My advice is to slightly warm the substrate solution before assay. Definitely it will work. The same was also happen when I worked.

The substrate was prepared in phosphate buffer

(90 ml) containing gum arabic (100 mg) and sodium deoxycholate

(207 mg). The substrate p-NPP (30 mg) was dissolved in

10 ml of isopropanol and mixed with buffer solution and warm gently. 

ALL D BEST...

-AKM

Thank you, Mr. Kumar Maharana

if I don't use Gum Arabic and Sodium Deoxycholate, it will work? (I dissolve Enzyme in Tris-HCl solution). 

DMSO is a good solvent to dissolve your substrate but must be

Thank you, Mr O'Neill

I also think about that. Maybe The concentration is so high, the kinetic data will be not reliable and the substrate can not be soluble. I will modify concentration and add some solvents or emulsifiers,

Gum Arabic and Sodium Deoxycholate both act as emulsifier therefore it should be added with the substrate pNPP. They will also help to dissolve the substrate in aquous solution.

I will try to do the way you showed. Thank you so much Mr KUMAR Maharana ^^

As pNPP is not soluble in buffer, one has to add a suitable detergent.

The original method was published by U.K. Winkler & M. Stuckmann (J. Bacteriol., Vol. 138, pp. 663-670 (1979)). Schmidt-Dannert et al. used a more suitable detergent, sodium taurocholate (C. Schmidt-Dannert et al., Biochim. Biophys. Acta, Vol. 1214, pp. 43-53 (1994)). 

Another alternative is to use the p-nitrophenyl ester of oleic acid. This has a suitable chain-length, but due to the double bond, it is better soluble and no detergent is required.

For example, using p-nitrophenyl butyrate (one of the commonly used p-nitrophenyl esters for lipase activity assays), a solution (0.62 M) in acetonitrile could be

prepared and then mixed with a solution of acetonitrile and buffer (50 mM, pH 4-7.5) to obtain the appropriate substrate solution. Then, the substrate solution could be incubated with lipase. After incubation at 60°C for 15 min., the lipase activity could be measured by monitoring the absorbance at 405 nm as experimental evidence of p-nitrophenol formation by hydrolysis reaction. 

Article Biosynthesis of ethyl butyrate with immobilized Candida rugo...

Dear Phúc Tô Đình,

I too use pNPP (30mM)  for lipase assay. I dissolve it in Isopropanol (Sigma). Add pNPP slowly in the solvent with appropriate mixing. Store at room temperature.

Once again my answer below. If you don't use a detergent, you might dissolve some of the pNPP, but not enough by just warming up the solution. This is definitely not sufficient to reach saturation concentrations of pNPP and hence all the kinetic data determined are wrong!

As pNPP is not soluble in buffer, one has to add a suitable detergent.

The original method was published by U.K. Winkler & M. Stuckmann (J. Bacteriol., Vol. 138, pp. 663-670 (1979)). Schmidt-Dannert et al. used a more suitable detergent, sodium taurocholate (C. Schmidt-Dannert et al., Biochim. Biophys. Acta, Vol. 1214, pp. 43-53 (1994)).

Another alternative is to use the p-nitrophenyl ester of oleic acid. This has a suitable chain-length, but due to the double bond, it is better soluble and no detergent is required.

Thank you so much Mr. Uwe T Bornscheuer

Your advise and information made me understand the problem. I found out the suitable detergent for my case.

Could you explain the wrong kinetic data to me if the pNPP concentration is unsaturated? Thank you

Dear Mr. Karthikumar Sankar 

I tried to do it. pNPP was very slowly soluble. I stored at room temp during 3 days but it still didn't solve completely. however, it's ok if I use a low concentration.

Thank you for your advise

Dear Mr. Daniele Spinelli

Thank your for your help ^^ before I read your comment, I had tried to use acetonitrile when I  had refered some journals.And It worked. I used Isopropanol : Acetonitrile with ratio 1:1. And 20mM pNPP completely solved. Your advise is so helpful.

Thank you so much

How to follow this question?

You see the upright corner "Follow this question". click that ^^

Thank you, Mr. Sachin Vyas ^^

I just prepared a solution for the lipase activity assay, but aiming at an end concentration of 0.1 mM of pNPP. I solved 3,8mg in 10ml isopropanol and it was compleatly solved after a few minutes. Then i diluted 10x in substrate buffer (50mM Sodium phosphate monobasic) and 5mM Sodium deoxycholate. And is see nice color development

Thank you, Mr. Thomassen :)

Can you tell me what you use sodium phosphate monobasic and Sodium deoxycholate for? What is function of this substrate buffer?

The Sodium phosphate is to buffer the solution to a stabile pH (of 7,5 in my case), the Sodium deoxycholate is a emuslifying bile salt to help emulsify the lipase substrate

thanks to all researchers for this important data

Hello everyone,

Firts thanks for this data.

I need your help if you don't mind, i use Maria G.Savviou et al method for the enzymatique assay of lipase using PNPB as substrat.

in this article they used a lipase boiled for 10min as blank for measuring the absorbance of the released p-nitrophenol after the enzyme reaction. I did the same procedure but when i boiled the lipase, it precipited and i can't used as blank. Can i use pNPB+buffer without enzyme as a blank?

Hello,

Yes you can use pnpp and buffer as blank you can add some calcium for better activity of lipase I will recommend you the following method which I have use in our lab, it works pretty well,

You can increase the substrate conc. And then plot the slope of reaction and compare them,

Hope it works and enjoy lipase assay

yahya

Reaction solution cosist of enzyme solution(0.005 mg/ml

of purified lipase in glycine buffer pH 8.75 100mM), buffer solution(glycine buffer pH 8.75

100mM) and substrate solution(2 mM p-NPP in 2-propanol). 10μl of enzyme solution was added

to 180μl of buffer solution and equilibrated for 5 min at 40°C in the multiplate reader then 10μl of substrate solution was added to the mixtur so final concentration in each well was 2.5 μg/ml and 0.1 mM respectivelly for lipase and p-NPP. final absorbance after 3 min used for activity

measurement. One activity unit define as the hydrolyzing of 1.0 μmol p-NPP per minute. the

extinction coefficient of p-Nitrophenol at 410 nm was 18.3 mM at pH 8.75 at 40 °C.

I will add our article associated to this method in advance.

Thank you for your answer.

I will try your method also.

Just i want to know what is the color of your substrate solution ? because for me, when i add the PNPB (14mM in 2-propanol) (clear) to (phosphate/citrate buffer (50mM; pH=7) + 100µL triton x-100 + 0.025g gum arbic ) it became a little yelow !? i don't know why?

Yes because pnpp might not be stable like any other molecules that form color, and you have prepared high concentration of it , gum Arabic make data reproducible but it consist of many carbohydrate and proteins so I didn’t use that.

Hello Yahya,

Thank you so much for your advices :)

how long can we stock the solution of PNPP ? or we should used it immediaetly after preparation?

Hello Yahya.

To test different methods of measurement of lipase activity, i used a commercial enzyme with 100000IU/g. I want to know how we can determine the concentration of my enzyme on mg/ml. because i didn't found the appropriate concentration on the bottle.

Should i prepare in this case, a solution stock of this enzyme and use different concentrations that i determine by my self, as we do it with BSA when we use it for protein assays?

Hello Adjout,

Sorry, I saw the question right now, Well. It would be better to use your substrate (in around 1 day ) and not more than a week,

secondly, you want to determine the concenteration of your commercial enzyme, well:

1- it might not be bad to ask te company which you buy the product they might give you the purity of their sample

2- if you couldn't get that information, you should check for purity of your smaple and see what are other commponents , for exmaple is the enzyme you are using is cloned in bacteria or fungi is very importnat,

3- you can use chromatography for farther purification of your enzyme, becuse if you want to study biochemical properties we should get around 90% purity ( most say 95%)

4- the first experiment should be electrophoresis, the best method is to run sds page/or native electrophoresis, then look for the bonds and see how many bonds you have , I recemmend 12 % gel , if you see multiple bonds around the gel it is an impure enzyme sample,

please run electrophoresis in differnt concenterations so that you could analyze your results, This the first step form my point of view :)

Dissolve substrate in isopropanol simply (20mM) in 5mL.

Hello, I am trying to do the lipase activity test and for this I prepare a stock solution of substrate (pNPP) at 50mM, I dissolve it in acetonitrile: isopropanol 1: 4 (v: v) and it dissolves completely after a few minutes. However to have a final substrate concentration of 1mM I prepare a working solution where I add a volume (100 μL) from the stock substrate and mix it with the reaction buffer but I have already tested with 0.3% Triton X-100, with 5mM sodium deoxycholate, but I still have turbidity in the work solution so I can not complete the trial, could someone please suggest a solution for this problem?

Make pNPP solution in isopropanol and slightly warm to dissolve it. I would like to add important tip here that use freshly prepared reagent .Because it is not stable.

more important is to make the solution of stock freshly.

The only scientific valid and verified method is to add a detergent such as taurodeoxycholate. See literature: U.K. Winkler, M. Stuckman, Glycogen, hyaluronate and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens, J. Bacteriol. 138 (1979) 663–679. See also: Gupta et al., Simplified para-nitrophenyl palmitate assay for lipases and esterases, Analytical Biochemistry 311 (2002) 98–99

I want to make a stock solution of p-NPA (20mM) in 1:1 acetonitrile and isopropanol for esterase activity assay. Uwe T Bornscheuer Phúc Tô Đình Khem Raj Meena Can I dilute it to desired concentration using 1:1 acetonitrile and isopropanol or can I use water too?