optimization of the enzyme activity can be separated from the search for the natural enzyme substrate. Don't forget that working with organic solvents or radioactive substances can be extremely hazardous and the required precautions should always be taken while performing this procedure.

Research

JoVE Journal

Biochemistry

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

High-throughput screening degli enzimi carboidrati di degradazione Utilizzando Novel insolubile Chromogenic Substrate Assay Kit

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

Ricerca

Biochimica

A Rapid Laser Probing Method Facilitates the Non-invasive and Contact-free Determination of Leaf Thermal Properties

Un laser Rapid Probing Metodo Facilita la determinazione non invasiva e senza contatto di Leaf Proprietà termiche

Plants can produce valuable substances such as secondary metabolites and recombinant proteins. The purification of the latter from plant biomass can be ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Caratterizzazione biochimica e strutturale del carboidrato trasporto substrato-proteina-SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

Definizione attività di Chaperone Redox-regolati di Hsp33 e mappatura cambiamenti conformazionali su Hsp33 usando la spettrometria totale di scambio di idrogeno-deuterio

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

Rilevamento di attività della fosfolipasi C in omogeneato del cervello dall'ape

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Misurare e interpretare i tassi di consumo di ossigeno in tutto Vola testa segmenti

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein

KRAS4b ricombinante completamente elaborato: isolamento e caratterizzazione delle proteine di Farnesylated e Metylated

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Analisi metabolomica a base di cromomia liquida non mirata del grano di grano di grano

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

Visualization and Quantification of TGF&#946;/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay

Visualizzazione e quantificazione della segnalazione TGFβ/BMP/SMAD in diverse condizioni di sforzo di taglio del fluido utilizzando il test di prossimità-legatura

Transforming Growth Factor &#946; (TGF&#946;)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and ...