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

这项工作是由（在InvestigaciónCientíficaBASICA在Investigación连接国界德拉西恩西亚82614，153998，253549和178359以及118），并从提高妇女地位总署德从理事会全国国立科学城ŸTECNOLOGIA - 墨西哥（国家科学技术委员会和墨西哥）资助项目Asuntos去个人Académico-国立自治大学墨西哥（DGAPA，墨西哥国立自治大学; PAPIIT IN202616，IN203612）。

1.克隆和过度表达结构基因的脂肪酶预测<ol><li>使用聚合酶链反应（PCR）14和特异性寡核苷酸（ 表1）15，扩增感兴趣（smc01003，smc00930或smc00171）的基因，预测为脂肪酶编码或磷脂酶，从宿主生物的基因组DNA（ 即， 苜蓿根瘤菌 ）。 <ol><li>引入特定的限制性位点（有寡核苷酸的设计的序列）。消化与相应的限制性内切酶的扩增的DNA片段...

<ol>
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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. 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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. 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在过去的20年中，许多生物基因组研究已经被测序，虽然丰富的基因组序列数据已经产生，功能解释相对滞后，因此阻碍了我们的基因组功能的认识。在基因组中的基因功能的基础上相似已知功能或保守的基序的发生基因经常被分配。然而，给定基因的精确功能往往不是公知的。尤其，预计酶的结构基因不能由组学技术容易地探索由于这样的事实，大部分酶催化涉及两个基板和两个产品的复杂的?...

基于甘油脂质，如三酰甘油和（甘油）磷脂构成了重要的可能是最知名的类脂质1。三酰基甘油（变量）的脂肪或油，其通常用作存储脂类，因此作为潜在的能量和碳的来源。标签可以通过脂肪酶，这是经常被生产菌分泌的消化外部变量并使它们可作为碳源退化。另外，脂肪酶已经被广泛地研究，多年来，由于其重要的生物技术应用2。 由于它们的两亲性质及其近圆筒状的形状，（甘油）磷脂展览成膜性，通常构成了双层膜3的主要类脂组分。在简单的微生物，如细菌大肠杆菌中，只有三个主要头组的变体，磷脂酰甘油（PG），心磷脂（CL），和phosphatidylethanolamine（PE）遇到，尽管人们应该知道，他们中的每一个都可以用在sn相当数量的不同脂肪酰基链的-1或SN -2位置引起大量不同分子种类4的被取代。其他细菌可能具有除了或代替其他磷脂。例如， 苜蓿中华根瘤菌 ，土壤细菌，其能够形成与豆科苜蓿（ 紫花苜蓿 ）一个固氮根瘤共生，包含除了PE的第二两性离子磷脂，磷脂酰胆碱（PC），5。另外，不含有脂质磷或甘油可能是细胞膜的两亲性并形成部分。例如，在磷限制生长条件下，在S根瘤菌 ，（甘油）磷脂主要由膜脂质不包含磷， 即，硫脂，鸟氨酸脂质和diacylglyceryl trimethylho取代moserine（DGTS）6。在细菌中，DGTS从二酰甘油（DAG）在一个两步通路7形成，但是对于DAG生成源不明确。脉冲追踪实验表明个人电脑可能是DGTS 8的前体，并使用在这个手稿，我们可以识别磷脂酶C（PLCP，SMc00171）中描述的方法，该方法是磷限制性的条件下形成并且可PC转换为DAG和磷酸8。 在另一项研究中，我们发现，酰基-CoA合成酶（FADD）S的缺陷型突变体进入生长9的固定相时的根瘤菌或大肠杆菌积累游离脂肪酸。尽管这些脂肪酸似乎是从膜脂质衍生自游离脂肪酸或解放出来的酶的确切来源没有已知的。再次，采用的策略在这个手稿概述，二patatin启动类10（磷）的脂肪酶（SMc00930和SMc01003），该对S中形成的游离脂肪酸贡献根瘤菌 11进行了预测。令人惊讶地，SMc01003使用的DAG作为底物将其转换为单酰甘油和最后甘油和游离脂肪酸11。因此，SMc01003是DAG脂肪酶（DGLA）。 虽然用于预测潜在（磷）存在一些算法脂酶12,13，它们的精确的功能和生理作用通常是不知道的。在这里，我们勾勒出一个协议，克隆和过度预测的或潜在的（磷）的脂肪酶。这份手稿解释酶检测如何开发和利用人工色底物的过度表达（磷）脂肪酶进行了优化。我们提供的例子，这些研究结果可能如何与优化的酶测定可遇到的实际（磷酸），酶底物，以及如何丰富我们的微生物生理学的理解。

<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

微生物产生的那些，以使可用于生物体外部基板分泌（磷）的脂酶广泛光谱。另外，其他（磷酸）脂肪酶可以在物理上与生产菌引起的内在脂质的营业额，并经常引起细胞膜的重塑有关。虽然势（磷）的脂肪酶可以用数目的算法时该基因/蛋白质序列可用来预测，经常没有获得的酶的活性，底物特异性，和潜在的生理功能的实验证据。这个手稿描述的测定条件与未知底物特异性，以及如何利用这些优化条件下在搜索用于相应（磷）的脂肪酶的天然底物准（磷）的脂肪酶的最优化。采用人工发色底物，如对硝基苯衍生物，可有助于检测小为在标准条件下的预测（磷）的脂肪酶的酶活性。具有遇到这样的次要的酶活性，酶测定法的不同参数可以在为了获得人造基底的更有效的水解而变化。已确定在其下的酶工作良好的条件之后，各种潜在天然底物应测定其降解，其可以采用不同的层析方法进行随后的处理。底物特异性为新酶的定义，通常提供假设这些酶，然后可通过实验测试的潜在生理作用。遵循这些指导原则，我们能够确定降解磷脂酰胆碱磷酸胆碱到甘油二酯和，在膜中生长时的磷限制条件细菌根瘤菌重塑的关键一步磷脂酶C（SMc00171）。两个预测patatin-就像有机体一样的磷脂酶（SMc00930和SMc01003），我们可以重新确定自己的底物特异性和澄清SMc01003是甘油二酯脂肪酶。

PC专用磷脂酶C SMc00171的活性的双- 对 硝基苯磷酸酯 从大肠杆菌获得的无细胞提取物大肠杆菌 BL21（DE3）×pLysS中，其中有smc00171表达，进行了研究它们对水解二- 对硝基苯基磷酸酯的能力，使用分光光度酶促测定法中，测量形成p -NP。没有水解活性是?...

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.

定义底物特异性的脂肪酶和磷脂酶考生

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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使用新型不溶性生色底物检测试剂盒碳水化合物降解酶的高通量筛选

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

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