Dear Agnieszka, I have copy and paste the method from my PhD thesis, I had worked with this method for years with no problem. Please compare with what you are doing and let me know for any problem, good luck

Lipase activity assay

Determination of liberated free fatty acid was measured by colorimetry method (Kwon and Rhee, 1986) using olive oil as substrate. The reaction mixture, consisting of 1 ml crude enzyme (culture filtrate), 2.5 ml olive oil emulsion (properly mixed of an equal volume olive oil with 50 mM sodium phosphate buffer, pH 7.0) and 0.02 ml of 20 mM CaCl2, was incubated in a water bath shaker for 30 min at 50°C under 200 rpm agitation. The enzyme reaction in the emulsion system was stopped by adding 6 M HCl (1 ml) and isooctane (5 ml), followed by mixing for 1 min. The upper isooctane layer (4 ml) containing the free fatty acid was transferred to a test tube and properly mixed with 1 ml copper reagent.

The reagent was prepared by adjusting the solution of 5% (w/v) copper (II) acetate-1-hydrate to pH 6.1 with pyridine. The free fatty acid dissolved in isooctane was determined by measuring the absorbance of the upper layer at 715 nm after mixture settlement. Lipase activity was determined by measuring the amount of free fatty acid released based on the standard curve of oleic acid (0-50.0 µmole) in isooctane. One unit of lipase activity was defined as 1.0 μmol of free fatty acid liberated min−1 and reported as Uml−1.

It is 2-50 micromoles of fatty acid in 5 ml of isooctane. Also u need to be precise for the time duration of vortexing as it will affect extraction of fatty acids in solvent phase. Further details you can find from the paper itself. Find the attachment..

All d best.

Dear Mamta, Thank you very  much for your answer. I prepared 0.5 - 10 micromoles in 2,5 ml of isooctane. I have beauty orange halo on plates with Rhodamine B (pH=10) and olive oil, however  there is no lipase activity when I do lipase assay. Could you tell me, please,  how to prepare the oil emulsion for the assey? I take 1 ml of olive oil, 1 g of arabic gum and 20 ml of buffer pH=10 (homogenized 1 min).

Yours,

Agnieszka

Dear Agnieszka, I have copy and paste the method from my PhD thesis, I had worked with this method for years with no problem. Please compare with what you are doing and let me know for any problem, good luck

Lipase activity assay

Determination of liberated free fatty acid was measured by colorimetry method (Kwon and Rhee, 1986) using olive oil as substrate. The reaction mixture, consisting of 1 ml crude enzyme (culture filtrate), 2.5 ml olive oil emulsion (properly mixed of an equal volume olive oil with 50 mM sodium phosphate buffer, pH 7.0) and 0.02 ml of 20 mM CaCl2, was incubated in a water bath shaker for 30 min at 50°C under 200 rpm agitation. The enzyme reaction in the emulsion system was stopped by adding 6 M HCl (1 ml) and isooctane (5 ml), followed by mixing for 1 min. The upper isooctane layer (4 ml) containing the free fatty acid was transferred to a test tube and properly mixed with 1 ml copper reagent.

The reagent was prepared by adjusting the solution of 5% (w/v) copper (II) acetate-1-hydrate to pH 6.1 with pyridine. The free fatty acid dissolved in isooctane was determined by measuring the absorbance of the upper layer at 715 nm after mixture settlement. Lipase activity was determined by measuring the amount of free fatty acid released based on the standard curve of oleic acid (0-50.0 µmole) in isooctane. One unit of lipase activity was defined as 1.0 μmol of free fatty acid liberated min−1 and reported as Uml−1.

Dear Agnieszka, I forgot to change the assay temperature that will be based on your enzyme optimum T, since mine was thermostable lipase the assay T was 50C and above.

Dear Afshin, thank you very much for your help. I prepared calibration curve: 0.5 - 10 micromoles in 2,5 ml of isooctane. I have beauty orange halo on plates with Rhodamine B (pH=10) and olive oil, however there is no lipase activity when I do lipase assay. To prepare oil emulsion i used 1 ml of olive oil, 1 g of arabic gum and 20 ml of buffer pH=10 (homogenized 1 min).  I do not know what is wrong. Does my strain has no lipase activity?

Yours,

Agnieszka

Of course your organism has lipase activity, but you need to optimize the assay condition, please pay attention that, presence of Ca is very important since many of lipases are Ca dependent, hence you need Cacl2 in your assay mixture, moreover, some mesophiles produce thermozymes, therefore check the activity in 30, 40 and 50 C may give you some info about the best T, in addition to me is better to check the activity first in a neutral pH, generally Tris-HCl give higher activity compare to phosphate buffer that give higher stability. What I can see from your method, the oil ratio is low, please mix the buffer and oil in 1:1 ratio and add the CaCl2, then vortex it for about 30s until it is milky in color, then use 2.5ml + 1ml enzyme, incubate in water bath under 200rpm (agitation is very important), after stopping the reaction also Mamta mentioned the vortexing at least for 1min in high speed is help to extract the fatty acids released. Please follow the method I have paste and let me know for any problem.

One more thing, are you sure you are using the right lipase production medium for your organism?  

Good luck

Many thanks for your help !!!!

Dear Afshin,

One more question: did you use highly refined, low acidity olive oil (Sigma) as a substrat for enzyme, or cooking olive oil (I used the last one).

Agnieszka

This you need to find out yourself by trial and error. Primarily because after the enzyme extraction, you may get different levels of activity of the enzyme. Therefore, telling you to certain standards for your standard curve may or may not work.  Now you must be wondering why your enzymes will show different activities, again it is because the nucleotide sequence for most of the lipase are highly polymorphic.

Thank you

Dear Agnieszka, 

Same as you I have always used normal cooking olive oil (bertolli classic), for other oils also I used whatever I could find in supermarket. Don't worry about the substrate, your enzyme will do its job if it is in its working condition (pH & T) and has access to essential factors such as CaCl2.

What I'm going to ask again is about production medium, since most of the microorganisms need inducers for production the extracellular hydrolytic enzymes such as lipases, proteses, amylayses, ... In fact these organisms mostly produce these enzymes to solve their problem to find the food, if you give plenty of sugar and simple carbohydrates they don't  need to waste  their energy for production of this type of enzymes but if the source of easy food is limited they try to get new food based on their environment, if lipid and oil is available, the lipase gene (if is available) will be induced, if medium is contain protein the protease gene (if is available) will be induced and so on, therefore content of the production media is very important for production of wild type extracellular hydrolytic enzymes (Please have a look on my BMC Biotechnology paper 2008). If your organism has the lipase gene (that based on the rodamin plate it has) but you don't use the suitable production medium you never get the activity, whatever assay you use.

Good luck    

As precised by Afshin, to test extracellular products produced by bacteria you should use substrate or media enriched on specific product. To detect lipase production by bacteria strains I often use media enriched on Tween 20 or Tween 80 and spot on surface culture to test and reveal easier eventual production after incubation in appropriate conditions and time. This method is very intresting and easy to test. Please let me know if you need more details, I can send you the detailed protocol.

Dear Monia,

Could you please send me the detailed protocol?

Thank you

Agnieszka

Can you used pera nitrophenyl butarate as substrate? it gives good result for lipase assay

Can you used pera nitrophenyl butarate as substrate? it gives good result for lipase assay

Dear Kamlesh,

Thank you for your suggestion. Agnieszka

Dear Kamlesh,

Good suggestion, but as we know and based on the definition,  lipase vs. esterase is the enzyme that is able to cut the carbon chains with more than 10 C, where esterases are able to cut below 10 C carbon chains. Hence different size of carbon chains connected to nitrophenyl ring are used for different purposes. Nitrophenyl butarate  is suitable for esterase assay, where for lipase assay nitrophenyl palmitate or oleate is suitable. Anyway their structures are different than real lipids and oils and based on the research purposes not everybody prefer them. 

Dear Afshin, you are absolutely right.

Agnieszka

Dear Agnieszka, 

I"ve been away and will send you by today later the full protocol I still using to test lipase production

Keep in touch 

Monia 

Thank you Monia. Bye

Have you try with colorimetric assays using p-nitrophenylesters? In this case, one unit of activity was defined as the amount of enzyme to form 1 micromole of p-nitriphenol per minute under the assay conditions. For details of the procedure see Spinelli et al. (2014) Biocatalysis 1, 1-12.

Dear Agnieszka,

             We used  4-Nitrophenyl palmitate along with Triton X100 as substrate for alkaline lipase assay. This is rapid(2 min) and sensitive.You can determine the activity even at lower range at 410nm.For this no incubation is needed. The only thing is you have to do the assay in buffered condition.

Use of nitrophenyl palmitate may not be specific for lipase, right? It gives a measure of esterase activity.

You can use p-Nitrophenyle Acetate or Butarate as a substrate and phosphate buffer 6.5 You can determine the activity even at lower range at 410nm.

Thank to Afshin. The given protocol is very good and i also had need this.

Do you agree that 4-Nitrophenyl palmitate is not specific for "true" lipase?

May be as per my study.

Hi can you give me the protocol for pnp test for lipase