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

이 작품은 (Investigación 엉 Fronteras 드 라 Ciencia에서 Investigación Científica BASICA에서 82614, 153998, 253549 및 178359뿐만 아니라 118) 및 Dirección 일반 드에서 Consejo 나시 오날 드를 CIENCIAS Y TECNOLOGIA - 멕시코 (CONACYT 멕시코)에서 보조금에 의해 지원되었다 (DGAPA - UNAM, PAPIIT IN202616, IN203612) Asuntos 드 개인 Académico-대학교 나시 오날 자치시 드 멕시코.

예상 리파제 1. 복제 및 과발현 구조 유전자 <ol><li> 중합 효소 연쇄 반응 (PCR) (14) 및 특정 올리고 뉴클레오티드 (표 1 참조) (15)를 사용하여, 관심 (smc01003, smc00930 또는 smc00171)의 유전자를 증폭 숙주 생물체의 게놈 DNA로부터, 리파아제에 대한 코드 또는 트리스 포스페이트 예측 (즉, , S. meliloti). <ol><li> (올리고 뉴클레오티드의 설계 순서?...

<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>

지난 20 년 동안, 많은 생물의 게놈은 서열화되었다 및 게놈 시퀀스 데이터의 재산이 생성되었지만, 기능 해석은 뒤쳐 따라서 게놈 기능에 대한 우리의 이해를 방해한다. 게놈에서 유전자의 기능은 종종 알려진 기능 또는 보존 모티브의 발생 유전자의 유사성을 기준으로 할당됩니다. 그러나, 특정 유전자의 정확한 기능은 대개 알려져 있지 않다. 특히, 효소 구조 유전자를 쉽게 인해 대부분의 효소...

이러한 트리 아실 글리세롤과 (글리세) 인지질 등의 글리세롤 기반의 지질이 중요한 구성하고 아마 가장 잘 알려진 지질 클래스 1. 트리 아실 글리세롤 (태그) 지방 또는 일반적으로 저장 지질의 기능 오일, 따라서 에너지 및 탄소 소스로합니다. 태그는 자주 외부 태그를 소화하고 탄소 소스로이를 사용할 수 있도록 생산 미생물에 의해 분비되는 리파제에 의해 저하 될 수 있습니다. 또한, 리파제 널리 그들의 중요한 생명 공학 응용 프로그램을 2 년 동안 연구되어왔다. 때문에 자신의 양친 매성 자연과 가까운 원통 모양, (글리세) 인지질 전시 막 형성 특성과 일반적으로 이중 층 막 3의 주요 지질 구성 요소를 구성합니다. 이러한 박테리아, 대장균, 세 개의 주요 그룹 헤드 변형, 포스파티딜 글리세롤 (PG), 카디오 리핀 (CL) 및 phosphatid 간단한 미생물에서하나는 그들 각자가 서로 다른 분자 종 (4)를 다수로 상기 SN의 다른 지방산 아실 사슬의 상당수 -1 SN -2 위치주는 증가로 치환 될 수 있음을 알아야하지만 ylethanolamine (PE)가 발생하는 . 다른 박테리아는 다른 인지질 외에 또는 대신이있을 수 있습니다. 예를 들어, Sinorhizobium의 meliloti은, 콩과 식물 알팔파 (Medicago 사티)와 질소 고정 뿌리혹 공생 관계를 형성 할 수있는 토양 박테리아, 제 양쪽이 온성 인지질, 포스파티딜콜린 (PC)를 PE 이외에 포함 오. 또한, 지질이 포함되지 인 또는 글리세롤은 세포막의 양친과 형태의 일부가 될 수 있습니다. 예를 들어, 인 - 제한 성장 조건에 따라, S.의 meliloti, (글리세) 인지질은 크게, 즉, sulfolipids, 오르니 틴 지질 및 diacylglyceryl의 trimethylho 인을 포함하지 않는 막 지질로 대체moserine (DGTS) 6. 박테리아, DGTS은 두 단계 경로 7에서 디아 실 글리세롤 (DAG)로 형성되지만 DAG 생성하기위한 소스는 명확하지 않았다된다. 펄스 - 체이스 실험은 PC가 인 제한 조건에서 형성된다 및 DAG 및 포스 포 콜린에 PC를 변환 할 수있는 DGTS 8 전구체하고 우리는 포스 포 리파제 C (PLCP, SMc00171)을 식별 할 수있는이 원고에 기술 된 방법을 사용하여 수 있다는 제안 8. 별도의 연구에서 우리는 발견이 아 닐 -CoA 합성 효소 (FADD) S.의 결핍 돌연변이 성장 9 정지상 들어갈 때 meliloti 또는 대장균 유리 지방산 축적. 이들 지방산은 세포막의 지질로부터 유도 될 듯하지만, 유리 지방산 또는이를 유리화하는 효소 (들)에 대한 정확한 소스는 공지되지 않았다. 다시 말하지만, 전략을 사용하는 것은이 원고에 설명 된 두 파타 틴과 같은 10 (포스) 리파제 (S.에서 유리 지방산의 형성에 기여하고 SMc00930 SMc01003) meliloti (11)는 예측했다. 놀랍게도 SMc01003는 글리세롤 및 유리 지방산 및 모노 아실 글리세롤 마지막 11로 변환 기판으로서 DAG를 이용했다. 따라서, SMc01003는 DAG 리파제 (DGLA)입니다. 알고리즘의 숫자가 전위 (포스)를 예측하는 존재하지만 12, 13는, 리파제 자신의 정확한 기능과 생리 학적 역할은 일반적으로 알려져 있지 않다. 여기에서 우리는 복제, 프로토콜 개요 및 예측 또는 전위 (포스) 리파제를 과발현. 이 원고 효소 분석법 개발 인공 발색 기질을 사용하여 과발현 (포스) 리파제에 최적화 할 수있는 방법을 설명한다. 우리는이 연구 결과는 미생물 생리학에 대한 우리의 이해를 풍부하게하는 방법을 최적화 효소 분석과 실제 (포스) 리파제 기판이 발생할 수있는 방법과 예제를 제공합니다.

<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

미생물 유기체의 외부 기판을 사용할 수 있도록하기 위해 분비된다 (포스) 리파제의 넓은 스펙트럼을 생산하고 있습니다. 또한, 다른 (포스) 리파제는 물리적으로 생산 유기체가 고유 지질의 매출을 일으키는 원인이 자주 세포막의 리모델링에 상승을 제공과 연관 될 수있다. 전위 (포스)은 리파제 유전자 / 단백질 서열 사용할 알고리즘의 번호와 예측 될 수 있지만, 효소 활성, 기질 특이성, 잠재적 생리 기능의 실험적 증거는 종종 수득되지 않았다. 이 원고는 알 수없는 기질 특이성하는 방법과 각각의 (포스) 리파제의 자연 기판에 대한 검색이 최적의 조건을 사용하는 방법과 미래 (포스) 리파제에 대한 분석 조건의 최적화에 대해 설명합니다. 미성년자를 감지하는 데 도움이, 같은 페이지의 -nitrophenyl 유도체 인공 발색 기판을, 수 사용표준 조건에서 예측 (포스) 리파제에 대한 효소 활성. 이러한 보조 효소 활성을 발생하는 데, 효소 분석의 고유 파라미터는 인공 기판의보다 효율적인 가수 분해를 얻기 위해 변화 될 수있다. 효소가 잘 작동되는 조건을 판정 한 후, 자연 전위 다양한 기판은 그들의 열화 구별 크로마토 그래피 방법을 이용 하였다 될 수있는 프로세스를 분석한다. 새로운 효소 기질 특이성의 정의는 종종 후 실험적으로 테스트 될 수있는 이러한 효소의 전위 생리적 역할에 대한 가설을 제공한다. 이 가이드 라인에 따라, 우리는 성장의 인 제한 조건에 따라 세균 Sinorhizobium의 meliloti에서 세포막의 리모델링을위한 중요한 단계에서, 포스 포 콜린과 디아 실 글리세롤하는 포스파티딜콜린을 저하 포스 포 리파제 C (SMc00171)을 식별 할 수 있었다. 두 patatin- 예측을 위해같은 유기체의 포스 포 (SMc00930 및 SMc01003)처럼, 우리는 그들의 기질 특이성을 재정의하고 SMc01003은 디아 실 글리세롤 리파제 있음을 명확히 할 수있다.

비스 P는 -nitrophenyl 인산와 PC 고유의 포스 C SMc00171의 활동 E. 얻은 무 세포 추출물 대장균 BL21 (DE3)는 smc00171가 형성된 P는 -NP을 측정하는 분광 효소 분석을 이용하여, 비스 페이지 -nitrophenyl 인산 에스테르를 가수 분해하는 ?...

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.

리파제 및 포스 후보자에 대한 기질 특이성을 정의

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

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Biochemistry

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