Defining Substrate Specificities for Lipase and Phospholipase Candidates

1. Clone and Overexpress Structural Gene for Predicted Lipase

1. Using polymerase chain reaction (PCR)¹⁴ and specific oligonucleotides (Table 1)¹⁵, amplify the gene of interest (smc01003, smc00930, or smc00171), predicted to code for a lipase or phospholipase, from the genomic DNA of the host organism (i.e., S. meliloti).
   1. Introduce specific restriction sites (with the designed sequence of the oligonucleotides). Digest the amplified DNA fragment with the corresponding restriction enzymes and clone it into an expression vector such as plasmids of the pET series¹⁶.
   2. After verifying the correct DNA sequence for the cloned gene, transform the vector to an expression strain such as Escherichia coli BL21(DE3) pLysS¹⁶.
2. Prepare an overnight pre-culture of the expression host E. coli BL21(DE3) pLysS, harboring the respective pET vector with the cloned gene or the empty vector, in 100 ml culture flasks containing 20 ml of Luria Bertani broth (LB)¹⁷ plus the required antibiotics. Culture the cells at 30 °C (or at the usual growth temperature of the bacterium from which the lipase originates).
   1. Using the overnight pre-cultures, inoculate 500 ml of prewarmed LB medium (plus the required antibiotics) in 2 L culture flasks to obtain an initial optical density at 620 nm (OD₆₂₀) = 0.05. Follow growth of cultures and at an OD₆₂₀ = 0.3, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 100 µM, and incubate under agitation at 30 °C for a period of 4 hr.
   2. At the end of the incubation period, transfer each culture to a 500 ml centrifuge tube and centrifuge at 5,000 x g at 4 °C for 30 min. Resuspend bacterial cell pellets in 5 ml of suspension buffer (e.g., SMc00930- and SMc01003-expressing cells in 50 mM Tris-HCl pH 8.0 and SMc00171-expressing cells in 50 mM diethanolamine-HCl pH 9.8). Store the cell suspensions at -80 °C until use.

2. Prepare Cell-free Protein Extracts and Determine Protein Concentration

1. Defrost bacterial cell suspensions and store on ice. Pass cell suspensions three times through a cold pressure cell at 20,000 lb per in². Remove intact cells and cell debris by centrifugation at 5,000 x g for 30 min at 4 °C.
2. After centrifugation, prepare aliquots of 100 and 500 µl from the supernatant for subsequent analysis and store them at -80 °C until use.
3. Use one of the 100 µl aliquot to determine protein concentration of distinct cell-free extracts by a method of choice or as described¹⁸.

3. Use Artificial Substrates for Optimizing Enzyme Activities of (Phospho)lipases

1. For an initial coverage of distinct enzyme activities, use artificial substrates that yield a colored product upon hydrolysis, such as p-nitrophenol (p-NP).
   1. For enzyme assays optimized already with artificial p-nitrophenyl ester substrates (outlined for phospholipase C PlcP (SMc00171), as well as for the predicted patatin-like phospholipases SMc00930 and SMc01003), use pipetting schemes described in Table 2.
   2. When exploring a new potential (phospho)lipase, prepare a first standard enzyme assay containing 50 mM Tris-HCl, pH 8.5, 100 mM NaCl, 0.05% Triton X-100, 0.5 mM p-nitrophenyl-containing compound (p-nitrophenyl phosphate, bis-p-nitrophenyl phosphate, p-nitrophenyl decanoate, or p-nitrophenyl palmitate), and cell-free protein extract (check 1, 3, 10, 30, 100, 300, and 1,000 µg) in a total volume of 1 ml in 1 ml plastic cuvettes.
      ​NOTE: Use alkaline pH (Figure 1) when following p-nitrophenyl ester hydrolysis in a continuous assay. Alternatively, use single time-point assays for a range of pH values, adding NaOH at the end of the incubation period to terminate the enzyme reaction and to ensure that all p-NP is present in the phenolate form.
   3. Follow the time course for an increase of absorbance at 405 nm, due to the formation of p-NP, in a spectrophotometer at 30 °C over a 5 min period. Quantify the initially linear formation of p-NP by determining the initial slope of increase of absorbance per time.
   4. Calculate the change of concentration (Δc) for p-NP using the law of Lambert-Beer (ΔA = ε Δc d)¹.
      ​NOTE: ΔA is the linear change of absorbance determined, ε is the molar extinction coefficient at the respective wave length (in units of M^-1 cm^-1), d is the length of the light path (1 cm), and Δc is the change of concentration (in units of M) to be determined.
      1. Considering that the assay volume is 1 ml, calculate the amount of p-NP formed.
         ​NOTE: Amount = concentration x volume.
      2. Calculate the enzyme activity by dividing the amount of p-NP formed by the time in which it is formed. Determine the specific enzyme activity by dividing enzyme activity by the amount of protein (in mg) which was responsible for generating this activity.
   5. Compare absorbance changes provoked by protein extracts in which a candidate gene (smc00171, smc00930, or smc01003) had been expressed with extracts that harbor only an empty vector.
      NOTE: In order to continue with the following steps, the specific activities, caused by protein extracts in which a candidate gene had been expressed, should be at least twice or more than the values obtained for the specific activities caused by protein extracts that harbor only an empty vector.
   6. For further experiments, select those conditions in which hydrolysis of the p-nitrophenyl-containing compound is minimal with cell-free extracts (i.e., empty vector) and for which the most pronounced formation of p-NP and the p-nitrophenolate anion (Figure 1) can be observed when protein extracts are employed, in which a candidate gene had been expressed.
2. After determining the initial enzyme activity in 3.1, optimize the assay conditions for the respective enzyme by varying pH, type of buffer, buffer strength, concentrations of NaCl, detergents such as Triton X-100, and the absence or presence of different bivalent cations.
   1. For different concentrations of each variable, determine the specific enzyme activity (see 3.1.4.2) (the highest number obtained defines the condition of maximal enzyme activity). Use the combination of the optimal conditions encountered for each variable to define an optimized enzyme assay in which each variable is present in its optimal concentration.

Figure 1. p-Nitrophenyl esters as artificial substrates for (phospho)lipases in a spectrophotometric assay. Upon hydrolysis of p-nitrophenyl esters, an acid (R-OH) and p-nitrophenol (p-NP) are formed. Due to the pK_a = 7.2 for the dissociation of the phenolic H⁺ from p-NP, at a pH > 9.2 more than 99% are in the bright yellow p-nitrophenolate form and a molar extinction coefficient of 18,000 M^-1 cm^-1 can be used at a wave length of 405 nm for the quantification of free p-nitrophenolate²². When buffers with a pH of 8.5 were used, the absorbance was determined at 400 nm and a molar extinction coefficient of 14,500 M^-1 cm^-1 was employed²³. Please click here to view a larger version of this figure.

NOTE: After having defined the optimal conditions for the activity of the enzyme of interest, embark on the search for the real/physiological substrate of this lipase. In principle, take two, often complementary, approaches to achieve this goal, an in vivo approach or an in vitro approach.

4. In Vivo Identification of the Physiological Substrate of a Lipase

NOTE: In an in vivo approach, express the lipase of interest in a host organism^8,11 in order to register over time whether expression of the lipase alters the host´s lipid profile. In another in vivo approach, generate a mutant deficient of the gene of interest^8,11 and study whether its lipid profile is distinct from the wild type version^6,8,11. In order to obtain a quantitative assessment of an organism's lipid profile, a simple method consists in radiolabeling cellular compounds, extracting the lipids, separating them by chromatography, and quantifying the radioactively labeled separated lipids.

1. Radiolabeling of lipids.
   1. Prepare an overnight pre-culture of an organism of interest (E. coli or S. meliloti) in 5 ml of the desired culture medium (complex medium or defined minimal medium) and grow at 30 °C.
   2. From the pre-culture, inoculate into 20 ml of the same fresh medium in a 100 ml culture flask to obtain an initial OD₆₂₀ = 0.3 for the culture.
   3. Take an aliquot (1 ml) of the culture under sterile conditions and transfer to a 14 ml sterile polystyrene round-bottom tube.
   4. Add 1 µCi of [1-¹⁴C]acetate (60 mCi per mmol) to the 1 ml culture.
   5. Incubate the liquid culture under agitation at 30 °C for a period of 24 hr.
   6. At the end of the incubation period, transfer the culture to a 1.5 ml microcentrifuge tube and centrifuge at 12,000 x g at room temperature for 5 min.
   7. Resuspend the pellet in 100 µl of water. At this point, store the cell suspension at -20 °C or immediately continue with the extraction of polar lipids (section 4.2).
2. Extraction of polar lipids.
   ​NOTE: The method described here essentially follows the procedure reported by Bligh and Dyer¹⁹.
   1. To the 100 µl of aqueous cell suspension, add 375 µl of methanol:chloroform solution (2:1; vol/vol).
   2. Vortex for 30 sec and incubate for 5 min at room temperature.
   3. Centrifuge 5 min at 12,000 x g at room temperature.
   4. Transfer the supernatant to a new 1.5 ml microcentrifuge tube.
   5. Add 125 µl of chloroform and 125 µl of water, vortex 30 sec.
   6. Centrifuge 1 min at 12,000 x g at room temperature.
   7. Transfer the lower chloroform phase to a fresh tube and dry with a stream of nitrogen gas.
   8. Dissolve dried lipids in 100 µl of chloroform:methanol solution (1:1; vol/vol).
      ​NOTE: At this point, an aliquot of 5 µl of the lipid solution can be quantified by liquid scintillation counting.
   9. For thin-layer chromatographic (TLC) analysis, dry down the remaining 95 µl with a stream of nitrogen gas and redissolve dried lipids in 20 µl of chloroform:methanol solution (1:1; vol/vol). Use a 3 µl aliquot for TLC analysis.
3. Separation of polar lipids by thin-layer chromatography (TLC).
   ​NOTE: Depending on the lipid classes to be analyzed, different combinations of solid and mobile phases might be employed for separation. Here a typical separation for charged polar lipids and another, more suited for neutral polar lipids, using high performance thin-layer chromatography (HPTLC) silica gel aluminum sheets as the solid phase, are outlined.
   1. Separation of charged polar lipids by two-dimensional TLC (2D-TLC).
      1. Apply a 3 µl aliquot of lipid sample in one corner of a HPTLC silica gel aluminum sheet (10 x 10 cm), 2 cm from the edge of the plate.
      2. Prepare and mix the mobile phase (140 ml chloroform, 60 ml methanol, and 10 ml water) for separation in the first dimension.
      3. Coat a TLC developing chamber internally with chromatography paper.
         ​NOTE: This is to ensure that the gas phase of the chamber will be saturated rapidly (within 30 min) after the mobile phase for the first dimension has been added to the chamber and the chamber has been closed with a glass plate.
      4. Prepare and mix the mobile phase (130 ml chloroform, 50 ml methanol, and 20 ml glacial acetic acid) for separation in the second dimension and transfer to a second TLC developing chamber internally coated with chromatography paper and let the chamber saturate.
      5. Carefully transfer the HPTLC silica gel aluminum sheet with the dried lipid sample to the first chamber and develop (i.e., perform chromatography) the plate for 60 min in the closed chamber in the first dimension⁵.
      6. Remove the plate from the chamber and let solvents dry off in a flow hood for 30 min.
      7. After turning the plate by 90 degrees with regard to the previous chromatography, transfer the HPTLC silica gel aluminum sheet, on which the lipids have been separated in one dimension, to the second chamber and develop the plate for 60 min in the second dimension⁵.
      8. Remove the sheet from the chamber and let the solvents dry off in a flow hood for at least 2 hr.
   2. Separation of neutral polar lipids.
      1. Apply 3 µl aliquots of lipid samples on a HPTLC silica gel aluminum sheet starting 2 cm from the edges of the plate. If multiple samples are analyzed in a one-dimensional chromatography, keep a distance of at least 1.5 cm between the different sample application spots.
      2. Prepare and mix the mobile phase (140 ml hexane, 60 ml diethylether, and 8 ml acetic acid) and transfer to a TLC developing chamber internally coated with chromatography paper and covered with a glass plate to let the chamber saturate (30 min).
      3. Transfer the HPTLC silica gel aluminum sheet with the dried lipid samples to the chamber and develop the plate for 30 min in the closed chamber.
      4. Remove the plate from the chamber and let the solvents dry off in a flow hood for 2 hr.
4. Quantification and visualization of separated polar lipids.
   1. Once the developed TLC sheet is dry, incubate it with a photostimulable luminescence (PSL) screen in a closed cassette for 3 days.
   2. Expose the incubated screen to a PSL scanner and acquire a virtual image of the separated radiolabeled lipids.
   3. Perform quantification using PSL software²⁰.
5. Visualization and isolation of individual polar lipids classes.
   1. Incubate developed TLC sheet for 10 min in a chromatography chamber in the presence of 1 g of iodine crystals.
      ​NOTE: Separated lipidic compounds will dissolve the iodine and appear as brownish spots.
   2. Circle the spots with a pencil, compare them to the relative mobility (R_f) of standard compounds (i.e., 1,2-dipalmitoyl-sn-glycerol, dipalmitoyl-L-α-phosphatidylcholine, DL-α-monopalmitin, or palmitic acid), and identify to which lipid class they might belong.
   3. In a fume hood, let the iodine evaporate from the TLC sheet.
   4. With the help of a spatula, scrape the silica gel containing the compound of interest from the sheet, and extract the compound from the silica gel with a mixture of 100 µl of water and 375 µl of methanol:chloroform solution (2:1; vol/vol).
   5. Continue with extraction according to Bligh and Dyer as outlined (4.2.2 onwards).
   6. Store purified lipid class in 100 µl of chloroform:methanol solution (1:1; vol/vol) at -20 °C until use.

5. In Vitro Identification of the Physiological Substrate of a Lipase

NOTE: In an in vitro approach, study whether the lipase of interest can convert a mixture of isolated lipids or individual pure lipids to the corresponding hydrolysis products under the conditions defined as optimal in 3.2.

1. Use pipetting schemes for enzyme assays as per Table 3 for PC-specific phospholipase C SMc00171 (see 5.2), phospholipase A (see 5.3), and DAG lipase SMc01003 (see 5.4) activity.
2. Determination of PC-specific phospholipase C activity (Table 3).
   1. To a 1.5 ml microcentrifuge tube, add 5,000 counts per minute (cpm) of total ¹⁴C-labeled PC and a solution of Triton X-100.
   2. Mix and dry under a stream of nitrogen.
   3. Add diethanolamine-HCl, pH 9.8 buffer, as well as NaCl and MnCl₂ solutions and bidistilled water to obtain a final volume of 99.5 µl. Vortex for 5 sec.
   4. Add 0.5 µl of enzyme (5 µg protein) (i.e., a cell-free extract in which overexpressed SMc00171 is present) to initiate the reaction. Mix briefly.
   5. Incubate at 30 °C for 4 hr.
   6. Stop the reaction by the addition of 250 µl of methanol and 125 µl of chloroform.
   7. Extract lipids as described previously (see 4.2).
   8. Separate lipids by one-dimensional (1D)-TLC (see 4.3.2 and 4.4), and analyze them by PSL imaging.
3. Determination of phospholipase A activity (Table 3).
   1. To a 1.5 ml microcentrifuge tube, add 5,000 cpm of total ¹⁴C-labeled phospholipids and a solution of Triton X-100.
   2. Mix and dry under a stream of nitrogen.
   3. For a final 100 µl assay, add Tris-HCl, pH 8.5 buffer, NaCl solution and water. Vortex for 5 sec.
   4. Add 5 µl of enzyme (50 µg protein) (i.e., a cell-free extract in which overexpressed SMc00930 or SMc01003 is present).
   5. Incubate at 30 °C for 5 hr.
   6. Stop the reaction by the addition of 250 µl of methanol and 125 µl of chloroform.
   7. Extract lipids as described previously (see 4.2), separate them by 1D-TLC using 130 ml chloroform, 50 ml methanol, and 20 ml glacial acetic acid as the mobile phase, and analyze them by PSL imaging.
4. Determination of diacylglycerol (DAG) lipase activity.
   1. Preparation of ¹⁴C-labeled DAG.
      1. Radiolabel S. meliloti cultures (see 4.1) and extract polar lipids (see 4.2) as described. Separate S. meliloti total lipid extracts by 1D-TLC in chloroform:methanol:acetic acid (130:50:20; vol/vol) using conditions described for separation in second dimension in 4.3.1.
      2. Visualize PC by iodine staining and use a pencil to mark the localization of phosphatidylcholine (PC).
      3. Isolate radiolabeled PC as described in 4.5.
      4. Quantify extracted PC by scintillation counting.
         ​NOTE: About 320,000 cpm PC is expected.
      5. Treat PC (250,000 cpm) with 0.1 U of phospholipase C from Clostridium perfringens in 50 mM Tris-HCl, pH 7.2, 0.5% Triton X-100 and 10 mM CaCl₂ for 2 hr in a total volume of 100 µl and stop the reaction by adding 250 µl of methanol and 125 µl of chloroform.
      6. Extract lipids as described previously and separate by them by 1D-TLC (see 4.3.2).
      7. Isolate diacylglycerol from the silica plate and quantify by scintillation counting (as described in 4.2)
   2. Diacylglycerol lipase assay (Table 3).
      1. To a 1.5 ml microcentrifuge tube, add 5,000 cpm of ¹⁴C-labeled DAG and a solution of Triton X-100.
      2. Mix and dry under a stream of nitrogen.
      3. For a final 100 µl assay, add Tris-HCl (pH 9.0) buffer, a NaCl solution and bidistilled water. Vortex for 5 sec.
      4. Initiate the reaction by adding 5 µl of enzyme (50 µg protein of cell-free extract).
      5. Incubate at 30 °C for 4 hr.
      6. Stop the reaction by the addition of 250 µl of methanol and 125 µl of chloroform and extract lipids as described previously (see 4.2).
      7. Analyze neutral polar lipids by 1D-TLC (see 4.1.3.2) and subsequent PSL imaging.